SCHOOL OF SCIENCE BiomedicalResearchJournal APRIL 2015 | VOLUME 2 | ISSUE 1 pISSN: 2349-3666; eISSN: 2349-3674 SCHOOL OF SCIENCE BiomedicalResearchJournal APRIL 2015 | VOLUME 2 | ISSUE 1 EDITORS-IN-CHIEF EDITORIAL BOARD Dhananjaya Saranath (Mumbai, India) Ali Syed Arbab (Detroit, USA) Alpana Ray (Missouri, USA) Aparna Khanna (Mumbai, India) Amit Agarwal (Bangalore, India) Anandwardhan Hardikar (Sydney, Australia) Anjali A. Karande (Bangalore, India) Ashok B. Vaidya (Mumbai, India) Basuthkar J. Rao (Mumbai, India) Dhirendra Bahadur (Mumbai, India) SECTION EDITORS Hemant Malhotra (Jaipur, India) Karuna Shanker (Lucknow, India) Cancer Biology: Kirti S. Laddha (Mumbai, India) Mayur Yergeri (Mumbai, India) Girish Maru (Navi Mumbai, India) Mohan C. Vemuri (Frederick, USA) Naganand Rayapuram (Evry, France) Stem Cell Biology: Nancy Pandita (Mumbai, India) Partha Basu (Kolkata, India) Vaijayanti P. Kale (Pune, India) Paul J. Verma (Rosedale, Australia) Prasad S. Adusumilli (New York, USA) Nanotechnology: Pritish Bhattacharya (New Jersey, USA) Pulok Mukherjee (Kolkata, India) Vilas G. Gaikar (Mumbai, India) Purvish M. Parikh (Mumbai, India) Ramesh Goyal (Ahmedabad, India) Phytochemistry: Sai Yendamuri (New York, USA) Sukhinder Kaur Cheema (St. John's, Canada) Lokesh Bhatt (Mumbai, India) Sumitra Chanda (Rajkot, India) Sunita Saxena (New Delhi, India) Surinder K. Mehta (Chandigarh, India) Tania Fernandez (San Francisco, USA) Victoria M. Villaflor (Chicago, USA) EDITORIAL OFFICE EDITORIAL ASSISTANT School of Science, NMIMS (Deemed-to-be University) Bhaidas Sabhagriha Building, Bhaktivedanta Swami Marg, Vile Parle (W), Mumbai 400056, India. Email: brj.sos@nmims.edu Brijesh S. 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Contents April 2015, Volume 2, Issue 1 Editorial: Precision/personalized medicine in cancer Dhananjaya Saranath and Aparna Khanna ..........................................................................................1 Advances in omics technologies in GBM Uday B. Maachani, Uma Shankavaram, Kevin Camphausen and Anita Tandle ..................................6 Dendrimers based electrochemical biosensors Saumya Nigam, Sudeshna Chandra and Dhirendra Bahadur ...........................................................21 Developmental signalling in maintenance and regulation of cancer stem cells Sweta Dash, Raghava Reddy Sunkara and Sanjeev K. Waghmare ..................................................37 Diverging role of Nrf2 in cancer progression and prevention Lokesh Gambhir, Rahul Checker, Deepak Sharma and Santosh K. Sandur .....................................57 Physiology of embryo-endometrial cross talk Deepak N. Modi and Pradeep Bhartiya …………………..…………….............................………….....83 Human EGFR-2, EGFR and HDAC triple-inhibitor CUDC-101 enhances radiosensitivity of glioblastoma multiforme cells Cody D. Schlaff, W. Tristram Arscott, Ira Gordon, Kevin A. Camphausen and Anita Tandle ............105 Phenotypic and functional characterization of a marrow-derived stromal cell line, M210B4 and its comparison with primary marrow stromal cells Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale ........120 Editorial Precision/Personalized Medicine in Cancer Dhananjaya Saranath and Aparna Khanna Recent technological advances have provided decisions. unprecedented develop including 'Next Generation Sequencing' 'Precision/ (NGS) with both availability and affordability Personalized Medicine', with confluence of will enable understanding of cancer and other medicine and technology making significant diseases, a feasible proposition. platforms opportunities for to implementing Thus, advanced technology advances in treatment. The Cancer Genome Next Generation Sequencing is massively Atlas (TCGA), a large scale initiative started in parallel sequencing enabling rapid sequencing 2006, to generate a comprehensive landscape of the entire genome or exome sequencing on for identification of alterations in tumor types whole genome or cDNA (RNA-Seq), builds with a view to develop better therapies, is on a on the concept of 'NGS taking us into wind down. The next phase is use of the expanded genomic testing for risk prediction, information 'Precision/ diagnosis, prognosis, treatment response and Personalized Medicine'. Implementation of disease free survival or overall survival in real 'Precision/Personalized Medicine' requires time'.The understanding of the mechanisms understanding of the biology of each cancer and processes in cancer with single aberrations type with precise definition of the cancer or cumulative alterations including mutations, genome. The different genome alterations will rearrangements, amplifications, deletions, identify the 'Founder Mutations' involved in insertions and other alterations in cancer will the early phase, but may not be associated with be discerned. It is important to remember that a fully transformed phenotype; 'Driver several countries have initiated studies in this Mutations' required for fully transformed direction. Prime Minister David Cameroon, phenotype; Mutations' UK, endorsed the 'Genomes Project' for considered as collateral damage. Treatment collection of data for whole genome sequences against key oncogenic driver mutations in from 100,000 individuals, to be completed by individual cases with targeted drugs will be the 2017, sanctioning USD 475 million for benefit of the 'Precision Medicine' approach. sequencing studies, with a view to better Rational choices for treatment have to be understand preceded by full genomic data and expression cancer. Barack Obama, President, USA, data, launched the 'Precision Medicine' initiative generated and facilitating for 'Passenger combination therapy complex diseases including Biomed Res J 2015;2(1):1-5 Editorial: Precision/personalized medicine in cancer 2 with a USD 215 million for genomic data on examples are targeted drugs Dabrafenib and one million volunteers to accelerate patient Trametinib, mitogen activated protein kinase powered research that promises to accelerate 1/2 biomedical discoveries and provide clinicians BRAFV600E/K; with new tools, knowledge and therapies for Bevacizumab, against vascular endothelial individual patients. A result of better factor A is a targeted therapy in cancers of the understanding of cancer, is the current colon, lung, breast, kidney and brain; whereas repertoire of targeted therapy drugs and Ramicirumab, a monoclonal antibody against personalized medicine on the global scene. vascular endothelial growth factor receptor 2, (MAPK1/2) for melanoma angiogenesis with inhibitor, In the past decade the advent of targeted is used in gastric cancer and Non-small cell therapy and new tailored drugs has led to a lung cancer, and in combination with revolution in treatment of lung cancer, with Docetaxel improves outcomes in bladder larger benefit, lower toxicity and better quality cancer. HER2 gene antibody – Herceptin, of life for the patient. The treatment is often shows substantial survival benefits in all based on molecular profiling of individual newly diagnosed and recurrent breast cancer patients with identified cancer, as also patients indicated in other cancers with a similar expression of the gene. Development of molecular profile. Thus, Tyrosine kinase companion inhibitors including Erlotinib, Gefitinib, pathogenic molecular alterations and new Afatinib targeting epidermal growth factor targeted receptors (EGFR), and ALK Inhibitors guidelines for several molecular diagnostic including Crizotinib, Ceritinibare beneficially tests used in patients with aberration of the genes in Personalized Medicine' will enable the current Non-small cell lung cancer, and indicated for oncologists to 'Win the War against Cancer'. with diagnostics drugs are amplification go and indicating hand-in-hand, available. Thus, overthe and 'Precision/ additional cancers with the appropriate The current issue includes a review paper molecular profile. Involvement of Kras, and an original article on glioblastoma EGFR, ALK, HER2, Braf, MET, AKTI, multiforme MAP2KI, PI3KC genes have been identified aggressive brain tumor, a cancer with bad in lung cancers, opening possibilities of prognosis and median survival of 15 months. targeted therapy with consequential benefits. The conventional treatment of GBM using the Thus, an additional aspect which has emerged strategy is combination therapy using two or more chemotherapy, targeted drugs, or targeted drugs plus the incrementally in the last 30 years. With the conventional chemotherapeutic drugs. A few advent of molecular biology and consequent Biomed Res J 2015;2(1):1-5 (GBM), of one surgery, has of the radiation advanced most and only Saranath and Khanna 3 improved understanding of basic tumor for better contemporary treatment. biology, targeted therapies have become Nanomaterials and nanoparticles cornerstones for cancer treatment. Several including dendrimers, polymers, nanotubes, signaling pathways including RTKs/PI3K/ oxides, and enzymes and their hybrids as AKT/mTOR/VEGF/VEGFR are deregulated catalysts for various sensors such as glucose in in sensors, DNA sensors, neurotransmitters and sensors, are another facet of technological progression of GBM. Dr. Anita Tandle and advances with tremendous applications in colleagues from National Cancer Institute, health sciences. Dendrimers are synthetic Bethesda, Maryland, USA, discuss the Omics nanoscale compounds with unique properties, of GBM and applications in novel therapies, in resulting the article, ‘Advances in Omics Technologies applications. Dendrimers have a number of in GBM’. The authors survey the technologies features that make them ideally suited for of genomics, transcriptomics, epigenomics, sensor applications, such as, its high surface proteomics, post area, high reactivity, easy dispersability and transcriptional modifications of microRNAs rapid fabrication. Dr. Saumya Nigam and Dr. in GBM. A comprehensive information in Dhirendra Bahadur, Indian Institute of GBM will lead to better understanding of the Technology Bombay, Mumbai, along with Dr. cancer, signal Sudeshna Chandra, NMIMs (Deemed-to-be) transduction pathways, and identify key University, present a review on ‘Dendrimers molecules associated with the pathogenesis, based electrochemical biosensors’. The culminating in development of new drugs and review highlights the advanced development 'Personalized treatment'. The original article of by the group, ‘EGFR 2, EGFR and HDAC electrochemical triple dendrimers. A must read review for all to GBM, playing tumorigenesis, a treatment major resistance metabolomics highlight inhibitor the role and various CUDC-101 enhances radiosensitivity of GBM cells’, convincingly in effective, biomedical rapid and and biosensors industrial versatile based on understand the technology. shows enhancement of in vitro radiosensitivity The concept of cancer stem cells (CSC) of GBM and breast cancer cell lines proposed earlier in the year 2000, are now well selectively, with no effect on normal human accepted to play a critical role in cancers. The lung fibroblast cell line. The radiosensitization CSCs are more of an enigma and relatively of the cancer cell lines was attributed to more difficult to decode the biology of CSCs. inhibition of DNA double stranded break The conserved Wnt/β-Catenin, Notch and repair and modulation of cell cycle. A better Sonic Hedgehog pathways regulate stem cell understanding of the cancer will open avenues pluripotency and cell fate decisions during Biomed Res J 2015;2(1):1-5 Editorial: Precision/personalized medicine in cancer 4 normal embryonic development and adult cells’ by Dr. Vaijayanti Kale and colleagues tissue homeostasis, and aberrant activity from the National Centre for Cell Science, within these pathwaysis displayed in several Pune, emphasizes importance of alternative cancers. Human cancers contain a relatively systems for investigating regulation of dormant hematopoietic stem cells (HSCs). The authors cell population, CSCs, with characteristics similar to normal stem cells. showed Convincing evidence indicates that CSCs are unequivocally responsible adipogenic lineage, and exhibited a higher therapy for chemotherapy/radiation resistance, maintenance that the cell line M210B4 differentiated towards and HSC-supportive ability and conclude that the consequent recurrence of the cancer. The roles cell line M210B4 is an appropriate substitute of Wnt, Notch and Hedgehog pathways in to study HSC regulation in vitro. cancers and their deregulation are of critical The transcription factor Nrf2 containing significance, directly linked to CSCs. In order the conserved basic leucine zipper structure to target the CSCs therapeutically, it is belongs to the Cap 'N' Collar family, and plays imperative to understand the molecular a critical role in cell defense and survival mechanisms regulating CSCs responsible for pathways. Nrf2 often protects cells and tissues maintenance and recurrence of cancer, and from develop combination therapies to target CSCs transcription of cytoprotective genes, and inhibiting the cumulative action of the hence deregulated genes. Dr. Sanjeev Waghmare and protecting against redox-mediated injury and his colleagues from Advanced Centre for carcinogenesis. Paradoxically, the flip side of Treatment, Research and Education in Cancer, Nrf2 is protection of cancer cells from Navi the chemotherapeutic agents and/or radiotherapy intricately complex signalling cascades of resulting in resistance to the therapy and Wnt, Notch and Hedgehog genes, regulation cancer and maintenance of normal developmental upregulated in several cancer types, and processes, and their association in cancer, in associated with poor prognosis in cancer the article, ‘Developmental signalling in patients. The dilemma of the dual action of maintenance and regulation of cancer stem Nrf2 has been well reviewed in the article, cells’. Whereas, the article, ‘Phenotypic and ‘Divergent functional the progression and prevention’ by Dr. Santosh maintenance and regulation of a marrow- Sandur and colleagues from Bhabha Atomic derived stromal cell line, M210B4 and its Research Centre, Mumbai. The review comparison with primary marrow stromal indicates a wider approach with better Mumbai, succinctly characterization Biomed Res J 2015;2(1):1-5 review in toxicants and considered progression. role of carcinogens via chemopreventive, Nrf2 Nrf2 is aberrantly in cancer Saranath and Khanna 5 comprehension of the mechanisms of action of infertility and intra-uterine growth retardation Nrf2 and consequent design and development are minimized or avoided in normal fetal of drugs to handle the upregulation or growth. The highly orchestrated embryo- downregulation of Nrf2 in the preventive, endometrial cross talk involves a plethora of protective or destructive niche of normalcy molecules including hormones, cytokines, and diseases. Thus, 'One fit for all' is not a growth factors, specific immune modulating feasible solution in all conditions indicating factors, to create the appropriate micromileu importance for establishing pregnancy. Dr. Deepak Modi of 'Precision/Personalized Medicine'. and Mr. Pradeep Bhartiya from the National The mechanisms of embryo implantation Institute for Research in Reproductive Health, and development resulting in pregnancy are Mumbai, take us through the ‘Physiology of comparable to cancer with respect to the embryo-endometrial cross talk’ lucidly growth of highlighting the various processes and A receptive endometrium, interactions. The networking interactions and normal blastocyst, cross talk between fetal and intricate physiology in a pregnancy is very maternal compartments remodeling uterine well explained. The applications in a clinical vasculature, and selector activity comprise scenario innate requirements for successful pregnancy. infertility treatment and development of Adverse contraceptive drugs are discussed. processes development. events and such mechanisms as preeclampsia, for successful implantation, Biomed Res J 2015;2(1):1-5 Review Advances in OmicsTechnologies in GBM Uday B. Maachani, Uma Shankavaram, Kevin Camphausen, Anita Tandle* Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda MD,USA Glioblastoma multiforme (GBM) is one of the most lethal human cancers and poses a great challenge in the therapeutic interventions of GBM patients worldwide. Despite prominent recent advances in oncology, on an average GBM patients survive 12–15 months with conventional standard of care treatment. To understand the pathophysiology of this disease, recently the research focus has been on omics-based approaches. Advances in high-throughput assay development and bioinformatic techniques have provided new opportunities in the molecular analysis of cancer omics technologies including genomics, transcriptomics, epigenomics, proteomics, and metabolomics. Further, the enormous addition and accessibility of public databases with associated clinical demographic information including tumor histology, patient response and outcome, have profoundly improved our knowledge of the molecular mechanisms driving cancer. In GBM, omics have significantly aided in defining the molecular architecture of tumorigenesis, uncovering relevant subsets of patients whose disease may require different treatments. In this review, we focus on the unique advantages of multifaceted omics technologies and discuss the implications on translational GBM research. INTRODUCTION Glioblastoma (GBM) recurrence. As the name infers, it is Brain tumors account for about 85–90% of all multiforme microscopically showing regions primary central nervous system (CNS) tumors. of Worldwide, approximately 343,175 new cases multiforme genetically with various genetic of brain and other CNS tumors were diagnosed alterations leading to its aggressive nature. pseudopalisading and hemorrhage, in the year 2012 (http://www.cbtrus.org/). The standard of care for treatment of GBM Glioblastoma or Glioblastoma multiforme includes surgical resection followed by (GBM) is the most lethal and clinically radiation and chemotherapy. The addition of a challenging of brain tumors. Most patients die chemotherapeutic agent, Temozolamide in of their ailment in less than a year (Stupp et al., recent years changed the median survival of 2005). Some of the reasons for high fatality for GBM patients to 14.6 months from 12.1 are the complex nature and diffuse character of months with surgery and radiotherapy (Stupp the tumor itself and the high rate of disease et al., 2005). Also, currently there is no Key words: Glioblastoma, Omics, Genomics, Transcriptomics, Epigenomics, Proteomics, Metabolomics. *Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive Magnuson Clinical Center Room B3-B100, Bethesda MD 20892, USA. Email: tandlea@mail.nih.gov Biomed Res J 2015;2(1):6-20 7 Maachani et al. standard of care available for recurrent disease instrumentation and bioinformatics data and most of the patients die. Hence there is an analysis have reshaped how we view the urgent need to develop molecular targeted cancer genome (Vucic et al., 2012). “Omics” therapy for this devastating disease. refers to the study of cancer as a whole entity Some responsible of the for molecular molecules. It includes (but not limited to) therapeutic resistance includes genetic and DNA mutations, copy number changes, epigenetic alterations, activation of stem cell epigenetic changes like DNA methylation, pathways, tumor transcriptome analysis and whole-genome microenvironment and cellular metabolism. DNA/RNA sequencing. The omics-based However, the functional consequences of recent many of these alterations are largely unknown transcriptomics, epigenomics, proteomics and in GBM tumorigenesis (Frattini et al., 2013; metabolomics have unveiled the molecular Schonberg et al., 2013). mechanisms behind various cancers and changes progression focusing on the various micro- and macro- and and GBM alterations in the approaches including genomics, assisted in identification of next-generation Omics molecular markers for early diagnosis, With the sequencing of the human genome, the prognosis, predictive of response to treatments study of biological systems underwent a major and predisposition to gliomas (Cho, 2010; genomic revolution. The major technological Chin, 2013) (Fig. 1). The publically available breakthroughs multi-omics in high-throughput assay development, technological advancements in databases collected by International Cancer Genome Consortium Figure 1: Omics in Glioblastoma. Biomed Res J 2015;2(1):6-20 8 Advances in omics technologies in GBM (ICGC) and The Cancer Genome Atlas attempted to correlate mRNA signatures with (http://cancergenome.nih.gov/) network the grades of gliomas and their clinical group using a sample cohort of several behavior to aid in overall prognosis and hundred clinical specimens of GBM further treatment response of patients (Mischel et al., elaborated the molecular processes funda- 2004; Kim et al., 2002). Transcriptomics is the mental to GBM pathogenesis (Hudson et al., study of RNA transcripts that are produced by 2010; Verhaak et al., 2010). the genome, under specific circumstances or a in a specific cell using high-throughput Genomics/Transcriptomics methods, such as microarray analysis, Early work on gene expression analysis of allowing the identification of genes that are gliomas employed DNA microarrays and differentially expressed in distinct cell Table 1: Role of Omics in biomarkers identification and disease prognosis GBM Biomarkers Role in GBM prognosis EGFR amplification EGFR amplification is the most common event in primary GBM , with EGFRvIII being the most EGFRvIII mutation prominent mutated receptor tyrosine kinase receptor occurring in ~50% of GBM cases that overexpress EGFR (Verhaak et al., 2010). A potential predictive biomarker for molecular therapies. PDGFRA PDGFRA is mainly mutated and expressed in abnormally high amounts in proneural tumors (Verhaak et al., 2010) and associated with poor prognosis in IDH1 mutant GBM (Brennan et al., 2013). TP53 mutation TP53 gene although mutated, has no predictive or prognostic role. Can distinguish tumor grade (Brennan et al., 2013). 1p/19q Co-deletion 1p/19q co-deletion is the most common genetic alteration in oligodendroglioma tumors and is associated with favorable response to chemotherapy, radiation and survival (Alaminos et al., 2005). MGMT promoter Promoter methylation of MGMT gene, inactivates DNA repair function (Esteller et al., 1999). It is methylation the first predictive epigenetic biomarker with a putative diagnostic role in detecting pseudoprogression. MGMT methylation helps in molecular stratification of patients for Temozolomide therapy (Malstrom et al., 2012). VEGF VEGF is considered to be the driving factor of tumor angiogenesis and has been identified in 64.1% GBMs. It is a strong predictor of survival , in patients with gliomas (Reynes et al., 2011) PTEN A gene level biomarker with poor survival outcomes for GBM (Baeza et al., 2003). PTEN is deleted in 50–70% of primary and 54%–63% of secondary GBM. Also mutated in 14%–47% primary GBM. Mutation is linked to resistance to targeted EGFR inhibitors in GBM (Deberardinis et al., 2008). IDH1/2 mutation IDH1 mutation is now recognized as an important driver in the etiology of low-grade and secondary brain tumors (48). Has prognostic value in WHO grade III and IV GBM. Accumulation of oncometabolite 2-hydroxygluatrate (2HG) considered as metabolomic imaging biomarker for mutant IDH1 gliomas (Chen et al., 2014). Biomed Res J 2015;2(1):6-20 9 Maachani et al. Table 2: Molecular targeted therapies for glioblastoma Other current strategies tested in GBMs Pathways targeted Agents Molecular targets Erlotinib (Roche) Kinase inhibitors of Epidermal Growth Factor Pathway EGFR is amplified and frequently mutated in ~50% of GBMs and is overexpressed in many malignant gliomas. Therefore could be used Gefitinib (AstraZeneca) EGFR as a therapeutic targeted agent in GBM patients. VEGF Pathway Targeting vascular endothelial growth factor (VEGF) pathways to Bevacizumab (Avastin; Recombinant human induce anti-angiogenic effects in the treatment of malignant gliomas Genentech) neutralizing monoclonal has been in focus for past few years. antibody to VEGF Vatalanib (Novartis) Kinase inhibitor of VEGFR/PDGFR Cediranib (AstraZeneca) pan-VEGFR inhibitor TGF-β is a multifunctional cytokine, which regulates gliomacell Trabedersen (Antisense Anti-sense TGF-β2 motility, invasion, and immune surveillance. Several small molecule Pharma) mRNA Rapamycin (Sirolimus) inhibitors of m-TOR Transforming Growth Factor β(TGF-β) Pathway inhibitors of TGF-β receptors have shown antitumor efficacy in preclinical models of gliomas. PI3K–AKT–mTOR Pathways PI3K pathways regulate several malignant phenotypes including antiapoptosis, cell growth, proliferation, and invasion. Activated PI3K Temsirolimus (Sirolimus) phosphorylates several downstream effectors, including AKT. mTOR is a major player connecting multiple pathways downstream from Everolimus (Novartis) AKT. PKC Pathways Protein kinase C (PKC) is a serine/threonine kinase that regulates enzastaurin (Eli-Lilly) PKC-β inhibitor with cell proliferation, invasion, and angiogenesis. activity against glycogen synthase kinase 3β Note: Several of the above agents are being evaluated in clinical trials as monotherapies or in combination with other treatment modalities such as chemotherapy or radiation in patients with malignant gliomas. populations. Recent multi-omics (genomics, (Verhaak et al., 2010). These subtypes were transcriptomics data defined on the basis of distinct gene signatures integration studies have utilized patient and also characterized by different molecular derived samples and cell lines to reveal alterations and activated pathways (Verhaak et heterogeneity among the primary GBM, al., 2010; Brennan et al., 2013). The proneural suggesting additional molecular subclasses: subtype neural, proneural, classical and mesenchymal abnormalities in platelet-derived growth and proteomics) was mostly characterized by Biomed Res J 2015;2(1):6-20 10 Advances in omics technologies in GBM factor receptor-α (PDGFRA) or in isocitrate methylation dehydrogenase 1 (IDH1); whereas mutation of promoter-associated the epidermal growth factor receptor (EGFR) specific loci accompanying tumor suppression was found in the classical subgroup, and in GBM (Sturm et al., 2014), such as mutations in neurofibromin 1 (NF1) were (CDKN2A), RB1, PTEN, TP53 (Costello et common in mesenchymal tumors. The neural al., 1996; Nakamura et al., 2001; Baeza et al., subtype seemed to be similar to the classical 2003; Amatya et al., 2005) and other subtype but with a higher frequency of TP53 previously unrecognized regulatory genes mutations (Brennan et al., 2013). Cytogenetic EMP3, PDGFB (Alaminos et al., 2005; Bruna and molecular studies have also identified a et al., 2007). Most significantly, O-6-methyl- number chromosomal guanine-DNA methyltransferase (MGMT) abnormalities and genetic alterations in promoter hypermethylation was identified malignant novel occurring in ~45% of adult patients with GBM candidates, particularly in GBMs. The (Esteller et al., 1999; Brennan et al., 2013). identification of molecular subtypes has MGMT hypermethylation leads to gene revealed a set of core signaling pathways silencing, and reduced gene expression levels commonly activated in GBM (Table1) which compromises its ability to repair (Furnari et al., 2007) and could be used in damaged DNA by alkylating agents like molecular targeted therapies (Table 2). Temozolomide (Felsberg et al., 2011). Thus, of recurrent gliomas, as well as have identified frequent hypermethylation of gene methylation could be used as a biomarker to predict sensitivity to chemo- radiotherapy Epigenomics enzymatic (Wick et al., 2012; Malmström et al., 2012). modifications of DNA and associated histone Further, based on DNA methylation patterns, proteins to regulate gene expression. In recent proneural subtype is classified into CpG island years these changes have been recognized as methylator phenotype (CIMP) positive and important causes of phenotypic changes in CpG–CIMP-negative GBM subsets which human The strongly correlates with IDH1 gene mutation epigenetic changes are dynamic in nature and status (Noushmehr et al., 2010; Turcan et al., play an important role in gene expression and 2012). Glioma CIMP (G-CIMP) is a powerful DNA structure. determinant Epigenetic changes cancers involve (Esteller, 2007). Epigenetic alterations, of tumor aggressiveness especially those related to changes in histone (Riemenschneider et al., 2010; Brennan et al., acetylation, are a recent focus for therapeutic 2013). These epigenomic and other multi- drug targeting in clinical trials. Genomic-array omic (microarray) mutations, techniques Biomed Res J 2015;2(1):6-20 studying DNA analyses have altered revealed proteins, several miRNA 11 Maachani et al. expressions and pathways associated with efficient biomarker validation, treatment GBM pathogenesis and prognosis. monitoring and can be translated into clinical applications in an affordable manner. Various Proteomics plasma/serum Proteomic profiling represents the large-scale identified earlier for GBM including YKL-40, examination of protein expression, post- GFAP translational modification, and understanding (Jayaram et al., 2014). Reynes et al. (2011) how different proteins interact with each other. reported inflammatory markers (C-reactive Using various bioinformatics techniques, the protein, IL-6 and TNF-) and angiogenesis information can be unified into protein markers such as VEGF and soluble VEGF networks. histopathology receptor 1 to be significantly elevated in the represents the gold standard for the typing and plasma of GBM patients. Jung et al. (2007) grading of gliomas and depends largely on identified GFAP as a discriminatory serum certain architectural similarities of tumor cells biomarker with normal glial cells (Tohma et al., 1998; osteopontin (OPN), validated using IHC and Riemenschneider et al., 2010). We feel that the ELISA in GBM patients, was shown to underlying disease pathology would result correlate into differential proteomic profiling of (Sreekanthreddy et al., 2010). In an extended diseased tissue and the surrounding disease- effort, the TCGA group also generated protein free normal tissue. Recent technological expression data from 214 GBM patient advances in proteomics has allowed analysis samples using a high throughput antibody- of glioma patient biopsies, proximal fluids, based reverse phase protein arrays (RPPAs) cerebrospinal fluid (CSF) and cyst fluid, (Brennan et al., 2013) revealing several plasma, glioma cell lines. This has allowed a mutations, altered genes, proteins and their comprehensive proteomic profiling of glioma pathways underlying GBM pathophysiology biology to aid the traditional histopathology in (Dong et al., 2010). Currently, biomarkers and matrix for been metalloproteinase-9 GBM. with have Similarly, poor serum prognosis improving our understanding of glioma Some of the challenges in using protein processes and to better evaluation of drug profiling more commonly in characterizing responses to treatment (Somasundaram et al., and quantifying accepted protein biomarkers 2009). The techniques involve evaluation of includes high costs, lengthy production times protein arrays, including antibody and aptamer and most importantly lack of high specificity arrays. This allows simultaneous detection of antibodies. multiple approach has the potential to identify novel proteins/phosphoproteins. These high throughput techniques can be used for diagnostic, Moreover, prognostic, the and proteomic therapeutic Biomed Res J 2015;2(1):6-20 12 Advances in omics technologies in GBM biomarkers for human gliomas. The Spectroscopy (MRS), Nuclear Magnetic application of proteomics in neuro-oncology Resonance (NMR), have helped in profiling is still in its developing stage. Please refer global metabolomic signatures in cancers recent reviews by Whittle et al. (2007) and including glioma (Dunn et al., 2005; Serkova Niclou et al. (2010) for more on the current and Niemann, 2006). Several key differences status of glioma proteomics and its clinical in metabolite profiles have been identified in applications. GBM cancer cells when compared to normal controls, providing a novel insight into GBM Metabolomics tumorigenesis (Spratlin et al., 2009). As Nearly a century ago, Otto Warburg made a metabolomics reflect underlying altered seminal observation that even in the presence genotype-phenotype, it can be used as a of adequate oxygen cancer cells metabolize predictive biomarker for measure of efficacy glucose by aerobic glycolysis, termed as and as a pharmacodynamic marker, for both Warburg effect (Warburg et al., 1924; 1927). traditional Moreover, disease-related agents. Using the 1H-NMR spectra and neural altered cellular metabolism has come into networks, human glioma cell cultures can be forefront of cancer research. Now, there is separated into drug-resistant and drug- increasing evidence that the underlying sensitive groups before treatment with genetic alterations contributing to glioma nitrosourea treatment (El-Deredy et al., 1997). pathogenesis is also responsible for altered Frequent genetic alterations in glioma such as cellular metabolism (Parsons et al., 2008). MYC amplification, PTEN deletion or protein Metabolomics refers to the global quantitative loss and EFGR amplification are associated assessment of endogenous enzyme kinetics, with multiple downstream metabolic targets cellular biochemical reactions, and synthesis (Deberardinis et al., 2008). IDH1 and IDH2 of cellular metabolites within a biologic metabolic genes are mutated in ~12% of system, (Griffin and Shockcor, 2004; Boros et primary gliomas, 86% of grade II and III al., 2005). Although considerable progress has gliomas and secondary glioblastoma through a been made in understanding GBM biology gain-of-function mutation that alters the through genetic analysis, little is known about enzymatic activity of the protein product, the underlying metabolic alterations in glioma. which results in the production of 2- In recent years, several biochemical and hydroxyglutarate (Dang et al., 2009). The biophysical very Spectrometry recently techniques (MS), chromatography, such liquid- Magnetic Biomed Res J 2015;2(1):6-20 chemotherapy and hormonal as Mass detection of 2-HG metabolic product has been and gel- proposed to be a potential tool for in vivo Resonance distinction of secondary from primary 13 Maachani et al. glioblastomas (Esmaeili et al., 2013). More Srinivasan et al., 2011). More recently several recently Chen et al. (2014) showed that lines of evidence have implicated over- IDH1-mutant glioma growth is facilitated by expression of miR21 with chemo and overexpression of glutamate dehydrogenase 2 radioresistance of GBM cells. Its expression gene (GLUD2) and it could be targeted for levels have been associated with glioma grade growth Hence, and as a candidate independent marker for metabolomics applications in a clinical overall survival (Chao et al., 2013; Wu et al., perspective may have a favorable impact on 2013). Thus, integrative omics analysis has glioma grade, metabolic state and treatment revealed the importance and scope of stratification of glioma patients. translational inhibitory effects. repression in microRNA- mediated GBM pathogenesis. Please refer to Other Omics: microRNAs additional reviews (Sana et al., 2011; Karsy et In recent years, microRNAs (miRNAs) have al., 2012; Nikaki et al., 2012) for more emerged in the forefront of cancer molecular detailed coverage on miRNA expression and biology. function in GBM. MicroRNAs are key post- transcriptional regulators that inhibit gene expression by promoting mRNA decay or Omics data integration methods suppressing translation (Iorio and Croce, The post-human genome project era has 2012). Experimental and clinical evidence generated enormous heterogeneous and large supports that miRNAs play pivotal role in data sets. As vast gene profiling datasets and cancer proliferation, technologies are being developed, they have apoptosis and metastasis (Cho, 2011; Iorio and created an unprecedented need to develop Croce, 2012). The functional role of miRNAs technologies to process the data in a was first discovered in human gliomas (Li et meaningful way. The efforts have yielded al., 2013). Several miRNA expressions are meaningful results in cancer biomarker found to be dysregulated in GBM. TCGA discovery, protein interactions and genotype group identified alterations in 149 miRNAs to phenotype correlations (Park et al., 2005). (Dong et al., 2010) and an expression However, current omics technologies cannot signature comprising 10 miRNAs with model prognostic prediction (Srinivasan et al., 2011). molecules by analyzing individual genes, miR-128, miR-342 and miR-21 are known to proteins or metabolites. This is often not very play both oncogenic and tumor suppressive effective roles and are being explored as possible heterogeneous nature of human cancers. markers for GBM (Dong et al., 2010; Cancer is a complex biological system and gene regulation, interactions due to between the multiple complex and Biomed Res J 2015;2(1):6-20 14 Advances in omics technologies in GBM requires a better understanding of the disease's network and analyze experimental data in the complexity at systems-level (Faratian et al., context of pathways using multiple source 2009; Hu et al., 2013). Pathway and network omics data (Wang et al., 2012; Blazier and based methods have taken more important role Papin, 2012; Federici et al., 2013). Although in analysis of high-throughput data, that can currently there are tools available to process provide a global and systematical way to large datasets generated by one platform, it is explore the relationships between biomarkers expected that soon tools combining data and their interacting partners (Wang et al., across multiple platforms will be available to 2015). Integration of data from multiple omic researchers. This will help in integrating studies can not only help unravel the research results into a framework of whole underlying biological systems to support translation of molecular mechanism of carcinogenesis but also identify the signature research into clinical applications. of signaling pathway/networks characteristic for specific cancer types that can be used for Omics Advantages in GBM therapy diagnosis, prognosis and designing tumor So far clinical translation of an effective GBM targeted therapy. therapy has been hindered by multiple factors, Most recently, attempts at integration of including diffuse infiltration at the time of multiple high-throughput omics data have diagnosis, significant cellular heterogeneity concentrated on comparing data acquired (both using conditions/ difficulty in crossing the blood-brain barrier platforms to explore functional and regulatory by effective drugs, and the role of tumor associations between genes and proteins progenitor cells in reestablishment of resistant (Faith et al., 2007; McDermott et al., 2009). disease following chemo and radiotherapy. This has culminated into combining functional Current standard treatment of GBM consists characterization and quantitative interactions of attempted gross total surgical resection extracted from various biomolecules such as followed by concurrent temozolomide and DNA, mRNA, proteins and metabolites (Chen radiation therapy (RT) (Clarke et al., 2010). et al., 2011; Coban and Barton, 2012; Mitchell Although, RT provides good local control, it is et al., 2013) (Fig. 1). Some analysis utilizes not very beneficial in controlling the disease pathways in the form of connected routes recurrence. In case of GBM, majority of through a graph-based representation of the patients die from recurrent disease, as metabolic network (Blum and Kohlbacher, currently there is no effective therapy for 2008). Other approaches focus on the recurrent GBM. Therefore, the addition of functional module of protein interaction systemic chemotherapy to RT can help in various experimental Biomed Res J 2015;2(1):6-20 intratumoral and intertumoral), 15 Maachani et al. controlling recurrence and offering an methyl group to the middle guanine in a GGG additional radiosensitization benefits in GBM, sequence to convert it to O6-methylguanine. benefiting both definitive and palliative Temozolomideexerts strategies for disease management (Stupp R et activity by interfering with repair of damaged al., 2006; Clarke et al., 2010). So far non- DNA after radiation treatment (Mrugala and omics studies have identified few GBM targets Chamberlain, 2008). In a recent randomized at the protein level, but fail to see an overall trial, role of molecules in signaling pathways, Temozolomide chemotherapy with radiation, protein-protein interactions, and role in significantly metabolic processes. Unfortunately so far only survival from 12.1 months to 14.6 months, for one drug has been identified (Temozolomide) GBM patients (Stupp et al., 2005, Clarke et al., which can radiosensitize GBM patients. Thus, 2010). The consequent analysis of these non-omics techniques will compliment whole patients by Hegi et al. (2005) reported that genomic/epigenomics/metabolomics approach patients with methylated MGMT gene of omics technologies. Without publically promoter were benefited from this treatment available databases, the surge of preclinical compared to patients with unmethylated and clinical information seen in the GBM field MGMT promoter. The MGMT promoter over last few years, would have not been methylation silences the gene function possible. As omics studies expand our required understanding of the molecular pathways methylation and therefore cannot counteract driving GBM tumorigenesis, more druggable the action of Temozolomide. Thus, omics has targets will be identified to treat GBM patients. been helpful in predicting tumor response to Also, understanding of ionizing radiation at Temozolomide and to guide clinical decision the level of molecular biology will lead to making. The other most common types of development and production of targeted chemotherapies for GBM under investigation radiosensitizers. Temozolomide is currently include the only radiosensitizing agent used for GBM antiangiogenic therapies, immunotherapies, with class I evidence of benefit (Mrugala and gene Chamberlain, 2008). It is a novel oral therapies and drugs to overcome resistance bioavailable (Table 2). second-generation alkylating its concomitant to and improved reverse targeted therapies, antineoplastic adjuvant progression the molecular free O6-guanine therapies, radiation-enhancement agent. At physiologic pH it undergoes hydrolysis to its active form methyltraizeno- Challenges and Prospective imidazoleoarboxamid The Oncogenic transformation is a complex, mechanism of action of MTIC, is to transfer a multistep process that differs widely between (MTIC). Biomed Res J 2015;2(1):6-20 16 Advances in omics technologies in GBM and even within cancer types. Advances in the therapeutic agents for GBM. The advances large scale omics technologies have led to with respect togene expression profiling, identification of promising GBM disease signaling pathway characterization, glioma biomarkers. The major challenge is how to stem cell identification, regulatory RNA bring omics research into accurate and reliable studies, clinical use. In case of GBM, omics immunomodulation approaches, have resulted technologies have their limitations due to late in several ongoing clinical studies evaluating diagnosis of disease, intrinsic molecular new therapeutic agents for GBM (Table 2). It is complexity and genetic heterogeneity of evident that omics based cancer research is GBMs. To find consistencies that can be going to play a pivotal role in diagnosis, therapeutically targeted on the basis of treatment and monitoring of GBM patients. metabolomic changes and molecular analysis, poses a major problem. However, we are optimistic that the wealth of CONFLICT OF INTEREST information generated by omics techniques The authors claim no conflict of interest. has paved a roadmap for designing new REFERENCES Alaminos M, Davalos V, Ropero S, SetienF, Paz relevant metabolic routes for interactive MF, Herranz M, et al. EMP3, a myelin-related network gene located in the critical 19q13.3 region, is Bioinformatics 2008;24:2108–2109. navigation and visualization. epigenetically silenced and exhibits features Boros LG, Lerner MR, Morgan DL, Taylor SL, of a candidate tumor suppressor in glioma and Smith BJ, Postier RG, Brackett DJ, et al. 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MicroRNA-21 expression is associated with overall survival in patients with glioma. Diagn Pathol 2013; 8. Review Dendrimers based Electrochemical Biosensors Saumya Nigam1, Sudeshna Chandra2, Dhirendra Bahadur1* 1 Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai, Mumbai, India 2 Department of Chemical Sciences, School of Science, NMIMS (Deemed-to-be) University, Vile Parle (W), Mumbai, India Electrochemical biosensors are portable devices that permit rapid detection and monitoring of biological, chemical and toxic substances. In the electrochemical biosensors, the bioreceptor is incorporated into the transducer surface; and when in contact with the analyte, generates measurable signals proportional to the analyte concentration. Materials with high surface area, high reactivity, and easy dispersability, are most suited for use in biosensors. Dendrimers are nanomaterial gaining importance for fabrication of electrochemical biosensors. These are synthetic macromolecules with regularly branched tree-like and globular structure. The potential applications of dendrimers as biosensors are explored due to their geometric symmetrical structure, chemical stability, controlled shape and size, and varied surface functionalities, with adequate functional groups for chemical fixation. The current review provides multifaceted use of dendrimers for developing effective, rapid, and versatile electrochemical sensors for biomolecules. The redox centers in the dendrimers play an important role in the electron transfer process during immobilization of biomolecules on the electrodes. This has led to an intensive use of dendrimer based materials for fabrication of electrochemical sensors with improved analytical parameters. The review emphasizes development of new methods and applications of electrochemical biosensors based on novel nanomaterials. INTRODUCTION Dendrimers are globular repetitive and radial branching units and the macromolecules with well-defined, mono- terminal functional groups. To achieve a high disperse, spatial degree of precision and structural order, conformations, and a wide spectrum of dendrimers are synthesized in a stepwise chemical and physical properties (Tomalia et fashion. The number of repeat branching al., 1985). These characteristics indicate molecules used during the synthesis refers to significant differences from the classical the generation of dendrimers, which also polymeric molecules. Structurally, these governs the shape and size of the dendrimers. macromolecules are divided into three Generally, two different methods namely, architectural divergent and convergent, are adopted for the three hyperbranched dimensional regions: the central core, Key words: Dendrimers, Biosensors, Polyamidoamine, Polypropylene imine, Bioreceptors, DNA sensor. *Corresponding Author: Dhirendra Bahadur, Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai, Mumbai, India. Email: dhirenb@iitb.ac.in Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 22 synthesis of dendrimers, and classified into geometric progression with every generation different “generations”. It is the hyper- followed by increase in molecular weight. branching of the molecule from the centre of This compromises the reaction kinetics, the dendrimer towards the periphery that making it slower and synthesis of high results in homostructural layers between the generation dendrimers becomes difficult, focal points (branching points). The number of further lowering the yield of desired product. focal points from the core towards the outer Addition of each branching unit requires care surface is the generation number. Thus, and precision to prevent structural defects and generation refers to the number of repeated asymmetry in the dendrimer structure. branching cycles performed during the Secondly, the separation of desired dendrimer synthesis. The core part of the dendrimer is from the by-products is hindered due to denoted generation “zero” (G0). For example molecular similarity exhibited by the by- if a dendrimer is made by convergent product as well as the desired dendrimer. On synthesis, and the branching reactions are the other hand, convergent method employs performed onto the core molecule three times, synthesis of small dendrites from the exterior the resulting dendrimer is considered a third and the reaction proceeds inwards to the generation central core. The convergent procedure results dendrimer. Each successive generation results in a dendrimer roughly in lesser twice the molecular weight of the previous purification of dendrimers resulting in high generation. degree of structural defects monodispersity. and easy Despite the The two synthetic methods have inherent possibility of purer and flawless dendrimers, advantages and disadvantages. Using the the convergent method falls short in synthesis divergent synthesis method, the dendritic of higher generation dendrimers. This choice molecule is formed from a central core which is limited due to the steric forces crowding the then extends radially outwards through dendrites around the central core molecule. addition of branching molecules. The main Despite the difficulties, these macro- advantage of the divergent method is that high molecules have gained interest over classical molecular is polymers due to the varied options presented attained with desired repetitive branching by dendritic macromolecules. The freedom of monomers. Thus, the dendrimer can be tailor choice of central core, branching monomeric made to achieve maximum functionalities and units and surface functional groups from the properties. However, two major challenges are vast pool of molecules gives rise to a encountered in divergent synthesis. First, the multivalent system. Ethylenediamine, 1,4- number of reaction points increase in diaminobutane, nanoscaffold Biomed Res J 2015;2(1):21-36 architecture 1,12-diaminododecane, 23 Nigam et al. cystamine, 1,6-diaminohexane and ammonia sensor response to be used in biosensing are the most common core molecules. The applications. Various varied core and branching monomers affect ferrocene, polystyrene, the internal chemical environment, three carbohydrates, etc. have been explored for dimensional structures and size of internal surface modification (Ashton et al., 1997; cavities in the dendrimer. Due to the different Chen et al., 2014; Hung et al., 2013; Yoon et structural and chemical properties, these user- al., 2000). The conductivity of the moieties customized dendrimers find applications in plays an important role in enhancing response the fields of drug delivery, gene delivery, of the dendritic scaffold in sensing various antimicrobials, magnetic resonance imaging, biomolecules. The most common modifying immunosensing and biosensing. molecule is ferrocene which exhibits multi- molecules like polyaniline, Methyl acrylate alternating with ethylene electron transfer in various redox interactions. diamine forms the most widely synthesized, Ferrocene has been exploited as central core, studied and used class of polyamidoamine branching monomer as well as for surface (PAMAM) dendrimers (Esfand et al., 2002), groups in various dendritic systems (Mehmet with the internal amide groups providing an et al., 2013; Villalonga-Barber et al., 2013). abundance of lone pairs of electrons. Another They popular class of amine terminated dendrimers moieties undergoing redox processes without is (PPI) decomposition while maintaining the desired synthesized by Michael's addition of primary electrochemical reversibility (Sun et al., amines 2014). the poly to (propylene acrylonitrile imine) followed by subsequent hydrogenation by Raney cobalt or behave The as non-interacting molecular redox recognition of Raney nickel catalyst (de Brabander-van den biomolecules by dendrimers is primarily Berg et al., 2003). The interiors of PPI governed dendrimers are the tertiary nitrogen atoms with conformation in higher generations. The lone pairs of electron contributing to their branches of lower generation dendrimers tend reactive cavities. Both the classes of to radiate out towards the periphery and exist dendrimers have primary amine groups on the in open conformation. On the other hand, as surfaces governing the surface properties, the number of generation is increased, the reactivity and surface charge. Thus, any kind branches tend to retract and adopt globular of detection response observed in these conformations in a three dimensional space dendrimers is attributed to the amine groups. with The surface of dendrimers is further modified governing the structures. The generation to enhance the reactivity/interaction and dependent conformational changes confirmed by the intramolecular three dimensional hydrogen bonding Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 24 by X-ray analysis, demonstrated that the applicable in several biomedical and higher generations are more spherical as environmental analyses. compared to lower linear generations (Percec a) Peroxide sensor: Copolymers of pyrrole- et al., 1998). The globular conformations PAMAM closely resemble morphology of globular electrochemical sensing of hydrogen proteins and are useful in several biosensing peroxide. applications associated with the biomimetic pyrrole-PAMAM with branched amine macromolecular architecture. A vast variety of periphery and focal pyrrole functionality biomolecular species have been detected using are synthesized by divergent method. The dendrimer scaffolds. In the following sections, conjugate is covalently attached to the details of the various sensors using different electrode types of dendrimers are discussed. peroxidase (HRP) immobilized on it to dendrimer Different surface are used generations and for of horseradish form conducting films for H2O2 sensing. Dendrimers in electrochemical biosensing The steady state amperometric response is By definition, a biosensor is an analytical measured device that makes use of bioreceptor molecule concentration at +0.35V vs. Ag/AgCl, and immobilized onto a transducer (recognition) demonstrated that the dendritic wedge surface and produces measurable signals in the played presence of an analyte, due to the bio- immobilization of the HRP enzyme recognition the (Mehmet et al., 2012). Yang et al. (2014) concentration of the analyte. Biosensors are described a magnetic electrochemical classified based on either the bioreceptor or sensor comprising Fe3O4 nanoparticles transduction method or both. Common with graphene oxide (GO) and subsequent bioreceptors include enzymes, antibodies and modification by PAMAM dendrimers. DNA, while transducers include electro- The chemical, piezoelectrical, optical techniques. modification of the gold electrode acting The transducer techniques using electro- as the working electrode and used for the chemical biosensors have an edge over other detection of H2O2 in phosphate buffer methods due to excellent selectivity and solution by the method of amperometric sensitivity, and precise detection of the desired i-t curve. The cyclic voltammograms of species. These are relatively cheaper, faster Fe3O4/GO and more user friendly as compared to other showed an increase in current while techniques. The exceptional features render displaying steady redox peaks which the electrochemical biosensors increasingly confirmed occurrence of a catalytic event proportional Biomed Res J 2015;2(1):21-36 to as an a function important platform and was of role employed H2O2 for for Fe3O4/GO–PAMAM 25 Nigam et al. reaction on the electrode interface. H2O2 was detected in a linear calibration range -5 (Chandra et al., 2009). b) Glucose Sensor: A dendritic wedge based -3 of 2.0 × 10 –1.0 × 10 M with a correlation on pyrrole-PAMAM dendrimer was used coefficient of 0.9950 and detection limit of to immobilize glucose oxidase (GOx) for -6 2.0 × 10 M. The sensor platform also the construction of an amperometric displayed excellent recovery ratios of glucose sensor (Mehmet and Cevdet, 96.9–108.1% H2O2 added to milk and juice 2012). Nanobiocomposite based glucose samples. Another amperometric electro- biosensor was prepared by electro- chemical was polymerization of pyrrole containing developed by modifying gold bead PAMAM encapsulated Pt nanoparticles electrodes PAMAM (Pt-PAMAM), and GOx. The developed dendrimers of different generations of 2, 3 sensor had a sensitivity of 164 µA mM-1 and 4, followed by absorption of Prussian cm-1 and a detection limit of 10 nM within blue bonded a wide working range from 0.2−600 µM. dendrimer/PB modified electrodes offered Pyrrole provided electrical conductivity, enhanced sensitivity and lower detection stability and homogeneity to the thin film, limits (Bustos et al., 2006). Metallic while PAMAM provided a favorable (Rhodium) nanoparticles stabilized with microenvironment to maintain bioactivity N, N-bis-succinamide-based dendrimer of GOx (Tang et al., 2007). Yoon and were immobilized on glassy carbon colleagues used varying degrees of redox- electrode (GCE) and electrocatalytic active ferrocenyl in combination with activity PAMAM biosensor (PB). reduction with The towards for H2O2 starburst covalently hydrogen investigated using peroxide dendrimers (Fc-D) as cyclic recognition unit for fabrication of a voltammetry and chronoamperometry. glucose sensor (Yoon et al., 2000). GOx The dendrimer stabilized nanoparticles was deposited layer-by-layer on Au- showed excellent electrocatalytic activity surface to form an enzymatically active for H2O2 reduction reactions and a steady- GOx/Fc-D multilayered assembly. The state cathodic current response was bio-electrocatalytic signals from the observed at −0.3 V (vs SCE) in phosphate multilayer were directly correlated to the buffer (pH 7.0). The electrochemical number of layers deposited, confirming sensor displayed a linear response to H2O2 the tunable sensitivity of the electrode and concentrations ranging from 8 to 30 μM hence a potential microbiosensor. Cyclic with a detection limit and sensitivity of 5 voltammetry μM and 0.031 × 10−6 A μM−1, respectively resonance (SPR) was used to investigate and surface plasmon Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 26 the redox-orientation changes of ferrocene-tethered dendrimers and GOx. SPR monitors change in the refractive index of the medium next to the Au sensing surface and are used to monitor immobilization of GOx onto the Au surface (Frasconi et al., 2009). Redoxactive dendrimer fabricated using different generations of poly (propylene imine) core with peripheral octamethyl ferrocenyl units (Fig. 1) and deposited on Pt electrodes for immobilizing GOx has been used for detection of glucose (Armada et al., 2006). The amperometric response of all the dendritic mediators towards glucose was determined at several Figure 1: Structures of varying generations of octamethyl ferrocenyl dendrimers for use as electrode material for determination of glucose (Armada et al., 2006). applied potentials. Glucose biosensor has been developed based on bioactive the surface of PGLD and distribution of polyglycerol PANINT's (Santos et al., 2010). (PGLD) and chitosan dendrimer (CHD). Both the dendrimers Ferrocenyl dendrimer (PAMAM-Fc) were conjugated with GOx to form has PGLD-GOx amperometric glucose biosensor. Series of entrapped and in CHD-GOx polyaniline and nanotubes also asymmetric used for fabricating PAMAM an dendrimers (PANINT's) during template electro- containing a single ferrocene unit located chemical polymerization of aniline. The in the focal point have been synthesized. prepared and The transducer consisted of a gold biosensors electrode covalently modified with 3- PGLD-GOx/PANINT's CHD-GOx/PANINT's exhibited strong amperometric response mercaptopropionic to glucose concentrations in ranges dendrimers and GOx enzyme. The observed PAMAM-Fc/GOx in human GOx/PANINT's was blood. more PGLD- acid, PAMAM-Fc biosensor showed sensitive excellent performance for recognizing (10.41 nA.mM ) as compared to CHD- glucose at +0.25V with a high sensitivity -1 -1 GOx/PANINT's (7.04 nA.mM ), due to (6.54μA/mM) and low response time specific organization of the GOx layer at (~3s) in the concentration range of 1–22 Biomed Res J 2015;2(1):21-36 27 Nigam et al. mM (Mehmet et al., 2013). c) DNA Sensor: Dendrimers were also exploited for their possible use in fabricating DNA sensors. An electrochemical DNA nanobiosensor was Figure 2: Proposed charge transfer scheme between PBS, DNA and PPI-AuNP (Arotiba et al., 2008). developed by immobilization of 20-mer thiolated probe ssDNA on electro- sequence, immobilized on a multinuclear deposited nickel poly (propyleneimine) (II) salicylaldimine metallo- dendrimer (PPI) of generation 4 (G4), dendrimer on GCE has been reported doped with gold nanoparticles (AuNP) (Arotiba et al., 2007). The authors studied (Arotiba et al., 2008). Cyclic voltammetry electrochemical showed that the designed platform immobilization layer of the PPI derivative (GCE/PPI-AuNP) exhibited reversible by electrochemical behavior in pH 7.2 methods. The metallo-dendrimer was phosphate buffer saline (PBS) solution electroactive with two reversible redox due to PPI. The redox chemistry of PPI centers and was a strong DNA adsorbant. involves a two electron and one proton The sensor responded to 10 µL of 5 nM process and is pH-dependent. PPI-AuNP target DNA with detection limit as low as was able to amperometrically detect target 3.4 × 10-12 M. Gold electrode has been DNA concentrations at 0.05 nM in PBS. modified with 3-mercaptopropionic acid Using and electrochemical spectroscopy (EIS), the impedance biosensor -12 characterization impedimetric reacted and with on amperometric amino-terminated PAMAM G-4 dendrimer to obtain a thin -9 film (Li et al., 2009). Recognition layer of M for target DNA. The probe immobiliza- single-stranded 3´-biotin-avidin combina- tion effectiveness is apparently attributed tion was immobilized onto the thin film to to the AuNP's ability to connect to the detect the complimentary target. Cyclic thiolatedssDNA on the GCE surface via voltammetry (CV), differential pulse Au-S linkages. Further, the electrostatic voltammetry (DPV) and electrochemical interaction between the cationic platform impedance spectroscopy (EIS) has been and the anionic DNA probe improved the used immobilization process. Proposed charge hybridization of DNA. The dynamic transfer scheme between the electrolyte, detection range of the sequence-specific DNA and PPI-AuNP is shown in Fig. 2. DNA was 1.4 × 10-11–2.7×10-14 M with a exhibited a dynamic linearity of 10 –10 A DNA biosensor with probe DNA to study immobilization and detection limit of 1.4 × 10-14 M. Sahoo et Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 28 al. (2013), demonstrated a label free generation impedimetric DNA biosensor based on dendrimer was covalently functionalized third generation G3 PAMAM dendrimer onto functionalized GaN nanowires (NWs). (MWNT) The developed nanosystem provided large transducer docking sites to immobilize probe (p-) confinement of probe DNA. Impedance DNA covalently. The biosensor was spectroscopy revealed occurrence of ultrasensitive and showed detection limit hybridization between surface confined as low as attomolar (aM) concentration of ssDNA probe with target DNA in solution complementary DNA. to form double stranded DNA (dsDNA). Impedance spectroscopy revealed an The interfacial charge-transfer resistance increase in the resistance polarization (Rp) of the electrode towards the redox indicating efficient charge transfer due to electrolyte changed due to occurrence of strong covalent binding on NWs surface. hybridization. The large number of amino Zhu et al. (2006) modified gold electrodes groups of the dendrimer enhanced the with sub-monolayers of mercaptoacetic surface binding of the probe DNA which acid (RSH) and reacted with G-4 PAMAM in turn resulted in increase in the dendrimers to obtain thin films of sensitivity of the impedimetric biosensor PAMAM/RSH. DNA probe was then for the target DNA. The interfacial charge- immobilized onto the thin films to afford transfer resistance responded linearly to stable recognition layers. DPV was used to the logarithmic concentration of the target monitor with DNA within a concentration range from daunomycin (DNR) as indicator. The 0.5 to 500 pM with a detection limit of 0.1 PAMAM-modified Au electrodes without pM (S/N = 3) (Zhu et al., 2010). Single- ssDNA showed good electrochemical use electrochemical DNA biosensor has response in DNR solution, while on been fabricated based on pencil graphite attachment with ssDNA the modified electrode modified with succinamic acid electrode showed a decrease in the DPV and response of DNR. This is attributed to less PS/GCE). Calf thymus double stranded accessibility of DNR molecules to ssDNA DNA (ctDNA) and DNA oligonucleotide probe on the electrode surface. Besides (DNA ODN) immobilized on surface of high low G2-PS/GCE under optimum conditions, generation dendrimers are also used to showed a detection limit of 4.2 µg/mL develop DNA biosensor. A second (Congur et al., 2014). Besides dendrimers, DNA target (t-) hybridization generation Biomed Res J 2015;2(1):21-36 dendrimers, PAMAM multi-walled G2 and and (G2-PAMAM) carbon used as tether PAMAM nanotube electronic for dendrimer surface (G2- 29 Nigam et al. Figure 3: SEM images of Den-Au electrodes by electrodeposition in 2.8 mM HAuCl4 and 0.1 M H2SO4 at different time points (A) 20s, (B) 100s, (C) 300s and (D) 600s (Li et al. 2011). dendritic nanostructures have been used as based on amino-terminated PAMAM electrode biosensing dendrimer. Thrombin aptamer probe was applications. Li et al. (2011) described immobilized onto activated dendrimer dendrimer-gold (Den-Au) nanostructure monolayer film and detection of thrombin modified electrode by directly placing the was investigated in the presence of the electrode into 2.8 mM HAuCl4 and 0.1 M reversible [Fe(CN)6]3−/4− redox couple H2SO4 solution at -1.5 V. Scanning using impedance technique. The results electron microscopic images show growth showed that the charge-transfer resistance evolution of Den-Au at different time (Rct) value had a linear relationship within period (Fig. 3). The Den-Au modified concentrations electrode respond to 1 fM complimentary thrombin, and detection limit (S/N = 3) of target DNA within a wide detection range. 0.01 material in Aptamers, as single-stranded DNA or nM Impedimetric range (Zhang of et 1–50 al., aptasensor nM 2009). based on RNA sequences that bind to specific target succinamic molecules was determined by a label-free dendrimer was developed for monitoring highly sensitive impedimetric aptasensor interaction between DNA aptamer (DNA- acid-terminated PAMAM Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 30 APT) and its cognate protein, human and their films, PDAMS with shorter activated protein C (APC), a key enzyme branches form rougher films and exhibit in the protein C pathway. The dendrimer higher rate constants (Kobs) and sensitivity modified aptasensor showed detection and smaller Michaelis constants (K'M), limits of 1.81 µg/mL in buffer solution and than 0.02 µg/mL in diluted FBS (Erdem et al., electrocatalytic activity towards NADH 2014). oxidation (Jiménez et al., 2014). PMDUS indicating better Hyperbranched e) Other biomolecules: Tang et al. (2007) carbosilane polymers, polydiallyl methyl reported enzyme based amperometric silane biosensor for determination of glutamate. d) Coenzyme Sensor: (PDAMS) and polymethyl with A self-assembly of glutamate dehydro- for genase (GLDH) and PAMAM dendrimer stabilization of Pt nanoparticles and as encapsulated Pt nanoparticles on carbon electrode material for NADH oxidation. nanotubes The modified electrodes worked in wide were used as electroactive material (Fig. linear concentration ranges for NADH 4). The electrochemical activity was with a detection limit of 4.78 µM for reported to be attractive with large PDAMS/PtNPs/Pt and 6.18 µM for determination range of glutamate (2-250 PMDUS/PtNPs/Pt. With regard to the µM), short response time (< 3 s), high structure of the two carbosilane polymers sensitivity (433 µA/mM-1 cm2) and diundecenyl ferrocene silane moieties (PMDUS) were used (GLDH/Pt-PAMAM)n/CNT) Figure 4: Schematic showing the procedure of immobilizing Pt-PAMAM onto CNTs (a) layer-by-layer self-assembly of GLDH and Pt-PAMAM onto CNTs (b) Pt-PAMAM/CNTs heterostructures were covalently attached via EDC (Tang et al. 2007). Biomed Res J 2015;2(1):21-36 31 Nigam et al. stability. PDATT/Den Pt-PAMAM and GLDH were (AuNPs)/laccase probe (Rahman et al., 2008). The modified alternately deposited until suitable layers electrode were obtained. PAMAM G-4 dendrimers transfer (DET) process of laccase and a crosslinked with reduced graphene oxide catechin biosensor was fabricated based were tested for performance as electro- on the electrocatalytic process of laccase. chemical biosensors by immobilizing The linear range and detection limit for enzyme tyrosinase (Araque et al., 2013). catechin sensing was 0.1–10 and 0.05 µM, The respectively. bioelectrode electrocatalytic showed An direct electron- electrochemical towards biosensor based on PAMAM dendrimers determination of catechol with a response was developed for the detection of time of about 6s, linear range of 10 nM to fructose in food samples by immobilizing 22 µM, sensitivity of 424 mAM-1 and a low fructose detection limit of 6 nM (Fig. 5). cysteamine and PAMAM dendrimers. The PAMAM behavior excellent displayed dendrimer dehydrogenase (FDH) on encapsulated concentration range of the enzymatic AuNPs were first immobilized to a biosensor was 0.25–5.0 mM fructose conducting polymer with two amine (Damar et al., 2011). PAMAM dendrimers groups (3',4'-diamine-2,2',5',2''-terthio- were also used to enhance signal response phene (PDATT) through covalent bonding of a nanobiocomposite fabricated to between –COOH group of PAMAM and obtain an immunosensor for alpha-feto –NH2 group of PDATT. Laccase was protein (AFP) in human serum (Giannetto subsequently covalently bonded to the et al., 2011). The binding of the dendrimer –COOH of PAMAM dendrimers to form with biologically active molecules like Figure 5: (A) Amperometric response obtained with Tyr/PAMAM-Sil-rGO/GCE for different catechol concentrations at Eapp= -150 mV (B) FE-SEM image of Tyr/PAMAM-Sil-rGO (Araqueet al., 2013). Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 32 antibodies can improve the activity and was in accordance with referenced sensitivity of the system. Response range enzyme-linked and precision were evaluated using cyclic (ELISA) method. immunosorbent assay voltammetry (CV) and double step A multi-analyte sensing device based chronoamperometry (DSCA) with limit of on PAMAM dendrimer for simultaneous detection of 3 ng/mL and limit of at-line monitoring of glucose, ethanol, quantification of 15 ng/mL. The enhanced pO2- and cell density was fabricated (Akin immunosensor could be useful for et al., 2011). The device consisted of a monitoring prognosis of pregnancy and dual biosensor, a modified microscope occurrence diseases. and a fiber optical pO2-sensor integrated Recently, a redox-active silver-PAMAM into a flow analysis (FA) system. The dendrimer nanostructure was synthesized electrochemical transducer consisted of in situ by using wet chemistry (Xiaomei et self-assembly of cysteamine on gold al., 2013), and functionalized with mono- surface. Alcohol oxidase and pyranose clonal mouse anti-human antibody for free oxidase were immobilized onto the gold prostate specific antigen (fPSA). Using, surface by means of PAMAM (poly- graphite as the working electrode, a layer amidoamine) of gold nanoparticles modified with aldehyde cross-linking. The responses for prostate-specific antibody (mAb2). In glucose and ethanol were linear up to 0.5 presence of the fPSA, specific immuno- mM. The biosensor was used for complex was formed on the functionalized simultaneous determination of ethanol antibody modified electrode. The Ag- and glucose in yeast fermentation process. mediated PAMAM dendrimer directly A highly stable and sensitive ampero- catalyzed reduction of H2O2 in the metric biosensor was developed by detection solution. Thus, PSA was immobilizing alcohol oxidase (AOX) detected primarily due to the antigen- through PAMAM dendrimers on a antibody immunocoupling. Under optimal cysteamine-modified conditions, the developed immunoassay surface for determination of ethanol (Akin could determine target fPSA in the et al., 2009). The optimized ethanol dynamic range of 0.005–5.0 ng/mL with a biosensor showed a linearity from detection limit (LOD) of 1.0 pg/mL (S/N = 0.025–1.0 mM with 100 s response time 3). In addition, the accuracy of the and detection limit (LOD) of 0.016 mM. electrochemical immunoassay evaluated The analytical characteristics of the for detection of clinical serum specimens, system were also evaluated for alcohol of neoplastic Biomed Res J 2015;2(1):21-36 dendrimer gold via glutar- electrode 33 Nigam et al. determination in flow injection analysis films. The multilayer film assembled with (FIA) mode for analysis of ethanol in the dendrimer stabilized Au nanoparticles, various alcoholic beverage as well as provided a new approach to fabricate offline monitoring of alcohol production biosensors and bioreactors based on direct through yeast cultivation (Yuksel et al., electrochemistry of proteins and enzymes. 2012). PAMAM dendrimer (generation G4) CONCLUSIONS stabilized with 1-hexadecanethiol was Contemporary studies indicate that the most used for immobilization of acetylcholin elementary chemical reaction of electron esterase from electric eel, and choline transfer is widely prevalent in several oxidase from Alcaligenes sp. was used as biological systems and more importantly in electrode material for fabrication of an nanosystems with redox dendrimers. This is amperometric pesticides possible by tailoring the nature and topology (Snejdarkova et al., 2004). On similar of the dendrimers to precisely control location lines, urea electrochemical biosensor was of the redox sites within the macromolecule developed based on an electro-co- and study its electron-transfer processes. The deposited dendrimer increase in efforts to combine dendrimers with modified screen printed carbon electrode. other molecules like pyrrole, ferrocene, Urease enzyme was immobilized onto enzymes, etc. is promising in biosensing electrodes and an amperometric response applications. sensor for zirconia-PPI in urea concentration from 0.01 mM to 2.99 mM was obtained with sensitivity of -1 -2 3.89µA mM cm (Shukla et al., 2014). ACKNOWLEDGEMENTS The authors acknowledge Department of PPI dendrimers have also been used to Science and Technology, Government of reduce HAuCl4 to form core-shell PPI-Au India, New Delhi, for providing financial nanoclusters with several PPI molecules support. The authors also acknowledge the attached on the surface of one gold publishers nanoparticles (Zhang et al., 2007). PPI-Au permissions for the figures. for providing copyright nanoclusters and myoglobin (Mb) were alternately adsorbed on the surface of CONFLICT OF INTEREST pyrolytic The authors claim no conflict of interest. graphite (PG) electrodes forming {PPI-Au/Mb}n layer-by-layer Biomed Res J 2015;2(1):21-36 Dendrimers based electrochemical biosensors 34 REFERENCES Akin M, Prediger A, Yuksel M, Höpfner T, modification of poly(propylene imine) Demirkol DO, Beutel S, Timur S, Scheper T. A dendrimers. Chem – A Europ J 1997;3: new set up for multi-analyte sensing: At-line 974–984. bio-process monitoring. Biosens Bioelectron Bustos E, Chapman TW, Rodríguez-Valadez F, Godínez LA. Amperometric detection of H2O2 2011;26:4532–4537. Akin M, Yuksel M, Geyik C, Odaci D, Bluma A, using gold electrodes modified with starburst Höpfner T, Beutel S, Scheper T, Timur S. PAMAM dendrimers and prussian blue. 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Waghmare* Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India Tissue stem cells self-renew throughout the life of an organism thereby maintaining tissue homeostasis and prevent cancer. The major signalling pathways such as Wnt, Notch and Sonic hedgehog control the stem cell regulation and their deregulation leads to cancer. Recent evidences showed that there exists a subset of cells within tumour termed as cancer stem cells (CSCs). These CSCs escape the conventional chemoradiotherapy and further lead to tumour relapse followed by metastasis. This review focuses on the developmental signalling pathways that are involved in the regulation and maintenance of normal stem cells and CSCs. Understanding the molecular mechanism may be useful to specifically target the CSCs while sparing the normal stem cells to reduce tumorigenecity. INTRODUCTION Tumour is composed of a heterogeneous group implantation in mice (Furth et al., 1937). This of cells with different morphologies and suggested that certain cells within a tumour behaviour. Research in cancer biology may have the ability to give rise to tumour indicates that several cancers are supported by growth (Furth et al., 1937). Later, in 1994, a small subset of cells with stem cell like John Dick's group identified human acute properties and are termed as cancer stem cells myeloid leukaemia-initiating cells using (CSCs) or tumour initiating cells (TIC). CD34+CD38- markers and showed that these Evidences of CSCs involved in resistance to cells initiated tumour (Lapidot et al., 1994). In conventional therapies, leading to metastasis 1997, Bonnet and Dick showed for the first and tumour recurrence is abundant (Beck and time that the CD34 CD38 population of cells Blanpain, 2013; Chandler and Lagasse, 2010; had the self-renewal property. The authors Prince and Ailles, 2008). performed limiting dilution assay to show that + - + - As early as 1937, Furth and colleagues low numbers of CD34 CD38 cells were able demonstrated that a single cell was able to to form tumours in NOD/SCID mice, identical produce a haematopoietic malignancy on to donors; whereas considerably higher Key words: Cancer stem cells (CSCs); EMT, Epithelial to mesenchymal transition; Lineage tracing; β-catenin; NICD, Notch intracellular domain. *Corresponding Author: Sanjeev K. Waghmare, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India. Email: swaghmare@actrec.gov.in Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 38 numbers of non-CSCs (CD34+CD38+) were of CSCs has come from the lineage tracing unable to form tumours (Bonnet and Dick, experiments in mice model for various cancers 1997). These cells were coined as cancer stem such as glioblastoma, skin and colon cancers. cells. In 2003, Michael Clarke's group The assay showed that the individual reported the first isolation of CSCs from breast fluorescent tagged cells have the capability to tumour (Al Hajj et al., 2003). Subsequently, give rise to a tumour (Chen et al., 2012; the presence of CSCs in other solid tumours Driessens et al., 2012; Schepers et al., 2012). like melanomas, hepatocellular carcinoma, Although many different markers for glioblastoma, pancreatic cancer, colorectal CSCs have been identified in tumours of cancer and head and neck cancer have been different tissues, cells isolated by using these identified (Keshet et al., 2008; Li et al., 2007; markers are not a pure CSC population. Hence, Ma et al., 2007; Prince et al., 2007; Ricci- one of the major challenges is the isolation of a Vitiani et al., 2007; Singh et al., 2004). The pure population of CSCs. Recent study on CSC markers from various cancers are listed in quantitative proliferation dynamics of hair the Table 1. The characterisation of CSCs uses follicle stem cells showed the isolation of stem various assays that include: sphere-forming cells based on their cell division. This suggests assay, serial transplantation assay in NOD/ that it may be possible to isolate pure stem cell SCID mice and in vivo lineage tracing. Serial population (Waghmare and Tumbar, 2013; transplantation assay, is considered as 'gold Waghmare et al., 2008). Another challenge is standard' assay, and measures self-renewal as to understand how these CSC populations are well as the tumorigenic property of CSCs in regulated and maintained. Therefore, it is vivo (Al Hajj et al., 2003; Beck and Blanpain, important to study the various signalling 2013; Bonnet, 1997; Prince et al., 2007). pathways that are crucial for survival of CSC Recently the strongest evidence for existence population. Table 1: Cancer stem cell markers in various cancers Cancer Cancer stem cell markers Leukaemia CD34 CD38 (Bonnet, 1997) + - + Breast Cancer CD44 CD24- (Hajj et al., 2003); ALDH1+ (Ginesteir et al., 2007); CD133+ (Wright et al., 2008) Head and Neck CD44 Lin (Prince et al., 2007); A1DH1+ (Clay et al., 2010; Krishnamurthy et al., 2010); CD133 + - + + Cancer (Zhang et al., 2010); CD10+ (Fukusumi et al., 2014); CD98 (Matens de Kemp et al., 2013) Pancreatic Cancer CD44 +CD24-ESA+ (Li et al., 2007); c-Met (Li et al., 2011) Liver Cancer CD133+ (Ma et al., 2007); CD90+ (Yang et al., 2008); CD13+ (Haraguchi et al., 2010); OV6+ (Yang et al., 2008) Glioblastoma CD133+ (Singh et al., 2004); SSEA1+ (Son et al., 2009), MET (De Bacco et al., 2009) Melanoma ABCB5+ (Keshet et al., 2008) Colorectal Cancer CD133+ (Ricci-Vitiani et al., 2007); CD166+ (Dalerba et al., 2007; Vermeulen et al., 2008); Lgr5+ (Barker et al., 2007; Vermeulen et al., 2008), CD44+ (Haraguchi et al., 2008), CD44v6+ (Todaro et al., 2014) Biomed Res J 2015;2(1):37-56 Dash et al. 39 Embryonic developmental process and (Boumahdi et al., 2014). Another important phenomena common cancer stem cells Development of an organism is regulated at to both the CSCs as wells as the embryonic the molecular level by various signalling stem cells is the occurrence of epithelial to pathways, and deregulation in these molecular mesenchymal transition (EMT). During EMT, mechanisms leads to cancer formation. Recent the cells lose their polarity and acquire studies have shown various similarities migration capabilities that results in loss of between cancer and development. During the epithelial marker E-cadherin and simulta- normal developmental process, undifferentia- neous increase in mesenchymal marker N- ted embryonic stem cells further differentiate cadherin. During embryogenesis, EMT is and give rise to the differentiated tissues of an associated with gastrulation required for the organism. Similarly in cancer, undifferentia- formation of the three germ layers. In cancer, ted CSCs are involved in tumour progression EMT leads to invasion, metastasis and cancer that leads to metastasis (Bellacosa, 2013). stem cell-like phenotype (Kalluri, 2009; The embryonic stem cells have a core Singh, 2010). A recent study showed that of Twist1, an EMT promoter protein, is expressed transcription factors like Oct4, Sox2 and during early stages of tumorigenesis and is Nanog that contribute to self-renewal and required for the initiation of skin tumours pluripotency (Boyer et al., 2005). Similarly, (Beck et al., 2015). transcriptional network comprising lung CSCs showed elevated levels of Oct4 and All these indicate that regulation of Nanog transcription factors (Chiou et al., embryonic stem cells and CSCs share similar 2010). In head and neck cancer, CD44 variant mechanisms. Therefore, it suggests that CD44v3 was shown to interact with Oct4- deregulation Sox2-Nanog leading to CSC like properties pathways are involved in cancer formation and such as self-renewal and cisplatin resistance CSC regulation and maintenance. Hence, (Bourguignon et al., 2012). Recently, it was studying shown that the lineage ablation of Sox2- pathways will shed light on the regulation of expressing cells in both benign and malignant CSCs. the of various developmental developmental signalling skin squamous cell carcinomas resulted in tumour regression indicating an important role Developmental signalling pathways and of Sox2 in tumour initiation and CSC CSCs functions. Moreover, chromatin immuno- The various pathways which are deregulated precipitation analysis identified Sox2 target in cancer include Wnt, Notch, Hedgehog, genes involved in controlling tumour stemness EGFR, PI3K, NFκB, etc. Among these, three Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 40 Figure 1: Wnt Pathway. A) In the absence of the Wnt ligand, β-catenin is phosphorylated by destruction complex (APC, CK1α, GSK3 and Axin) and is subjected to proteasomal degradation resulting in no transcription of the Wnt target genes.B) In the presence of the Wnt ligand, the destruction complex is disrupted and thereby β-catenin enters the nucleus and brings about the transcription of Wnt target genes. APC: Adenomatous Polyposis Coli; CK1α: Casein kinase 1α; GSK3: Glycogen synthase kinase 3; TCF: T cell factor; LEF: Lymphoid enhancing factor; Dsh: Dishevelled; LRP: Low-density lipoprotein like receptors. well-known pathways such as Wnt, Notch and (Nusse et al., 1984; Rijswijk et al., 1987; Hedgehog play an important role in the Sharma, 2013). development and normal homeostasis. There are 19 highly conserved Wnt Conversely, deregulation of these pathways is ligands discovered till date. These ligands are shown in CSC regulation and maintenance secreted hydrophobic glycoproteins found to (Ailles, 2012; Purow, 2012). be associated with cell membranes and extracellular matrix. In Wnt producing cells, the Wnt Pathway endoplasmic reticulum produces Wnt ligands, Wnt pathway is evolutionarily conserved and lipid modified by porcupine (Mikels, 2006; is involved in various organisms. It was first Willert et al., 2003). Wnt ligands can act discovered in Drosophila, when a mutation in through two general categories of pathways: wingless (wg) gene led to a distinct phenotype canonical and non-canonical. The canonical including absence of wings and halters. Later, pathway is β-catenin dependent, while the Nusse's group showed that the insertion of non-canonical pathways include Wnt/Ca2+ and Mouse Mammary Tumour Virus (MMTV) in Wnt/JNK pathways. In the canonical pathway mice led to mammary tumour by proviral shown in Fig. 1, Wnt ligands bind to the activation of the int oncogene. The int conserved cysteine rich domain (CRD) oncogene was later demonstrated as the mouse domain of the frizzled receptors (Fz) which in homologue of the Drosophila wg gene. From turn forms co-receptors complexes with low- these two studies, a new nomenclature Wnt density lipoprotein like receptors (Lrp5/6). (combination of wg and int) was obtained Further, Biomed Res J 2015;2(1):37-56 this interaction recruits the Dash et al. 41 Dishevelled (Dsh) protein to the cytoplasmic gastrulation defect and perturbations in the tail of Fz receptor and brings about inhibition establishment of apical ectodermal ridge of destruction complex surrounding β-catenin. during development (Liu et al., 1999). Further, The components of the destruction complex absence of Wnt4 ligand led to defects in female comprise of scaffold protein Axin, Glycogen development, while Wnt7a deletion led to synthase kinase 3β (GSK3β), Casein kinase 1α female infertility in mice (Jeays-Ward et al., (CK1α) and adenomatous polyposis coli 2004; Parr et al., 1998). Axin1 knockout in (APC). In the absence of the Wnt ligands, the mice led to neuro-ectodermal and cardiac destruction complex hyper-phosphorylates β- abnormalities (Zeng et al., 1997). Wnt catenin and targets it for proteasomal signalling was shown to be crucial in hair degradation by ubiquitination. The binding of follicle development as targeted deletion of β- Wnt ligand to Lrp5/6 causes phosphorylation catenin in the epidermis led to failure in of the cytoplasmic tail of Lrp6, which in turn placode morphogenesis (Huelsken et al., recruits Axin to the receptor complex that 2000). Absence of Lef1 led to defects in the disrupts the destruction complex and stabilises pro-B-cell proliferation and abnormalities in β-catenin. The stable β-catenin translocates to several organs like teeth, mammary glands, the nucleus and binds to the lymphoid whiskers and hair (Reya et al., 2000; enhancing factor/T-cell factor (LEF/TCF) VanGenderen et al., 1994); while the knockout thereby the of Tcf1 led to thymocyte proliferation and different target genes involved in cell fate differentiation defects (Schilham et al., 1998). determination during embryonic development Using the Wnt reporter, Axin2-LacZ, Wnt and tissue homeostasis (Mikels, 2006; Willert responsive cells were localised to the sub et al., 2003). ventricular zone (SVZ) of the developing brain transcriptionally activating and basal layer of the mammary ducts, which Wnt signalling in normal development and are the stem cells niches. Furthermore, these cancer Wnt responsive cells showed high sphere Wnt pathway is involved in different biological forming ability and were able to differentiate. processes such as embryonic development, Hence, the Wnt pathway plays an important self-renewal, proliferation, morphogenesis, role in normal development and tissue etc. Wnt3a and Wnt1 knock out in mice led to homeostasis (Logan, 2004; VanAmerongen et deficiencies in neural crest derivatives and al., 2009). neural tube formation during the development There are strong evidences showing (McMahon et al., 1990; Yoshikawa et al., involvement of Wnt pathway in regulation of 1997). Wnt3 knock out in mice led to early various cancers. Frequent somatic mutations Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 42 in β-catenin were observed in both mice and (HSC), overexpression of β-catenin increases human hepatocarcinomas (Coste et al., 1998), the stem cell pool size suggesting that Wnt prostate cancers and colon cancers (Voeller et pathway al., adenoma hematopoietic stem cell homeostasis (Reya et initiation, the first step was APC inactivation al., 2003). In mice hair follicle stem cells, live followed by β-catenin stabilization, while cell imaging showed that β-catenin activation progression from adenoma to carcinoma in hair follicle stem cells was involved in hair required the synergistic action of k-ras follicle tissue growth (Deschene et al., 2014). activation and β-catenin nuclear localization Further, Wnt target gene Lgr5, a G-protein (Phelps et al., 2009). β-catenin was shown to coupled receptor was identified as an intestinal be essential for retaining tumorigenecity of stem cell marker indicating an important role MDA-MB-231 breast cancer cell lines both in of Wnt pathway in the regulation of intestinal vivo β-catenin stem cells (Ailles, 2012, Valkenburg, 2011). knockdown cells implanted into mice showed The deletion of Tcf4, a Wnt downstream gene decrease in the tumour size. In addition, an in showed loss of stem cell activity and reduced vitro study in breast cancer cell lines showed proliferation of the intestinal epithelium reduction in aldehyde dehydrogenase 1 (Korinek et al., 1998). In addition, Lgr5 was (ALDH1) positive cells (Xu et al., 2015). identified as a marker of hair follicle stem cells Wnt3a expression was associated with EMT (Jaks et al., 2008) with multipotent properties. and promoted colon cancer progression (Qi et Moreover, Wnt inhibitor SFRP1 was shown to al., 2014). Moreover, deletion of Axin1 was play an important role in hematopoietic stem reported in sporadic medulloblastomas and cell maintenance through extrinsic regulation hepatocellular carcinomas (Dahmen et al., (Renstrom et al., 2009). Over-expression of 2001). Increased expression of Dsh protein in Sfrp1 led to enhanced mesenchymal stem cell non-small cell lung carcinoma and meso- function in angiogenesis (Dufourcq et al., thelioma have been reported (Uematsu et al., 2008). Besides, Sfrp1 was over-expressed in 2003). hair follicle stem cells as compared to the non- 1998). and During in intestinal vitro. Further, is critical to maintain the stem cells (Tumbar et al., 2004; Zhang et al., Wnt signalling in normal and cancer stem 2009). Recently, it was shown that Sfrp1 gene cell regulation and maintenance is critical for maintaining proper mammary Wnt signalling is important in adult stem gland development wherein loss of Sfrp1 regulation and has been shown to be involved promotes mammosphere formation; however in stem cell proliferation, self-renewal and the role in mammary stem cells needs further maintenance. In hemato-poietic stem cells investigation (Gauger et al., 2012). Biomed Res J 2015;2(1):37-56 Dash et al. 43 In cancer, various reports have shown that Notch Pathway deregulation of Wnt pathway is crucial for the Notch gene was first discovered in Drosophila CSC regulation. Human head and neck CSCs by Morgan and Bridges where they showed treated with Wnt antagonist, secreted frizzled- that a mutation led to wings notching and related protein 4 (sFRP4), the CSCs showed hence the name “Notch” was coined (Morgan reduction in the sphere-forming capacity and and Bridges, 1916; Mohr, 1919; Poulson, decrease in the stemness markers like CD44 1940). There are four Notch genes, three and ALDH1 (Warrier et al., 2014). In another Delta-like and two Jagged genes in mammals, report, β-catenin was shown to be required for that are translated into different Notch ligands, maintenance of cutaneous CSCs since deletion Delta and Jagged. Recently, it was shown that of β-catenin led to reduction in the CSCs and for tumour regression (Malanchi et al., 2008). development, complex of Notch receptor- CSCs isolated from mammary tumours of Delta-Jagged acts in concert (Fiuza, 2004; radiation treated p53-null mice showed altered Boaretoa et al., 2015). cell fate determination during DNA repair in response to radiation as well as Since the Notch ligands such as Delta and β-catenin activation (Zhang et al., 2010). In Jagged proteins, as well as Notch receptors are prostate cancer, Wnt signalling induced transmembrane proteins, cell-cell contact is tumour initiation, EMT and metastasis. important for the signalling cascade. The Additionally, in prostate cancer cell lines and Notch receptors contain an extracellular primary cultures, Wnt3a treatment increased subunit, having multiple EGF-like repeats, the self-renewal capacity of putative prostate and a transmembrane subunit (Wharton et al., CSCs, emphasizing that Wnt signalling plays 1985). When the Notch ligand binds to its an important role in prostate cancer (Barker, receptor, the extracellular domain of the Notch 2006; Valkenburg, 2011; Verras et al., 2004). receptor is dissociated from the trans- Moreover, the inactivation of APC in Lgr5- membrane domain and the S2 cleavage site is positive stem cells at the intestinal crypts led to exposed (Fig. 2). This site is cleaved by transformation within days; while inactivation ADAM (a disintegrin and metalloprotease) of APC in progenitors or differentiated cells generating an intermediate that is further did not lead to tumour formation even after 30 cleaved by γ-secretase to generate Notch weeks (Barker et al., 2009). In addition, the Intracellular Domain (NICD). NICD then deletion of CD44, a CSC marker and a Wnt translocates to the nucleus where it binds to target gene in mice having heterogeneous APC ubiquitous transcription factor CSL (CBF-1, mutation (APC Min/+ ), attenuates intestinal tumorigenesis (Zeilstra et al., 2008). Suppressor of Hairless, Lag-1). This complex displaces a co-repressor complex containing Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 44 Figure 2: Notch Pathway. A) In the absence of the Notch ligands (Delta and Jagged), the S2 cleavage site remains hidden and inaccessible to ADAM. Hence, the NICD is not formed, with consequent no transcription of the Notch target genes. B) In the presence of the Notch ligands, the conformational change in the intracellular subunit of the Notch receptor takes place exposing the S2 cleavage site, thus leading to the formation of NICD. Further, NICD translocates the nucleus and brings about transcription of Notch target genes. NICD: Notch intracellular domain; ADAM: A disintegrin and metalloprotease; MAML: Mastermind-like protein; SKIP: Ski-interacting protein; SHARP: SMRT associated protein. SKIP, SHARP and histone deacetylases. establishment of the central and peripheral Further, it recruits a co-activator complex nervous systems, spermatogenesis, oogenesis, containing (Mastermind-like myogenesis and imaginal disc development protein), p300 and other chromatin modifying (Artavanis-Tsakonas et al., 1999). In normal enzymes, thereby bringing about transcription mammary development, Notch pathway of different Notch target genes (Ailles, 2012; activation is required for regulation of cell Andersson, 2011; Fiuza, 2004). fate, proliferation and stem cell self-renewal. a MAML The Notch pathway is also shown to be Notch signalling in normal development important for tip-cell formation during and cancer mammalian astrocyte differentiation and Notch pathway is also evolutionarily angiogenesis. In vertebrates, the Notch conserved and is important in cell to cell pathway leads to patterning during inner ear communication that regulates cell fate hair cell formation and insulin-secreting determination pancreatic β cell production (Ailles, 2012; during development, cell proliferation, differentiation and apoptosis. Fortini, 2009). The loss of Notch function in vertebrates is Notch signalling has been shown to be associated with disruption of neurogenesis, involved in various cancers. For instance, somite formation, angiogenesis, and lymphoid Notch1 regulates breast cancer cells by development. In Drosophila, Notch is shown inducing Slug expression (Shao et al., 2015). to control the fate of various cell types in the Notch4 promotes growth of gastric cancer eye. In vertebrates, Notch is involved in the cells through activation of Wnt1, β-catenin Biomed Res J 2015;2(1):37-56 Dash et al. 45 (Quian et al., 2015) and the downstream target expressed in stem cells and disruption led to such as c-myc and cyclin-D1. In mice, Notch1 defective stem cells proliferation (Kitamoto activation combined with p53 loss showed and Hanaoka, 2010). synergistic effect in the formation of Osteogenic sarcoma (Tao et al., 2014). Notch signalling plays an important role in a number of hematopoietic and solid tumours, but the strongest evidences for its role in CSC Notch signalling in normal and cancer stem regulation has been shown in breast cancer, cell regulation and maintenance embryonal brain tumours and gliomas (Fan et Notch has been shown to be involved in self- al., 2006; Pannuti et al., 2010). In various renewal, proliferation and differentiation of human breast cancer cell lines and primary adult stem cells in various tissues. In mice patient tissues, a significant decrease in mammary stem cells, the knockdown of Cbf-1, mammosphere a canonical Notch effector, showed increased inhibition has been demonstrated (Abel et al., stem cell activity in vivo suggesting a role in 2014). Further, studies on the human controlling mammary stem cells. (Bouras et mammary mammospheres have shown a al., 2008). Notch directly targets the crypt base feedback loop between Her2/Neu and Notch, columnar cells that maintain stem cell activity as well as promotion of a hypoxia resistant in mice (VanDussen et al., 2012). The Notch phenotype (Pannuti et al., 2010). In brain activation maintains the slow cycling property tumours, blockade of Notch led to a 5-fold of neural stem cells; however, blocking Notch reduction in the CD133+ cell fraction and total resulted in increased number of stem cell depletion of the side population cells. divisions followed by depletion of the stem Additionally, differentiated cell growth was cell pool (Chapouton et al., formation after Notch 2010). observed after Notch inhibition, but lacked Furthermore, constitutive activation of Notch formation of tumour xenografts efficiently, signalling promotes self-renewal in muscle indicating that the CSCs required for tumour stem cells through upregulation of Pax7 (Wen propagation were absent (Fan et al., 2006). A et al., 2012). Mice with satellite cell specific recent study on primary human pancreatic deletion of RBP-Jj (recombining binding xenografts showed upregulation of the notch protein-Jj), a nuclear factor required for Notch pathway components in pancreatic CSCs. signalling, showed depletion of the stem cell Additionally, inhibition of notch pathway pool and their muscles lacked ability to reduced CSC percentage and tumour-sphere regenerate in response to injury (Bjornson et formation significantly (Abel et al., 2014). al., 2012). Notch 3 has been shown to be Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 46 In Sonic-hedgehog Pathway Hedgehog pathway is delineated the Sonic-hedgehog pathway in elaborated in Fig. 3, absence of the hedgehog Drosophila and determines the anterior- ligand, Smoothened (Smo) is inhibited by posterior orientation of developing structures being bound to Patched (Ptc). When the (Nusslein-Volhard and Wieschaus, 1980). hedgehog ligand binds to Ptc, inhibition of Similar to Wnt and Notch, the key components Smo is released which acts on protein complex of the Hedgehog pathway are evolutionarily comprising of fused (Fu), suppressor of fused conserved, although differences are observed (Sufu) and cos-2-costa-2 (Wicking et al., in the mammalian and Drosophila Hedgehog 1999; Merchant, 2010). These proteins are signalling. While Drosophila has only one generally bound with Gli thereby inhibiting its hedgehog gene, three homologues have been action. Once the complex is disrupted, Gli identified in vertebrates namely, Sonic (Shh), translocates to the nucleus and brings about Desert (Dhh), and Indian hedgehog (Ihh). The transcription of different downstream targets Sonic Hedgehog pathway is extensively (Sasaki et al., 1999; Ruiz, 2007; Stecca, 2010). investigated in the vertebrate system (Chen et al., 2005; Varjosalo et al., 2006). The other Sonic-hedgehog components of the Hedgehog pathway include development and cancer patched (Smo), In vertebrates, the Sonic Hedgehog (Shh), is transmembrane expressed widely throughout the developing glycoprotein and a 7-pass transmembrane central nervous system (CNS), limb, gut, teeth protein and (Ptc) constituting a and smoothened 12-pass respectively Wicking et al., 1999). (Varjosalo, 2008; hair-follicle. signalling Dhh is in normal involved in development of the germline, while the Ihh is Figure 3: Hedgehog Pathway. A) In the absence of Hedgehog ligands (Indian, Sonic and Dessert), the Patched receptor (ptc) exerts inhibitory action on the Smoothened receptor (smo). The Gli complex (Gli1 and Gli2) remains in the cytoplasm followed by no transcription of Hedgehog target genes. B) In the presence of Hedgehog ligands, the inhibitory action of Patched (ptc) on Smoothened (smo) is released, and hence Gli complex translocates to the nucleus and brings about transcription of Hedgehog target genes.Fu: Fused; SuFu: Suppressor of Fused; Cos 2: cos-2costa-2. Biomed Res J 2015;2(1):37-56 Dash et al. 47 involved in development of the skeletal system Hedgehog almost completely blocks intestinal (Bitgood et al., 1996; Wicking et al., 1999). adenoma development (Buller et al., 2015). Shh also plays a role in neural stem cells, determining the neuronal cell fate (Merkle et Sonic-hedgehog signalling in normal and al., 2007). It was demonstrated that during cancer repair of acute airway injury, the Hedgehog maintenance pathway gets activated in the airway Hedgehog signalling is involved in stem cell epithelium (Watkins et al., 2003). Hedgehog regulation of various tissues. Shh regulates signalling components Ptc, Gli1, and Gli2 self-renewal of neural stem cells (Palma et al., were mammary 2005). The components of Hedgehog pathway progenitor cells grow as mammospheres (Liu such as Ptc, Gli1 and Gli2 are expressed in the et al., 2006). These reports indicate that the mammary stem cells and down regulated Hedgehog pathway plays a role in normal stem during differentiation (Liu et al., 2006). cell regulation (Ailles, 2012). Hedgehog is involved in controlling neural over-expressed when Hedgehog signalling is involved in various cancers. For example, Ptc1 mutation stem cell regulation and stem cells through the p53-independent regulation of Nanog (Po et al., 2005). was observed in patients with medullo- In colon carcinoma, Hedgehog signalling blastoma and rhabdomyosarcoma (Hahn et al., is activated in CSCs with higher expression of 1996; Johnson et al., 1996; Pietsch et al., Gli1 and Gli2. In non-small cell lung cancer, 1997). Sufu as well as Smo mutations were the malignant phenotype of the tumours is observed in medulloblastoma (Xie et al., 1998; maintained by ligand-dependent Hedgehog Taylor et al., 2002); Gli1 and Gli3 mutations pathway activation (Watkins et al., 2003). were seen in pancreatic adenocarcinoma; and Furthermore, Bmi1, which is a downstream Gli1 in target of the Hedgehog pathway was activated glioblastoma (Clement et al., 2007; Jones et in breast CSCs and is also shown to regulate al., 2008). Further, Kern et al showed that Gli normal and leukemic stem cells (Liu et al., and 2006; Takebe, 2011). gene amplification PI3K/AKT/mTOR was seen signalling act synergistically to initiate and maintain chronic lymphocytic leukemia (Kern et al., 2015). CONCLUSION Another report showed that Sonic hedgehog Tumour maintenance and progression is ligand over-expression led to increased regulated by a subset of cells that are known as number and size of intestinal adenomas in cancer stem cells (CSCs). Recently, due to APC (HET) mice, while loss of Indian increase in evidences on the existence of Biomed Res J 2015;2(1):37-56 Developmental signalling in cancer cells 48 CSCs, they have gained more attention but targeted drugs approach can be fruitful. how Hence, these CSCs escape the chemo- further detailed research on radiotherapy is still unknown. Moreover, how deregulation of the developmental pathways the CSCs are maintained in the tumour micro- in CSCs needs to be investigated. Eventually, environment remains elusive. Several reports elucidation of the signalling mechanisms will showed signalling pathways such as Wnt, enable to specifically target CSCs without Notch and Sonic-hedgehog are deregulated in affecting the normal cells. cancer, and also involved in the CSC regulation and maintenance. In addition, ACKNOWLEDGEMENTS evidence of cross-talk between the signalling The authors acknowledge Mr. Rahul Sarate pathways exists. 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Activation of Nrf2 dependent proteins is crucial in maintaining cellular redox homeostasis and combating toxicity of carcinogens. Thus, employing natural or synthetic activators of Nrf2 pathway is a promising approach for development of chemopreventive modalities. Intriguingly, recent reports have highlighted the dark side of Nrf2 suggesting that multiple cancer cells demonstrate constitutive activation of Nrf2 caused by mutations in Nrf2 or Keap-1 proteins, offering survival advantage. Additionally, Nrf2 pathway is also up-regulated in chemoresistant cells and may be a major contributor in acquired chemoresistance. Thus, targeting Nrf2 pathway has emerged as a novel strategy to improve efficacy of chemotherapeutic drugs. This review discusses the dark and bright sides of this transcription factor in line with the recent literature. INTRODUCTION The transcription factor, nuclear factor potentially [erythroid-derived 2]-like 2 (Nrf2) was transactivation domain (Moi et al., 1994). identified as NF-E2-like basic leucine zipper Further characterization demonstrated it as transcriptional activator that binds to the Cap`n`Collar (CNC) protein involved in the tandem NF-E2/AP1 repeat of the β-globin control locus control regions (Moi et al., 1994). The development by basic leucine zipper DNA Nrf2 gene was cloned and characterized by binding domain (bZip) homeotic gene. The using the tandem repeats of nuclear factor like CNC family comprises four members, namely erythroid factor-2 (NF-E2)/activator protein-1 Nrf1, Nrf2, Nrf3 and p45NF-E2. Nrf1 and (AP1) of the β-globulin locus as a recognition Nrf2 are ubiquitously expressed and are site probe. Nrf2 contains a basic leucine zipper essential for normal development in mice. The DNA binding domain at the C-terminus and an expression of Nrf3 is restricted to placenta and N-terminal acidic domain (rich in glutamic liver, while p45NF-E2 expression is restricted and aspartic acid residues), which could to erythrocytes (Ikeda et al., 2004; Motohashi of function Drosophila as an head acidic segment Key words: Cancer, Nrf-2 transcription factor, Keap-1 protein, β-TrCP protein. *Corresponding Author: Santosh K Sandur, Free Radical Biology Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Modular Laboratories,Bhabha Atomic Research Centre, Trombay, Mumbai, India. Email: sskumar@barc.gov.in Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 58 et al., 2002). Expression of Nrf1 is essential for seven lysine residues that direct ubiquitin embryonic development and its deficiency mediated proteasomal degradation of Nrf2 leads to hepatic abnormality. The Nrf2 (Fig. 1) (Itoh et al., 1999; Zhang et al., 2004). knockout mice are viable and exhibit no Neh3 is essential for interaction of Nrf2 with phenotypic defects, but are sensitive to CHD6 (a chromo-ATPase/helicase DNA oxidative stress (Chan and Kwong, 2000; binding protein) suggesting involvement in Chan et al., 1998; Leung et al., 2003; Ohtsuji interaction with co-transcription factors (Nioi et al., 2008; Ramos-Gomez et al., 2001; Xu et et al., 2005). Human Nrf2 is homologus to transactivation domains that interact with the mouse and contains six highly conserved CREB-binding protein (CBP) (Katoh et al., domains called Nrf2-ECH homology domains 2001). Neh6 domain interacts with β- (Neh). Neh1 domain has a nuclear localisation transducin repeat-containing protein (β-TrCP) signal and CNC-type basic leucine zipper (Jain and Jaiswal, 2007). Binding of Keap1 to necessary for DNA binding and dimerization. Nrf2 brings it close to E3 ligase complex The Neh2 domain contains a Keap1 (Kelch- through two major domains: BTB (Bric a like ECH-associated protein 1a, negative Brac, tramtrack, broad complex) domain regulator of Nrf2) binding pocket and has which interacts with Cul3; and kelch domain al., 2005). Neh4 and Neh5 are Figure 1: Structures and functions of Nrf2 and its repressors Keap1 and β-TrCP1. The relative position of the Neh domains is shown. The DLG and ETGE motifs present in Neh2 domain that bind to Keap1 are represented above with the numbering of amino acids based on the human cap'n'collar (CNC)-basic-region leucine zipper (bZIP) protein. Biomed Res J 2015;2(1):57-82 59 Gambhir et al. which binds to Nrf2. Interaction of Neh2 reductase (TrxR), peroxiredoxin (Prx), heme domain with Keap1 depends on low-affinity oxygenase-1 (HO-1) and transporters like binding via DLG motif and high-affinity multidrug binding of an ETGE motif which results in a (MRP) (Banning et al., 2005; Ishii et al., 2000; hinge and latch mechanism of binding. The N- Ishii and Yanagawa, 2007; Kim et al., 2001; terminal BTB/POZ (Pox virus Zinc finger) Maher et al., 2005; Moinova and Mulcahy domain enabling 1999; Sakurai et al., 2005). Phase II enzymes Keap1–Nrf2 interaction (Adams et al., 2000; reduce the toxicity of xenobiotics by making Kensler et al., 2007, Li et al., 2004; Lo et al., them water soluble, thereby facilitating their 2006; Padmanabhan et al., 2005). elimination. Efflux of endogenous molecules forms homodimers resistance-associated protein and xenobiotics is also governed by Nrf2 Activation of Nrf2 dependent genes mediated expression Exposure of cells to low levels of oxidative Constitutive expression of Nrf2 by tumor cells stress, electrophiles or chemopreventive may offer an advantage for ambient growth compounds leads to activation of Nrf2. Upon and activation, Nrf2 dissociates from inhibitory phenomena coined as “dark side of Nrf2” (Lau protein Keap1 and translocates to the nucleus. et al., 2008; Wang et al., 2008c). The present In the nucleus it forms a heterodimer with co- review emphasizes the putative dual role of transcription factor Maf and binds to the anti- Nrf2 pathway during cancer progression and oxidant response element (ARE) sequence to highlights its potential as a target for induce transcription of several different genes chemoprevention. detoxification of of transporters. xenobiotics, the (Zhang, 2006). ARE sequence is the 'core' sequence of 5´-RTGACnnnGCR-3´ identified Mechanism of Nrf2 Activation using murine GST-Ya ARE. The sequence was Nrf2 is sequestered in the cytoplasm by Keap1 used to identify genes present in the promoter which regulates Nrf2 stabilization and levels region (Rushmore et al., 1991). The Nrf2 inside the cell. The interaction between the downstream II two proteins is a dynamic process regulated in detoxifying enzymes like glutathione S- such a manner that enables Nrf2 to control transferase both the basal and inducible expression of genes (GST), oxidoreductase-1 include phase NAD(P)H (NQO1), quinone and UDP- dependent genes. Under homeostasis glucuronosyltransferase (UGT), intracellular conditions, Nrf2 is maintained at low basal cytoprotective glutamate levels for expression of cytoprotective genes cysteine ligase (GCL), glutathione peroxidase (Fig. 2) (Itoh et al., 1999). Nrf2 is at low levels (GPx), when bound to Keap1 homodimer through its proteins thioredoxin like (Trx), thioredoxin Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 60 Figure 2: Schematic model of Nrf2 activation under normal and oxidative stress conditions. and in vivo kelch repeats domains at C terminal, leading to alkylation site-directed Cullin3/Rbx1-mediated polyubiquitination mutagenesis, cys151 was identified as the and subsequent proteasomal degradation. major site directly alkylated by Nrf2 inducers Keap1 protein contains numerous cysteine along with critical residues cys273 and cys288 (cys) residues with potential to act as a redox (Dinkova-Kostova et al., 2002; Eggler et al., sensor (Hong et al., 2005b). 2005; Hong et al., 2005a; Levonen et al., 2004). Mutation at cys151 abolished induction Role of cys residues in Nrf2 activation of Nrf2 by activators like sulforaphane and The significance of Keap1 as a central tert-butylhydroquinone but had no impact on regulator of Nrf2 activation was revealed Keap1:Nrf2 binding. Keap1-cys151 restores while addressing the negative regulation of phenotypes like over-expression of Nrf2 and antioxidant machinery by Keap1 dependent post-natal lethality as observed in Keap1 null proteasomal degradation of Nrf2 (McMahon mice (Wakabayashi et al., 2004). However, et al., 2003). The half life of Nrf2 increases activation of Nrf2 by arsenite in cys151 Keap1 from 15 min to 30 min in cells expressing mutant MDA-MB231 cells, indicated a mutated ETGE motif containing Nrf2 and possible redox independent mode of Nrf2 Keap1 (Du et al., 2008). Using in vitro induction (Wand et al., 2008b). Further Biomed Res J 2015;2(1):57-82 61 Gambhir et al. cys273ser and cys288ser mutations showed release of Nrf2 from the low affinity binding abrogated repression of Nrf2 by Keap1 motif (Cullinan et al., 2004; Kobayashi et al., (Levonen et al., 2004; Wakabayashi et al., 2006). The change confers stabilisation and 2004). These observations demonstrated that accumulation of Nrf2 in the cytosol followed cys151 is required for the activation of Nrf2, by nuclear translocation. According to hinge whereas cys273 and cys288 are needed for and latch model, ETGE motif remains bound Nrf2 significant to the Keap1 following activation. This results contribution of the critical cysteine residues in saturation of Keap1 which is no longer able during Nrf2 activation and regulation under to oxidative stress was indicated. Several cellular translocation to the nucleus and binding to redox modifiers modulate activation of Nrf2 ARE to induce expression of cytoprotective via modification of the critical cysteine machinery of the host cell (Jain and Jaiswal, residues in Keap1. Further, the afore- 2006). An alternate model of induction is mentioned three critical cysteine residues attributed to the polyubiquitination of Keap1 undergo thiol modifications leading to at lys63, leading to subverted Cullin3 conformational change in the Cul3–E3 ligase interaction and dissociation of Nrf2 from complex leading to loss of E3 ligase ubiquitin Keap1 (Zhang et al., 2005). The ubiquitin- activity. The cysteine residues act as redox specific protease-15 deubiquitinase restored sensors to further perturb the efficiency of Keap1 activity (Villeneuve et al., 2013). inhibition. Besides, a compete with free Nrf2 inducing nuclear export signal on Keap1, and mutant Apart from Keap1 and Cul3/Rbx1, other form of Keap1 at leu308 and leu310 was mediators also contribute in regulating the low unable to locate in the cytoplasm (Kobayashi basal levels of Nrf2. Phosphorylation status of et al., 2009; Nguyen et al., 2005; Velichkova tyr568 on Nrf2 is governed by Src subfamily and Hasson, 2005). These studies suggested kinases Fyn, Src and Fgr, which influence the that under normal conditions, the signals from nuclear export of Nrf2. Under oxidative stress nuclear export sequence (NES) of Keap1 conditions, glycogen synthase kinase-3 beta maintained the Keap1 dimer in association (GSK-3β), a serine/threonine protein kinase, with Nrf2 in the cytoplasm. plays an important role in the nuclear export of Exposure of cells to oxidative, xenobiotic Nrf2 by phosphorylating Fyn. Another Src or electrophilic stress abrogates Keap1 member Bach1 has been shown to govern induced degradation of Nrf2. Perturbation in export of Nrf2 from the nucleus, thereby the in negatively regulating expression of its modifications of critical cysteine residues in dependent genes. Bach1 competes with Nrf2 Keap1. The conformational change renders for binding to ARE sequence, resulting in cellular redox status results Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 62 suppression of ARE mediated expression of rylated ser40 residue in the Neh2 domain Nrf2 dependent genes (Jain and Jaiswal, 2006; leading Niture et al., 2011). interaction in response to oxidative stress to disruption of Keap1/Nrf2 induced by tBHQ and β-naphthoflavone. Keap1 independent activation of Nrf2 Mutation in the serine residue results in Multiple studies have highlighted Keap1 abrogation of PKC induced activation of Nrf2 independent activation of Nrf2. Along with (Huang Keap1 dependent degradation of Nrf2, an phosphorylation of ser40 was required for alternate mechanism controls activation and release of Nrf2 from Keap1, but does not play stabilisation of Nrf2 mediated by β-transducin a role in nuclear translocation (Bloom and repeat-containing protein (β-TrCP) (Rada et Jaiswal, 2003). Nuclear localisation sequence al., 2012). Mouse Nrf2 contains two binding (NLS) and nuclear export sequence in Nrf2 sites for β-TrCP which acts as an adapter for regulates localization in the cell. The NLS the ligase motifs are identified by adapter proteins like complex. GSK-3β phosphorylates serine importins that facilitate transfer inside to residue in SCF/β-TrCP destruction motif nucleus (Theodore et al., 2008). Another “DSGIS” in Neh6 domain leading to Keap1 conserved protein kinase that influences Nrf2 independent degradation (Jain and Jaiswal, activation is casein kinase II (CK2). CK2 2007). Post translational modification also possesses an array of potential targets and governs Nrf2 activation. Nrf2 contains plays a role in complex cellular processes multiple serine, threonine and tyrosine including cytoprotection. Nrf2 contains 13 residues which serve as potential sites for potential phosphorylation targets for CK2 phosphorylation. Different pathways for abundant activation of Nrf2 are identified including domains. Phosphorylation dependent nuclear protein kinase C (PKC), mitogen-activated translocation of Nrf2 is sensitive to Ck2 protein kinases (MAPK), phosphatidylinositol inhibitor (Apopa et al., 2008; Pi et al., 2007). Skp1-Cul1-Rbx1 ubiquitin et in al., 2002). Neh4/Neh5 Interestingly, transcriptional 3-kinase (PI3K), and RNA-dependant protein kinase-like endoplasmic reticulum kinase Role of MAPK in activation of Nrf2 (PERK) (Cullinan and Diehl, 2004; Lee et al., PI3K 2001; Yu et al., 2000). PKC has multiple protein kinase (ERK) are proposed to regulate isoforms which play essential roles in growth, Nrf2 pathway (Cullinan et al., 2003; Kang et differentiation, apoptosis, al., 2001). tBHQ enhances NQO1 protein survival and carcinogenesis and PKC can be expression and activity in a PI3K dependent activated by oxidative stimuli. PKC phospho- manner in human neuroblastoma cells. tBHQ cytoprotection, Biomed Res J 2015;2(1):57-82 and extracellular signal-regulated 63 Gambhir et al. elicited ARE mediated induction of GST in activating Nrf2. The authors further hepatoma cells in a PI3K dependent manner. demonstrated Raf-1 mediated activation of PI3K inhibitor (Ly294002) abrogated tBHQ Nrf2 attributing it to up-regulation of the co- mediated NQO1 induction, indicating a role of activator CREB binding protein. PI3K in Nrf2 activation (Lee et al., 2001). PERK, a transmembrane kinase, phospho- Pro-oncogenic Effects of Nrf2: The Dark rylates Nrf2 in vitro leading to dissociation Side from Keap1. A pivotal role of PERK mediated It is well documented that oxidative stress activation of Nrf2 was proposed as a plays a pivotal role in the initiation and mechanism for maintenance of glutathione progression of cancer, with magnitude of levels that act as a cytoprotective buffer oxidative stress a key determinant of the against oxidative insult (Cullinan et al., 2003). response of a cell towards the oncogenic An important role of MAPK in the activation stimuli. Chronic exposure of cells to oxidative of Nrf2 via phosphorylation has been reported insult causes cytotoxicity due to irreversible by several investigators. Yu et al. (2000) damage to vital macromolecules; whereas studied MAPK mediated activation of phase II transient increase leads to the activation of detoxification multiple redox sensitive pro-survival transcription inducers (Jeong et al., 2006). In hepatoma factors. Therefore, in order to survive and cells, sulforaphane and tBHQ induced proliferate, tumor cells maintain a moderate activation of ERK, MAPK kinase and Raf-1, oxidative intracellular niche achieved by to mediate induction of phase II detoxification taking advantage of the antioxidant defense enzymes via Nrf2/ARE pathway (Yuan et al., machinery of the cell like Nrf2 pathway. 2006). MAPK/ERK upon activation initiates Constitutive activation of Nrf2 and expression phosphorylation modulates of dependent cytoprotective genes, permits activity of multiple downstream transcription tumor cells to nurture and expand in an factors (Shen et al., 2004; Zipper and ambient redox niche. High levels of Nrf2 Mulcahy, 2000). Dithiolcarbamate was shown expression is reported in multiple cancers to activate ERK and p38 resulting in including cancers of lung, breast, gall bladder, transcriptional Nrf2 pancreatic, colorectal and head and neck synthetase (Jaramillo and Zhang, 2013; Lau et al., 2008; (Wild et al., 1999). Shen et al. (2004) Shelton and Jaiswal, 2013; Sporn and Liby, investigated the transactivation potential of 2012). Ikeda et al. (2004) demonstrated different observed constitutive up-regulation of Nrf2 and GSTP1 differential effects of multiple MAPKs in in hepatocellular carcinoma indicating role of dependent enzymes cascade using that up-regulation γ-glutamylcysteine Nrf2 domains and of Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 64 Nrf2 in cancer promotion. Nrf2 regulates classified based on their functional impact into expression of an exclusive neoplastic lesion passenger mutations, null mutations and marker GSTP1 in an ARE dependent hypomorphic mutations. Passenger mutations mechanism. Higher levels of Nrf2 have been do not have any effect on Keap1/Nrf2 associated with poor clinical outcome and interaction, poor responsiveness in pancreatic, cervical diminished the ability of Keap1 to repress and lung cancer (Geismann et al., 2014; Sporn Nrf2. Most of the mutations do not affect the and Liby, 2012). Nrf2 levels, but impact the activity as Keap1 is whereas null mutations unable to act as a negative regulator (Hast et Dysregulation of Nrf2 pathway in cancer al., 2014; Hayes and McMahon, 2009; Shibata Persistent Nrf2 activation is responsible for the et al., 2008). Japanese patients with lung pro-tumorogenic effect due to genetic and adenocarcinoma epigenetic Nrf2/Keap1 mutations (Ohta et al., 2008). Dysregulated (frequencies of up to 30% in lung or ovarian suppression of Nrf2 by Keap1 in breast cancer cancer). Copy number loss in a member of E3 resulted due to mutation in cys23 residue (Nioi ubiquitin ligase complex or oncogenic and pathways or persistent exposure to oxidative reoxygenation stress leads to persistent activation (Barbano et upregulated and protected cancer cells from al., 2013; Martinez et al., 2014; Zhang et al., deleterious effects of oxidative stress (Kim et 2010). A mutation in the Keap1 protein or loss al., 2007). alterations in Nguyen, demonstrated 2007). Under conditions Keap1 hypoxic/ Nrf2 was of heterozygosity has been reported to result in Mutations in the DLG motifs of the DC persistent Nrf2 activation in multiple cancers domain in Keap1 show highest frequency in (Padmanabhan et al., 2006; Singh et al., 2006). lung cancers (Ganan-Gomez et al., 2013). DNA methylation of CpG sites in the promoter Interestingly frame shift mutations in Keap1 region of Keap1 was observed in 51% of are frequent in DGR domain (65%) essential breast, 20% of colorectal, and 12% of lung for interaction with Nrf2 (Taguchi et al., cancers, accounting for decreased levels of 2011). Other mutations in the intervening Keap1 Nrf2 region and BTB domain of Keap1 occur in activation (Bryan et al., 2013; Wang et al., prostate, lung and ovarian cancers. Mutation 2008a). Approximately 15% patients with in these domains influence the critical cysteine lung cancer posses somatic mutations in residues that inhibit its interaction with Keap1, resulting in impaired and inefficient Cullin3, leading to inhibition of poly- Nrf2 repression (Hayes and McMahon, 2009). ubiquitination of Nrf2. Mutation in other The amino acids like ser104, gly186,423 and and consequent prevailing Keap1 Biomed Res J 2015;2(1):57-82 enhanced mutations were 65 Gambhir et al. arg320 within the DC, BTB and IVR domains Nrf2 from ubiquitination, whereas mutations are cancer derived mutations that results in in the DLG motif disrupt low affinity binding impaired homo-dimerization of Keap1 needed but Nrf2 remains bound to Keap1. Both these for repression of Nrf2 (Hast et al., 2014). A mutations result in Nrf2 stabilisation and single nucleotide deletion in Keap1 gene was accumulation in nucleus (Taguchi et al., 2011). associated with marked drug resistance Along with the somatic mutations in against BRAF and cisplatin in melanoma cells Keap1/Nrf2, an alternate mechanism for (Miura et al., 2014). Although mutations in activation of Nrf2 in tumorigenesis is Keap1 play a central role in constitutive Nrf2 mediated by oncogenic signalling. Expression activation, deficiency of Keap1 per se does not of oncogenes like Kras, Braf and Myc activate result Nrf2, in cancer. Interestingly, Keap1 elevating antioxidant machinery knockdown mice (floxed Keap1 allele) did not resulting in depletion of the intracellular ROS develop spontaneous cancer and survived for 2 levels, thus providing a conducive reduced years. Keap1 knockdown mice showed environment for tumor growth (DeNicola et constitutive activation of Nrf2 in multiple al., 2011). tissues including lung and liver. These studies indicated that impaired Nrf2/Keap1 pathway Nrf2 in chemoresistance may result in cancer cell proliferation or A resistance to anti-cancer modalities, but it does activation not set off cancer initiation (Taguchi et al., protecting cancer cells from anti-cancer drugs 2010). In addition, mutations in Nrf2 gene are used in chemotherapy. Several studies have focussed in Keap1 binding domain near ETGE highlighted the pivotal role of Nrf2 in and DLG motifs termed as hot spot regions. chemoresistance such as cisplatin in ovarian Mutations in Nrf2 were observed in lung, head cancer, cervical cancer or endometrial serous and neck, oesophagus and skin cancers, but are carcinoma; gemcitabine in pancreatic cancer; less abundant (Kim et al., 2010; Shibata et al., doxorubicin in liver cancer and 5-fluorouracil 2008). Nrf2 deficient mice were more in gastric cancer (Chen et al., 2012; Duong et susceptible to urethane induced lung cancer al., 2014; Jiang et al., 2010; Ma et al., 2012). compared to Nrf2 wild type (Bauer et al., Elevated Nrf2 induces autophagy in ovarian 2013). The Nrf2 mutations are clustered carcinoma within ETGE (57%) and DLG (43%) moti, cisplatin and tamoxefin (Bao et al., 2014). Due which were indispensable for Keap1 binding. to Mutations in ETGE motif disrupt the high potential, several Nrf2 dependent genes are affinity binding with Keap1 and thus prevent implicated in conferring Nrf2 mediated distinctive the of property of Nrf2 chemoresistance, is imparting cytoprotective constitutive resistance and against detoxifying Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 66 chemoresistance, e.g., HO-1 is over-expressed inhibition in TrxR activity abrogates in multiple cancers. Due to the cytoprotective resistance against cisplatin (Sasada et al., nature, over-expression is undesirable in 1999). NQO1 is another Nrf2 dependent gene cancer cells. Over-expression of HO-1 was over-expressed in adrenal gland, bladder, associated with increased cell proliferation breast, colon, liver, lung, ovary, and thyroid and endothelial cell division leading to cancers (Basu et al., 2004; Siegel and Ross, angiogenesis (Was et al., 2006). Other Nrf2 2000). Suppression in NQO1 expression dependent genes including NQO1, GPX, TrxR sensitizes A549 cells to etoposide, cisplatin and Prx1 were shown to be up-regulated in and doxorubicin (Wang et al., 2008c). multiple cancer cells. GPx, a selenoprotein that detoxifies H2O2, is implicated in the Chemopreventive Effects of Nrf2: The control of malignant growth. Elevated GPx Bright Side levels were observed in advanced stages of Several compounds derived from natural or colorectal synthetic mucosa cancer, and Barrett`s origin with chemopreventive cancers activity act via Nrf2. Administration of associated with cell proliferation, growth and methylcholanthrene reduced cancer incidence inhibition of apoptosis (Banning et al., 2005; in rats caused by carcinogenic azo dyes, served Chu et al., 2004; Was et al., 2006). Peroxi- as a nucleation point for use of dietary redoxins (Prx) are thiol specific antioxidants compounds that detoxify peroxides and are elevated in non (Richardson and Borsos-Nachtnebel, 1951). small lung cancer (NSLC) and thyroid cancer, Multiple plant derived products possess a predictive factor for disease and associated chemopreventive effect by inducing Nrf2 with prognosis (Kim et al., 2007; Yanagawa et activation (Kelloff et al., 2000; Sporn and Suh, al., 1999). Trx and TrxR collectively form a 2000; Talalay and Fahey, 2001; Yang et al., redox couple with a pivotal role in maintaining 2001). Nrf2 activation results in increased cellular redox status in cellular functions expression (Brigelius-Flohe, 2008). Despite its protective preventing biomolecules from the damaging role as redox couple, TrxR1 was elevated in effects of oxidative and xenobiotic stress. Nrf2 gastrointestinal cancer tissues (Arner and knockout mice studies strengthened the notion Holmgren, 2006; Iida et al., 2004). TrxR of Nrf2 serving as a novel chemopreventive knockdown lung carcinoma cells showed factor controlling sensitivity to carcinogens reversal of tumorigenicity and invasion. (Slocum and Kensler, 2011). Ablation in Nrf2 Enhanced cellular expression of TrxR has been led to enhanced tissues damage caused by attributed cigarette to gastrointestinal esophageal cisplatin Biomed Res J 2015;2(1):57-82 resistance, and as chemopreventive of smoke, cytoprotective hyperoxia, agents proteins ischemic 67 Gambhir et al. reperfusion, portal vein embolization, and inhibition (Huang et al., 2015; Ying et al., chemical toxins (Chan et al., 1996; Cho and 2014). Nrf2 is also an anti-inflammatory Kleeberger, 2010; Kudoh et al., 2014; transcription factor and activation of Nrf2 and Shirasaki et al., 2014; Zhao et al., 2011). Mice dependent genes reduce chronic inflammation with Nrf2 over-expression resulting from associated Keap1 knockout shows increased resistance to pulmonary cancer. A protective role of Nrf2 is lung cancer cell metastasis (Satoh et al., 2010). supported by studies in mice with a single- Nrf2 ablation was associated with enhanced nucleotide polymorphism (SNP) in the sensitivity to mutagens and showed increased promoter region. The polymorphism was carcinogenesis in bladder, skin, hepatocytes associated with increased susceptibility to and colon on exposure to nitrosoamine, hyperoxia induced lung damage, due to low ultraviolet, aflatoxin, dextran sulphate sodium expression of Nrf2 (Cho et al., 2002; and azoxymethane (Iida et al., 2004; Khor et Yamamoto et al., 2004). cancers like colorectal or al., 2006; Osburn et al., 2007; Saw et al., 2011; Though higher levels of Nrf2 are observed Xu et al., 2006; Yates et al., 2006). Curcumin, in multiple malignancies, the role in initiation, sulforaphane, oltipraz and CDDO-imidazole promotion or transformation of normal cells activate Nrf2 while exerting chemopreventive remains contentious. Low levels of Nrf2 were effects and Nrf2 deficiency in mouse models essential for oncogenic transformation of abrogated their chemopreventive effects mesenchymal stem cells (Funes et al., 2014). (McMahon et al., 2001; Ramos-Gomez et al., Epigenetic reactivation of Nrf2 attenuated 2003; Shen et al., 2006; Slocum and Kensler, skin epidermal cell transformation (Su et al., 2011; Sussan et al., 2009). Nrf2 has also been 2014). Over-expression of Nrf2 in cancer cells implicated ROS may enable survival under conditions of dependent genetic lesions that promote oxidative stress, or detoxify xenobiotics metastasis (Satoh et al., 2010). Under leading to better survival. Nrf2 prevented conditions of increased ROS levels, Nrf2 initiation of lung cancer, but accelerated induced expression of Kruppel-like factor 9 progression through the Kras signalling (Klf9), which further enhanced oxidative pathway. Thus Nrf2 activators may pave the stress mediated cell death (Zucker et al., way for prevention of lung cancer (Satoh et al., 2014). The anti tumor potential of Klf9 in 2013). in protecting against different cancer types has been reported with inhibition of Glioblastoma stemness through Nrf2 transcriptional Interventions repression, and induced apoptosis in prostate cancer cells by Akt as Target for Therapeutic Nrf2 activation and enhanced expression of its Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 68 dependent genes associated with redox oleanane triterpenoids. Administration of regulating proteins, phase II detoxifying several Nrf2 activators in clinical trials was enzymes and transporters are exploited by well tolerated, resulting in elevated levels of cancer cells to survive and proliferate. cytoprotective enzymes (Kensler et al., 2012; Therefore, agents that inhibit Nrf2 expression Linker et al., 2011; Palsamy and Subramanian, in cancer cells may provide a novel strategy for 2011; Scannevin et al., 2012). In a Chinese therapeutic interventions to enhance efficacy study, aflatoxin intoxication as a risk factor of existing chemotherapeutic drugs. Brusatol, was reduced by oltipraz (Kensler et al., 2003). Bruceajavanica, Favourable effects of sulforaphane, a potent a plant extract from selectively inhibits Nrf2 by increasing Nrf2 activator, were observed ubiquitination and degradation. It reduced promotion or progression phase of cancer, and resistance towards cisplatin in cultured sulforaphane inhibited cancers of multiple xenografts (Ren et al., 2011). 6-Hydroxy-1- sites including skin, lung, bladder, breast, methylindole-3-acetonitrile (6-HMA) colon and stomach (Conaway et al., 2005; protected against cisplatin induced oxidative Dinkova-Kostova et al., 2006; Gills et al., nephrotoxicity by inhibiting Nrf2 activation 2006; Hu et al., 2006; Shen et al., 2007). (Moon et al., 2013). Luteolin, a plant derived Chemoprevention flavanoid, inhibited proliferation of tumor extracts of broccoli are in clinical trials in cells and reduced toxicity of cisplatin in a mice China (Egner et al., 2011). Similarly, synthetic model (Lin et al., 2010; Sun et al., 2012; Tang oleanane triterpenoids reduced progression of et al., 2011). All-trans retinoic acid (ATRA) lung, breast and pancreatic cancers, and inhibited Nrf2 by activating retinoic acid delayed onset of tumor driven by Kras, Trp53, receptor α, which directly interacts with Nrf2 Brca1 and Erbb2 oncogenes (Liby et al., and restrain binding to ARE (Wand et al., 2010). with in the sulforaphane-rich 2013a). However, the use of Nrf2 inhibitors in Salutary health effect of phytochemicals cancer therapy is at a nascent stage and that induce Nrf2 highlights the role of Nrf2- requires development of specific agents to activating foods and spices in human diet. minimize non-specific off-target effects. Food products like curcumin from turmeric Assuming Nrf2 as a target for cancer root, sulforaphane from broccoli, and prevention, several population-based clinical seaweed-based extracts from green alga Ulva trials diverse lactuca were shown to activate the Nrf2 chemopreventive drugs including phenethyl pathway in vivo. Extract with sulforaphane isothiocyanate, curcumin, concentration that is achieved by dietary resveratrol, fumaric acid esters and synthetic broccoli consumption, offered protection were conducted oltipraz, Biomed Res J 2015;2(1):57-82 with 69 Gambhir et al. against particulate pollution in humans Future perspective and conclusion: (Boddupalli et al., 2012; James et al., 2012; Given the dual role of Nrf2 in cancer, the prime Wang et al., 2013b). Similarly, phytochemical query is the role of Nrf2 in cancer initiation or constituents of garlic, tomatoes, grapes, green cell transformation. Transient activation of tea, coffee, and berries show Nrf2 activating Nrf2 by pharmacological activators is safe for properties, indicating beneficial effects by the purpose of chemoprevention as the dietary consumption (Kropat et al., 2013). activators do not seem to increase the tumor Numerous dietary supplement companies burden. A major concern of use of Nrf2 have developed mixtures of known Nrf2 activators is their cytotoxicity and non- activators to increase the antioxidant system in specific mechanism of action. The activators body. Protandim (LifeVantage, Inc, Sandy, show a tendency to modulate cellular redox UT, USA), reduced oxidative stress in humans and are reactive towards cysteine residues (Nelson et al., 2006). which may lead to modulation of signalling Apart of pathways. Thus, designing specific Nrf2 phytochemicals and dietary intake, life style of activators like ETGE and DLG mimetic, based an individual also plays an important role in on co-crystal structure of Neh2 domain and Nrf2 activation. A relationship between Keap1, may reduce the off target effects. physical activity and Nrf2 activation was Further, demonstration of miRNA mediated established in a mouse model and exercise- regulation Nrf2 pathway provides a new induced oxidative stress was higher in Nrf2 conduit to explore additional targets. Multiple knockout from mice the (Miller implication et al., 2012; studies highlight the cross talk of Nrf2 with Muthusamy et al., 2012; Zhao et al., 2013). other signalling pathways imperative for cell Evidence from several studies provide a strong survival. Results from our laboratory have incentive for development of novel Nrf2 demonstrated implication of Nrf2 cross talk activators as putative cancer chemopreventive with NF-κB as a prime target for anti- agents in normal healthy individuals without inflammatory effect (Gambhir et al., 2014). affecting pro-survival potential. However, Thus, novel agents targeting Nrf2 pathway caution must be exercised as a pro- specifically needs investigation. tumorogenic role of Nrf2 in various cancers indicates dual nature of Nrf2 activation. Nrf2 SUMMARY activation may provide a survival advantage to Nrf2 is a redox sensitive transcription factor, pre-existing cancer cells and also participate in maintained at low basal levels under normal resistance to chemotherapy or radiotherapy. conditions. Upon activation, it mediates expression of dependent cytoprotective genes, Biomed Res J 2015;2(1):57-82 Diverging role of Nrf2 in cancer 70 Figure 3: Dual role of Nrf2. The bright side is indicated by Nrf2 functions in normal cells where it acts as cytoprotective transcription factor inducing expression of an array of cytoprotective proteins, antioxidants and detoxifying enzymes leading to increased survival and cancer prevention. In tumor cells, constitutive high levels of Nrf2 provides an ambient niche for cancer cells to grow by reducing toxicity of endogenous ROS and xenobiotics. High levels of Nrf2 may increase cancer cell survival and imparts chemoresistance leading to poor clinical outcome. phase II detoxifying enzymes and antioxidant and makes them refractory to chemotherapy machinery. Evidences illustrating a positive and/or radiotherapy. Evidence to directly role of Nrf2 in cancer prevention has been implicate Nrf2 in cancer initiation needs documented. Thus, efforts are underway to confirmation. However, Nrf2 facilitates a identify novel agents that can activate Nrf2. reducing environment through up-regulation However, constitutive expression of Nrf2 may of prevent death of precancerous lesions and machinery. Thus providing armour for cancer promote survival of cancer cells under cell to create an ambient growth niche and oxidative stress suggesting a dual role (Fig. 3). resist toxicity of xenobiotics. Hence, Nrf2 may The transient activation of Nrf2 is beneficial, serve as an additional target for therapeutic in countering ill effects of xenobiotics, interventions, increasing susceptibility of oxidative cancer in conjuction with chemotherapy or stressors, carcinogens and mutagens. Whereas, persistent activation of Nrf2 in tumor cells confers survival advantage Biomed Res J 2015;2(1):57-82 the antioxidant and cytoprotective radiotherapy treatment modalities. 71 Gambhir et al. ACKNOWLEDGEMENTS Bioscience Group, Bhabha Atomic Research The authors would like to thank Dr. Anu Ghosh Centre. and Dr. S. Jayakumar for proofreading the review. 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Modi* and Pradeep Bhartiya Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health,Parel, Mumbai, India Implantation of the blastocyst stage embryo into the maternal endometrium is a critical determinant and a rate-limiting process for successful pregnancy. Embryo implantation requires synchronized changes in the endometrium before and after arrival of blastocyst into the uterine cavity. Extensive cross talks occur between the fetal and maternal compartments around the time of implantation which are reflected by morphologic, biochemical and molecular changes in the endometrial cells and the differentiating trophoblast cells. The embryo induced morphologic changes include occurrence of epithelial plaque reaction, stromal compaction and decidualization. Embryonic signals also alter the expression of a large number of transcription factors, growth factors and their receptors and integrins. Thus the embryo superimposes a unique signature on the receptive endometrium for successful implantation. Functionally, the embryo-endometrial cross talk is essential for endowing a “selector activity” to the receptive endometrium to ensure implantation of only a developmentally competent embryo. On selection, the decidua creates a conducive microenvironment for trophoblast invasion leading to placentation. Clinical evidences suggest that along with receptivity, a defective “selector” activity of the receptive uterus may be a cause of infertility and recurrent miscarriages. Defects in trophoblast invasion are associated with pregnancy complications like preeclampsia and intra-uterine growth retardation. It is envisaged that understanding of the embryo-endometrial dialogue leading to the “selector” activity, aids in development of appropriate therapeutic modalities for infertility related disorders and miscarriages. Conversely, it might also benefit the development of anti-implantation drugs for contraception. INTRODUCTION Human reproduction is an inefficient process. indicate that uterine implantation governs Epidemiological evidences suggest that only reproductive 30% of all conceptions get clinically incompetence at the endometrial level can be a recognized, and a large number of these are constraining factor. Thus, understanding the lost spontaneously. Furthermore, the success molecular of assisted reproduction is low, as reasonably interaction is of interest to reproductive good quality embryos fail to implant and there biologists, clinicians and couples affected by is a high frequency of spontaneous abortions. infertility. The understanding is also essential These epidemiologic and clinical evidences for designing rational management strategies success events of and maternal embryo-maternal Key words: Endometrium, Decidua, Implantation, Biosensor, Invasion, Trophoblast, Abortion. *Corresponding Author: Deepak N. Modi, Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health, Parel, Mumbai, India. Email: deepaknmodi@yahoo.com, modid@nirrh.res.in Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 84 for implantation failure and treatment of transformations. At the same time, the infertility. fertilized ovum undergoes several rounds of Our current understanding of the process cell division and transforms into blastocyst. of embryo implantation and the determinants The blastocyst has an outermost layer of of successful pregnancy have mainly stemmed specialized cells, trophoblast cells, that from animal models and in vitro studies using surround the pluripotent inner cell mass. The human tissues. Based on the data derived it is trophoblast cells come in direct contact with clear that endometrial receptivity and embryo the receptive endometrium establishing a firm implantation are complex processes involving attachment with the endometrial epithelial a delicately poised balance of maternal cells termed as apposition. Subsequently, the hormones, endometrial factors and embryonic trophoblasts invade the endometrium and influences. The current review focuses on establish contact with the maternal circulation cellular and molecular events associated with to form the placenta. endometrial receptivity and implantation to accomplish successful conception. The endometrium is refractory to embryo The implantation throughout the menstrual cycle embryo-endometrial cross talk at the time of except for a few days after ovulation. embryo apposition and implantation mainly in Approximately, on days 21–24 of the human the primates will be discussed. The general menstrual cycle (8–10 days post ovulation), understanding of the processes of endometrial the uterus becomes "receptive", enabling receptivity and implantation has been a subject blastocysts of recent reviews (Gellerson and Brosens, epithelium. 2014; Ozturk and Demir, 2010; Young, 2013; receptivity”, the achievement of this stage is Young and Lessey, 2010). highly dependent on the ovarian steroids, to adhere Termed to as the luminal “window of estrogen and progesterone. The estrogen in the follicular phase leads to proliferation of Endometrial receptivity A mutual communication the endometrial epithelial cells, the progesterone blastocyst and the uterus is indispensable for surge that occurs in response to ovulation leads implantation. Akin to many developmental to differentiation of the estrogen primed processes, it involves an elaborate sequence of endometrium to endow receptivity. Any genetic and cellular interactions, to be disturbance in the levels of these hormones executed within an optimal temporal frame for adversely affects endometrial physiology successful pregnancy. In order to receive a leading to failure of implantation. The developing embryo, the endometrium endures endocrine regulation of the menstrual cycle a series of morphological and physiological and role of hormones in endowing receptivity Biomed Res J 2015;2(1):83-104 between 85 Modi and Bhartiya to the endometrium has been a subject of series of cell divisions and reaches uterine extensive studies (Jabbour et al., 2006; Young, lumen at the blastocyst stage. At this time the 2013). trophoblast cells are differentiated and the Morphologically, the receptive phase embryo is ready to hatch. From rodents to endometrium is characterized by presence of humans, this embryo induces a second round columnar epithelium with microvilli, an of differentiation both at the morphologic and increase in stromal cell proliferation and biochemical level (Banerjee and Fazleabas, appearance of pinopod-like structures on the 2009). Distinct from the “receptive” stage luminal epithelium (Tu et al., 2014). endometrium, The the embryo induced morphological features of the “receptive” differentiation of the receptive endometrium is endometrium are associated with expression rather limited and largely derived from of a range of biochemical and molecular experimental studies in the non-human markers, crucial for endowment of this phase primates and endometrial biopsies obtained of like from conception cycles in humans. Three transcription factors, integrins and their major non-human primate models to identify ligands, cytokines and growth factors, have and dissect embryo induced morphological been associated with the receptive phase (Tu et and physiological changes in the receptive al., 2014; Wang and Dey 2006; Zhang et al., stage 2013,). A molecular signature of the “receptive hysterectomies window” profiling endometrium of baboons or rhesus macaques technologies have been identified that can sequentially mated with males of proven phenotype different phases of the menstrual fertility; 2) Endometria obtained from mated cycle including the receptive stage to bonnet monkeys where the presence of the objectively classify the implantation window embryo (Garrido-Gómez et al., 2013; Haouzi et al., preimplantation 2012). These findings open the field for the Endometrial tissue obtained from baboons diagnosis of the endometrial defects in assisted where human chorionic gonadotropin (hCG) reproductive technology programs (Ruiz- has been infused in the uterine lumen in a Alonso et al., 2013). manner that mimics the transit of blastocyst. the uterus. using Several global markers gene endometrium has and/or been factor are: 1) biopsies verified assay; Timed of the using a and 3) The models have inherent advantages and Embryo induced morphologic changes in disadvantages but are highly complementary the receptive endometrium and provide valuable information in terms of In a conception cycle, the egg that has identification and deciphering the functional fertilized in the fallopian tube undergoes a consequence of embryo induced changes in Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 86 uterine receptivity. Beyond the plaque reaction, thinning of the basal lamina and thickening and diffusion Epithelial changes: The earliest endometrial of the apical and lateral gap junctions in response prior to implantation is characterized luminal epithelial cells has been reported in by an increased proliferative activity of the pregnant luminal and glandular epithelium. Distinct endometrium (Demir et al., 2002; Rosario et from the epithelial proliferation observed in al., 2008). Along with these changes, a few the follicular phase, this proliferative activity granulocytic stromal cells are also observed in in the pregnant uterus is restricted to focal the luminal epithelium of pregnant bonnet and areas. In the luminal epithelium, there are large rhesus monkeys prior to implantation (Ghosh clump of nuclei with distinct entero- et al., 1993; reduplication and poorly packed chromatin conceivable that these changes occur to along with loss of basement membrane. These promote adhesiveness to the trophoblast cells changes are termed as “epithelial plaque at the time of apposition and invasion. human, bonnet and baboon Rosario et al., 2008). It is reaction” (Jones and Fazleabas, 2001; Rosario et al., 2005a). The formation of epithelial Stromal changes: An almost universal plaques is restricted to pregnant endometrium reaction of the endometrial stromal cells in and reported in a variety of primate species response to an embryo is decidualization. In its including humans (Rossman, 1940). While broadest sense, decidualization is defined as consistently detected in the conception cycle, the postovulatory endometrial remodeling the epithelial plaque reaction is hormonally which includes secretory transformation of regulated and does not require presence of an uterine stroma, influx of specialized uterine embryo, as infusion of hCG directly in the natural killer cells, and vascular remodeling. A uterus leads to formation of epithelial plaques more restricted definition of decidualization is similar to those observed in pregnant monkeys an (Fazleabas et al., 1999; Jones and Fazleabas, endometrial stromal 2001). specialized and The functional significance of epithelioid transformation cell of with distinctive the highly functions. epithelial plaques is not clear. It is speculated Decidualization only occurs in species in that the plaque may provide nutrition by means which placentation involves breaching of the of intracellular glycogen (Enders et al., 1985; luminal epithelium by the trophoblasts. The Rossman, 1940). The plaque response may extent of this differentiation process often stimulate precocious development of the correlates with the degree of trophoblast maternal vasculature below the epithelium invasion (Dunn et al., 2003; Gellersen et al., (Enders et al., 1985). 2007). Biomed Res J 2015;2(1):83-104 87 Modi and Bhartiya Morphologically, the elongated spindle functionalis region of the endometrial bed like stromal cells of the secretory phase (Rosario et al., 2005a). Increased vascularity endometrium transform into cobblestone like and angiogenesis at the implantation site of enlarged decidual cells with multiple club rhesus monkeys has been reported (Sengupta shaped projections arising from the cell and Ghosh, 2002). A similar increase in the surface and contain abundant glycogen stores number of small blood capillaries in the and lipid droplets (Welsh et al., 1985; Wynn et stroma of the endometrial functionalis have al., 1974). In humans, this transformation been demonstrated in baboons infused with occurs even in absence of an embryo and is physiological doses of hCG in the uterine referred to as the pre-decidual response. In a lumen (Banerjee and Fazleabas, 2009; Jones conception cycle, under the continuous and Fazleabas, 2001). These observations support of steroid hormones and blastocyst suggest that maternal tissues initiate neo- derived signals, decidualization of the entire vascularization which may be required for endometrium is observed (Brosens et al., immune cell differentiation and infiltration 2002; Gellersen and Brosens, 2014). The (See below). decidua forms a dense cellular matrix that allows coordinated trophoblast invasion while Immune cell infiltration: simultaneously protecting the conceptus from population in the endometrium consists of T maternal and environmental insults (Kliman, cells, 2000; Redhorse et al., 2004). In the non- lymphocytes. T cells and macrophages human primates, decidualization is observed account for a substantial proportion of the in conception cycle or on treatment with hCG leukocyte population in human endometrium (Jones and Fazleabas, 2001; Rosario et al., throughout the menstrual cycle (Jones et al., 2005a). These observations suggest that unlike 1998; King, 2000). The largest leukocyte in humans, embryo/embryonic factors are population in the human endometrium are the required for the endometrial stromal cells to large granulated lymphocytes which express undergo decidualization in monkeys. natural killer (NK) cell antigen CD56. The macrophages and The leukocyte large granular uterine NK (uNK) cell population is distinct Vascularization: A characteristic feature of from peripheral blood NK cells in phenotypic the endometrium in a conception cycle is the and molecular characteristics (Cooper et al., enhanced microvasculature. In the pregnant 2001; Fukui et al., 2011; King et al., 1991; bonnet monkeys, a large number of small Lysakova-Devine and O'Farrelly, 2014). blood vessels are detected in the stroma Around the time of implantation, uNK cells underlying luminal epithelium and the comprise 70–80% of the leukocyte population Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 88 in the endometrium and numbers increase if during early pregnancy. The following section conception occurs (King, 2000; Kodama et al., reviews the in situ molecular changes 1998). It remains to be identified whether the occurring in the primate endometrium in increase in cell number is solely the result of in response to embryonic signals. situ proliferation or homing from the peripheral circulation. In a conception cycle, Estrogen receptors (ER) and Progesterone the uNK cells differentiate into decidual NK receptors (PR) (dNK) cells, functionally distinct from non- Sex steroids exert their effects through their pregnant uterine counterparts (Kodama et al., receptors, 1998). The functional significance of uNK and progesterone receptor (PR). As compared to dNK cells in the primate endometrium is non-conception cycle, both ER and PR largely speculative. Based on mouse studies expression is higher in the conception cycle and clinical observations, it appears that NK around implantation in bonnets, baboons and cells are crucial for pregnancy and failure of rhesus (Ghosh and Sengupta, 1988, Rosario et uNK transformation to dNK cells leads to al., 2008). Post apposition ER expression is pregnancy loss (Fukui et al., 2011; Gong et al., lost in the epithelium and stroma but retained 2014; Quenby and Farquharson, 2006). in the wall of spiral arteries, blood vessels, and Whether myometrial smooth muscle cells (Hild-Petito this transformation occurs exclusively in response to embryo derived signals or due to decidualization of the stromal cells, needs to be investigated. estrogen receptor (ER) and et al., 1992; Perrot-Applanat et al., 1994). While PR is most abundantly expressed in the uterine glands and stroma in the receptive phase, expression of PR is down-regulated in Embryo induced molecular trans- the glands but present in the stroma formations in the receptive endometrium surrounding the glands and spiral arteries, wall The molecular dialogue between the embryo of spiral arteries, blood vessels, and smooth and endometrium involves a complex network muscle cells of the myometrium (Ghosh and of signaling molecules that mediate cell–cell Sengupta, 1988; Hild-Petito et al., 1992; or Perrot-Applanat et al., 1994). cell–extracellular matrix (ECM) interactions, and include factors such as cytokines, growth factors, cell-adhesion Homeobox genes HOXA10 and HOXA11 molecules and matrix metalloproteinases. HOX genes are essential for endometrial There is some evidence indicating that the growth, differentiation and receptivity by levels of steroid receptors, growth factors and mediating some functions of progesterone. cytokines are modulated in the endometrium Both HOXA10 and HOXA11 are expressed in Biomed Res J 2015;2(1):83-104 89 Modi and Bhartiya human endometrial epithelial and stromal transcriptome in luminal epithelium may be cells, and their expression is significantly inhibitory for implantation, and hence may be higher in mid- and late-secretory phases, down regulated by embryonic stimuli. As coinciding with time of embryo implantation transcription factors, HOX genes regulate and high levels of estrogen and progesterone other downstream target genes leading to (Daftary and Taylor, 2006; Godbole et al., proper development of endometrium and 2007; Modi and Godbole, 2009; Taylor et al., receptivity to implantation. A number of 1998; Xu et al., 2014). molecular and morphological markers specific Unlike steroid receptors, the expression of to the implantation window are regulated by HOXA10 is induced in the endometria of HOX genes, including pinopodes, β3 integrin bonnet monkeys in the conception cycle. and Abundant expression of HOXA10 protein is protein-1 (Daftary and Taylor, 2006; Modi and detectable in stromal and glandular cells of the Godbole, 2009). All the HOX targets are also pregnant bonnet monkeys (Godbole et al., modulated in the endometria in response to the 2007). Interestingly, treatment of endometrial embryo (Nimbkar-Joshi et al., 2009). insulin-like growth factor-binding cells with spent blastocyst culture medium and/or hCG resulted in increased transcription Cytokines and growth factors of HOXA10 (Blitek et al., 2011; Fogle et al., Leukemia inhibitory factor (LIF), Interleukin- 2010; Sakkas et al., 2003). However, unlike 6 and -11 (IL-6 and IL-11) are members of a the glands and the stroma, in luminal single family of cytokines that share the signal epithelium of the conception cycle, HOXA10 transducer receptor unit gp130 in target cells to expression is reduced and expression is elicit biologic effects. All these three cytokines virtually absent in the pre-epithelial plaques play key roles in implantation. First identified (Godbole et al., 2007; Modi and Godbole, in the mouse where targeted disruption of the 2009) These observations are surprising, as in LIF the mouse, suppression of HOXA10 in (Stewart epithelial cells leads to inhibition of embryo expression/secretion has been reported implantation; overexpression leads to increase infertile women with defects in implantation in litter size in mouse (Bagot et al., 2000). (Mikolajczyk et al., 2006; Tawfeek et al., While this might reflect the fundamental 2012). While LIF seems to be a critical differences in the mechanisms associated with requirement for implantation, the expression implantation primates, is not modulated by embryonic signals as the observations in the monkey indicates that levels do not alter in the implantation phase products endometria of bonnet monkeys in conception in of rodents and HOXA10-modulated gene showed et al., implantation 1992), failure reduced LIF in Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 90 as compared to non-conception cycle (Rosario conception of rhesus and bonnet monkeys et al., 2005b). However, in rhesus the (Rosario et al., 2005b; Sengupta et al., 2003). expression of LIF and its receptors are Several growth factors like Tumor Growth increased in the endometria of pregnant Factor (TGF) beta, Epidermal Growth Factor monkeys as compared to non-pregnant (EGF) and Tumor Necrosis Factor (TNF) controls (Sengupta et al., 2003). LIF is crucial alpha are pro-inflammatory cytokines that for implantation in primates as administration have emerged to be critical mediators for of an antagonist for LIF receptor or antibody implantation owing to their direct effects on against LIF directly in to the uterine cavity of immune cells (Dimitriadis et al., 2005; monkeys and mice, results in failure of Omwandho et al., 2010). In the window of pregnancy (Sengupta et al., 2006; Terakawa et implantation, al., 2011; White et al., 2007). The results endometrial TGF beta and its receptor occurs indicate that LIF is essential in the process of in the glandular epithelium of animals in the blastocyst implantation. LIF is also a promotor conception cycles as compared to non- of trophoblast invasion (Suman et al., 2013a; conception cycles (Rosario et al., 2005b; 2013b). Sachdeva et al., 2001). TNF alpha and its IL-6 and IL-11 are pleiotropic cytokines receptor a significant population increase increases in in the required for implantation. IL-11 expression is endometria of animals in the conception increased during decidualization (Godbole cycles as compared to non-conception cycles and Modi, 2010), recombinant IL-6 and IL-11 (Nimbkar-Joshi et al., 2009; Rosario et al., promote human 2005c). EGF and its receptors are detected in endometrial cells in vitro (Dimitriadis et al., both the glands and stromal compartments of 2005; Menkhorst et al., 2010). IL-6 and IL-11 the receptive phase endometrium; expression are also promoters of trophoblast invasion is increased mainly in the stromal cells of the (Champion et al., 2012; Modi et al., 2011; pregnant animals (Slowey et al., 1994). An Suman et al., 2009; 2013). IL-11 and the increase in endometrial LIF, EGF, TGF and receptor IL-11Rα are detected in the decidua TNF by endometrial cells in presence of mainly at implantation sites in cynomolgus embryonic stimuli prior to apposition suggests and rhesus monkeys (Champion et al., 2012; induction of an inflammatory like condition in Dimitriadis et al., 2005). It is also detected in the implantation phase endometrium, which the vascular endothelial cells and epithelial may be a requirement for initiation of plaques. Likewise, the expression of IL-6 is pregnancy; the increase in expression in significantly higher in endometria of animals stromal compartment implies involvement in the conception cycles as compared to non- with decidualization. decidualization Biomed Res J 2015;2(1):83-104 of 91 Modi and Bhartiya pregnancy using the bonnet model. The results Integrin and their ligands revealed that expression of alpha v increases in Integrins are heterodimeric glycoproteins luminal epithelial cells of pregnant animals, which undergo dynamic temporal and spatial show a shift in localization at the site of changes the attachment (Nimbkar-Joshi et al., 2012). At endometrium during the menstrual cycle in the non-attachment pole, the alpha(v) integrin women (Lessey and Arnold, 1998; Reddy and is mainly in the basal zone of the luminal Mangale, 2003). Likewise the extracellular epithelial cells. However, at the attachment matrix (ECM) ligands for these receptors are pole, alphav is redistributed and also detected likely to play a role in the establishment of a in the apical pole. The differential subcellular receptive endometrium. The integrins and distribution of integrin is directed by their cognate ligands show dynamic changes embryonic stimuli as treatment of epithelial in levels of expression and polarization during cultures with conditioned medium of human early pregnancy. In the baboon, the collagen embryos obtained at IVF leads to increased receptor alpha1beta1 and fibronectin receptor distribution of alpha v on the apical membrane alpha4beta1 expressed in glandular epithelium (Nimbkar-Joshi during window of receptivity are lost with the observations imply that embryonic stimuli not establishment of pregnancy. The vitronectin only directs cellular reprogramming by receptor, alpha4 beta3 is expressed in the changing gene expression, but also controls glandular epithelium in pregnant animals. The intracellular protein trafficking leading to osteopontin preferential sorting of proteins. in their distribution receptor, alphav in beta3, is et al., 2012). These expressed in both glandular epithelium, and From the above studies it is clear that decidualizing stromal cells of pregnant embryo induces distinct changes in the animals (Fazleabas et al., 1997; Mangale and receptive stage endometrium and affects Reddy, 2007). In the mouse decidua, almost all the compartments in preparation of interactions between integrin alphav beta3 and pregnancy. These changes seem to be induced vitronectin is required to maintain a balance in response to secretions by the embryonic between cell proliferation and apoptosis, along cells and are highly localized in nature. A with modulation of inflammatory responses summary of the morphological and molecular (Mangale et al., 2008). changes Recently the dynamics of integrin expression mainly alphav beta3 in the uterine that occur in the receptive endometrium in presence of an embryo are shown in Fig 1. epithelium has been detailed in early Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 92 Figure 1: Morphological and molecular changes in the endometrium in response to embryonic signals. The receptive endometrium senses the endometrium and undergoes extensive biochemical and morphological remodeling. The molecular changes that occur in the stromal and epithelial compartments are highlighted. Functional Consequence of the designed to decipher functional consequences Endometrial-Embryo Cross-Talk of embryo induced changes in endometrial From the discussion above it is clear that cells (Weimar et al., 2012). While it would be remarkable changes occur in the molecular beyond the scope of this article to review these profile of endometrium at the time of studies, the data derived from these studies, apposition and implantation, distinct from combined with changes seen in vivo, it appears those during the window of receptivity. The that the embryo signals the endometrial bed changes seem to be induced in response to prior to implantation making it competent for secretions by the embryonic cells and are embryo quality control and trophoblast localized in nature. However, the functional invasion. connotations of such observations remain far from clear. This is mainly due to our inability Decidua as a “Selector” for embryo quality to perform genetic manipulations in the control endometria of primates. Nevertheless, in The exceptional rate of early pregnancy loss recent years, elegant in vitro models have been may be due to the high prevalence of Biomed Res J 2015;2(1):83-104 93 Modi and Bhartiya chromosomal abnormalities in the embryo. decidual cells challenged with conditioned Genetic analysis of blastomeres taken from medium from good and poor quality embryos, good quality embryos obtained at in vitro identified 449 decidual genes deregulated in Fertilization (IVF) showed that around 70% response to medium conditioned by poor- harbor complex chromosomal abnormalities quality embryos (Brosens et al., 2014). One of (Chow et al., 2014; Mertzanidou et al., 2013). the down regulated genes in response to Such observations raise the question of how to signals in conditioned media derived from safeguard the mother against prolonged poor quality embryos was HSPA8. The protein investment in potentially developmentally functions in protein assembly and folding, abnormal embryos. One school of thought clatherin-mediated endocytosis, assembly of believes that abnormal embryos by themselves multiprotein complexes, transport of nascent are implantation polypeptides, and regulation of protein folding resulting in pregnancy failure. In recent years (Stritcher et al., 2013). The observation however, experimental evidence indicate that suggests spontaneous decidualization of endometrium developmentally impaired human embryos coupled to menstruation is a judicious strategy induce endoplasmic reticulum (ER) stress to meet the challenge. The decidua may play a response in decidualizing cells. An in vivo key role in discriminating normal and proof for the in vitro observations came from abnormal blastocysts to allow pregnancy. studies in uteri of mice flushed with Evidences to support this hypothesis were conditioned obtained from co-culture of decidual cells with developmentally competent and incompetent morphologically normal and abnormal human embryos. embryos obtained at IVF. While morpho- transcriptome revealed that medium derived logically normal embryos had no major effects from competent embryos evoked a supportive on production of a selected set of cytokines; intrauterine environment, whereas medium media derived from decidual cells co-cultured derived from poor quality embryos led to ER with morphologically arrested or abnormal stress (Brosens et al., 2014). Thus, it implies blastocysts led to down-regulation of IL-1b, - that the endometrium not only senses signals 6, -10, -17, -18, eotaxin and heparin-binding derived from the embryo and responds to EGF-like growth factor (Teklenburg et al., create a pro-implantation condition, but is also 2010a). Such down-regulation is associated capable of terminating the window of with closure of endometrial competence for endometrial receptivity to enable the mother to implantation and menstruation (Evans and dispose of compromised embryos. The Salamonsen, 2014). Microarray analysis of observation adds another dimension to the incompetent towards that soluble culture Analysis signal medium of the from of uterine Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 94 potential of decidualizing endometrial stromal and decidual cells or spent medium increases cells as sensors of embryo quality during trophoblast invasion (Godbole et al., 2011; implantation. Menkhorst Thus, we propose a dual-phase response of the endometrium. The steroid et al., 2012). We have demonstrated that decidual cell secretome primed enhances invasion of trophoblast cells through receptive phase endometrium responds to the altered expression of matrix metalloproteases incoming embryo creating an obligatory (MMPs) and tissue inhibitors of matrix environment for implantation. At the same metalloproteases (TIMPs) (Godbole et al., time the decidua gains a 'selector' activity to 2011). recognize developmental competence of the trophoblasts, the decidual cells also gain a implanting embryo. Based on the blastocyst migratory and invasive phenotype (Gellersen competence as judged by the decidua, either et al., 2010; Weimar et al., 2013). Thus, pregnancy is continued or the maternal decidualization and embryo driven changes in response is aborted and culminates in the uterine cells creates a microenvironment menstruation. favorable for implantation and placentation. Conversely, in response to Numerous growth factors that regulate the proliferation and invasion of trophoblast cells Regulation of trophoblast invasion Once the endometrium a have been identified at the fetal-maternal developmentally competent blastocyst and interface. The various factors secreted by the decides to continue with pregnancy, the decidual cells and/or the associated cell types embryo apposes and trophoblast cells begin to and their influence on trophoblast invasion has breach the luminal epithelium and invade in to been recently reviewed (Knöfler, 2010; Modi the maternal decidua to establish placentation. et al., 2012). Amongst the various factors, IL- Trophoblast cells are inherently invasive and 6, LIF and IL-11 are abundantly produced by can invade any tissue. However, in the the endometrial stromal and decidual cells, pregnant endometrium the invasion is highly and play a key role in trophoblast invasion controlled. It is believed that the decidualized (Fitzgerald et al., 2008; Modi et al., 2012; stromal cells secrete a complex array of Suman et al., 2013a; Suman and Gupta, 2014). molecules controlled IL-6 and LIF stimulates invasion of primary migration/invasion. While several of the trophoblast and JEG-3 choriocarcinoma cells molecules are already expressed by the via the STAT3 signaling pathway (Jovanović receptive endometrium, others are induced and Vićovac, 2009; Suman and Gupta, 2014). post decidualization and receiving of the The role of IL-11 in trophoblast invasion is embryonic signals. Co-culture of trophoblast less clear as it inhibits the invasion of primary that permit Biomed Res J 2015;2(1):83-104 encounters the 95 Modi and Bhartiya trophoblast and HTR-8/SVneo cells, but placenta formation, a major cause of increases invasion of the choriocarcinoma pregnancy related complications like fetal JEG-3 cells (Suman et al., 2009; 2012; 2013b). growth The hypertension leading to preeclampsia. discrepancy may originate from differences in the transcription factor content In restriction converse, and impaired gestational endometrial of the two cell lines. However, the data selectivity can result in superfertility. The suggests that locally produced IL-6, LIF and hypothesis stems from the observations that IL-11 act to finely tune invasion. While the women with recurrent miscarriages are highly cumulative effects of various factors and their fecund and time to pregnancy is reduced in roles under in vivo need those women with a history of five or more investigations, the observations together miscarriages (Teklenburg et al., 2010b). Since suggest developmentally that conditions decidualization driven incompetent blastocyst transformation of endometrial stromal cells implant (due to failure of selectivity), these creates a uterine microenvironment that would lead to late first trimester abortions. controls trophoblast invasion. Thus lack the “selector” activity in the endometrium may be a causative factor Clinical Repercussions of the Embryo- towards compromised pregnancy. Indeed, a Endometrial Cross-Talk loss of selection sensing has been observed in Endometrial receptivity is a major rate limiting endometrial stromal cells derived from step and bottleneck for the success of assisted women experiencing recurrent miscarriages reproductive technologies. The discovery that (Salker et al., 2010). Furthermore, when embryonic signals potentiate the already flushed through the mouse uterus, secreted primed uterus has opened several avenues for factors from decidualizing cultures of stromal understanding of the process of implantation cells derived from patients with recurrent and initiation of pregnancy. Given the miscarriages prolonged the window of experimental evidence demonstrating the receptivity and also increased the incidence of embryo-endometrial cross talk plays a key role pathological implantation sites, immune in endowing receptivity as well as selectivity defects and fetal demise (Brosens et al., 2014). to the endometrium, a logical consequence of a Additionally, endometrial stromal cells from reduced ability to recognize embryonic signals patients with recurrent miscarriages show is implantation failure and/or miscarriage. altered responses to hCG, and failure to Suboptimal response to signals of high quality discriminate between high and low quality embryos suboptimal human embryos (Salker et al., 2010; Weimar environment for subsequent development and et al., 2012). Thus, the “selector” activity of will result in a Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 96 the decidua may be a key to successful al., 2012). It is believed that 'poor placentation' pregnancy and defects in the process may along cause recurrent miscarriages. fetoplacental Once the embryo has implanted, the trophoblast cells invade to establish with hypoxic site, micromilieu shear stress of of uteroplacental blood flow, and aberrantly secreted proinflammatory substances into placentation. Multiple stages of placentation maternal could be compromised that can lead to contribute to progression of preeclampsia and diseases. Pre-eclampsia affecting 3–5% of IUGR (Furuya et al., 2008; Gourvas et al., pregnancies, which is characterized by 2012; Ji et al., 2013; Saito and Nakashima, gestational 2014). hypertension and severe circulation, Since trophoblast proteinuria, and is a major cause of fetal and placentation maternal deaths. decidualization, While pre-eclampsia is are synergistically invasion dependent defective on and proper embryo- detected later in gestation (20 weeks onwards), endometrial cross talk can lead to improper its pathogenesis is established early in decidual response thereby causing poor gestation where trophoblast invasion is trophoblast invasion, shallow placentation and defective. It has been shown that in women hence preeclampsia. Preliminary evidence with preeclampsia shallow placental invasion suggests that decidualization defects might and inadequate plugging of the spiral artery exist in decidua of women with pregnancies affects blood supply into the intervillous space complicated with preeclampsia (Saito and and alters the consistency of the blood flow. Nakashima, 2014). This can lead to fluctuations in the supply of Thus, there is a need for better oxygen to the placenta (Ji et al., 2013; Saito understanding of the basic processes of and Nakashima, 2014), triggering a maternal placentation and mechanisms that go awry in response by increasing blood pressure and women with preeclampsia and IUGR for compromising fetal development (Furuya et effective therapeutic approaches to these al., 2008). Another disorder caused by defects common disorders. in trophoblast invasion is intrauterine growth restriction (IUGR). IUGR arises as a result of CONCLUSIONS inadequate blood supply and/or inadequate The embryo-endometrial cross talk has transport of nutrients across the placenta to the evolved as a delicately poised mechanism to fetus, resulting in a range of mechanisms respond initially to the hormonal trigger to including reduced uteroplacental blood flow, achieve receptivity and then to amplify the compromised feto-placental angiogenesis and decision under embryonic signals to permit subsequent villous development (Gourvas et pregnancy. During this critical period, the Biomed Res J 2015;2(1):83-104 97 Modi and Bhartiya Figure 2: Biosensor activity of the implantation stage endometruim. A) In presence of a normal good quality embryo the endometrium undergoes extensive biochemical and molecular transformation allowing the apposition of the embryo to the luminal epithelium. Consequently, the secretory factors from the decidua promote invasion of trophoblast cells allowing plancetation. B) In presence of a poor quality embryo the endometrium responds by activating a strong inflammatory cascade thereby triggering closure of receptivity leading to menstruation. receptive endometrium gains the ability for survival ability. Once the receptive and the selectivity, where response of the luminal selective competence of the endometrium are epithelium serves to transduce and amplify ensured and the right blastocyst implants, the signals coming from competent embryos decidua creates a local microenvironment that renders the underlying decidual layer more is conducive for trophoblast invasion and receptive to invasion (Fig. 1). This permits placentation (Fig. 2). embryo apposition followed by trophoblast The significance of such bimodal and invasion to establish placentation (Fig. 2). In biphasic the event the endometrium experiences implanting embryo is potentially far reaching. presence of a poor-quality embryo, the To date, treatment of recurrent miscarriages supportive network is not activated, but the and implantation failure are inefficacious and decidua mount a stress response, leading to highly empirical. The recent understanding of withdrawal in the dual processes has revealed that recurrent menstruation and failure of pregnancy (Fig. 2). implantation failure may be caused by defects Such a selector mechanism of the decidua is in endometrial embryo cross talk. It will be highly desirous to avoid investing energy in necessary to unravel the molecular processes pregnancy with abnormal fetuses which may that control the timely transition of the not survive till term or have compromised receptive uterus to a selective decidua which of receptivity resulting endometrial response to the Biomed Res J 2015;2(1):83-104 Embryo-endometrial cross talk 98 subsequently permits trophoblast invasion to establish placentation. Once elucidated, ACKNOWLEDGEMENTS effective approaches to modulate implantation The authors acknowledge Indian Council of and treat pregnancy complications will be Medical Research (ICMR), New Delhi, for feasible proposition. 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Camphausen1 and Anita Tandle1* 1 Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda MD, USA 2 University of Vermont College of Medicine, Burlington, VT, USA 3 Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, MD, USA Radiotherapy remains the standard treatment for glioblastoma multiforme (GBM) following surgical resection. Given the aberrant expression of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) which may play a role in therapeutic resistance to receptor tyrosine kinase inhibitors, and the emerging use of histone deacetylase (HDAC) inhibitors as radiosensitizers, we defined the effects of CUDC-101, a triple inhibitor of HER2, EGFR and HDAC on the radiosensitivity of GBM cells. Clonogenic survival was used to determine the in vitro radiosensitizing potential of CUDC-101 on GBM, breast cancer, and normal fibroblast cell lines. Inhibitory activity was defined using immunoblots and DNA double strand breaks were evaluated using γH2AX foci. Effects of CUDC-101 on cell cycle and radiation-induced cell kill were determined using flow cytometry and fluorescent microscopy. CUDC-101 inhibited HER2, EGFR and HDAC and enhanced in vitro radiosensitivity of both GBM and breast cancer cell lines, with no effect on normal fibroblasts. Retention of γH2AX foci was increased by CUDC-101 alone and in combination with irradiation for 24 h. Treatment with CUDC-101 increased the number of cells in G2 and M phase, with only increase in M phase statistically significant. An increase in mitotic catastrophe was seen in a time-dependent fashion with combination treatment. The results indicate the tumor specific CUDC-101 enhanced radiosensitization in GBM, and suggest that the effect involves inhibition of DNA repair. INTRODUCTION The development of hematological malignancies; or using a single molecularly targeted radiosensitizers is crucial agent to target a specific pathway, such as for improving the efficacy of radiotherapy as a erlotinib, cancer main selectively inhibit receptor tyrosine kinases approaches exist regarding the incorporation (RTKs). The latter has historically been the of incorporating more popular approach in solid malignancies multiple drugs known to target multiple for the vast majority of newly investigated distinct pathways, such as, in the treatment of cancer treatment regimens, particularly when treatment molecular and application modality. inhibitors: Two gefetinib or lapatinib which Key words: Glioblastoma multiforme, Radiosensitization, HDAC, Multi-target therapy, HER2, EGFR. *Corresponding Author: Anita Tandle, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive Magnuson Clinical Center Room B3-B100, Bethesda MD 20892, USA. Email: tandlea@mail.nih.gov Biomed Res J 2015;2(1):105-119 CUDC-101 induced tumor cell radiosensitization 106 combined with radiotherapy. Single-target agents, despite their CUDC-101 has been shown to be an pharmacokinetic effective agent as a monotherapy for treatment simplicity, lower cost and reduced risk of of various tumor cell lines in vitro including adverse effects, are often clinically limited due non-small cell lung cancer, pancreatic, breast, to the genetic heterogeneity and myriad prostate, brain, and liver cancers (Lai et al., dysregulated pathways that exist not only 2010), and is currently undergoing phase I and among different malignancies, but from cell to Ib clinical trials as a monotherapy or in cell variations within the same histology (Lai combination with radiotherapy for multiple et al., 2010). cancers. As an initial step in evaluating the Recently, single small molecular inhibitors potential of CUDC-101 as a clinically have been designed to simultaneously target applicable radiosensitizer, we investigated the multiple critical cellular pathways to induce effects of CUDC-101 in a panel of cancer and cell death. The drug, CUDC-101, was normal cell lines. The data indicated that designed to target two members of the ERBB CUDC-101 selectively enhances tumor cell family, human epidermal growth factor radiosensitivity in vitro. Moreover, the receptor 2 (HER2) and epidermal growth mechanism appears to involve inhibition of factor receptor (EGFR), as well as function as DNA double strand break (DSB) repair and a pan-histone deacetylase inhibitor (HDACi) modulation of the cell-cycle. (Lai et al., 2010). The overexpression of these two RTKs has been associated with MATERIALS AND METHODS tumorigenesis and aggressiveness in many Cell lines and treatment cancers, including glioblastoma multiforme The human GBM cell line U251, breast (GBM) (Cancer Genome Atlas Research, carcinoma cell line MDA-MB-231 and 2008; Kesavabhotla et al., 2012; Lal et al., normal human lung fibroblast cell line MRC9 2002; Mittapalli et al., 2013; Pierga et al., were obtained from American Type Culture 2013; Wang et al., 2013). Additionally, histone Collection (ATCC). U251 and MDA-MB-231 acetyltransferase cells inactivation has been were grown and maintained in associated with oncogenesis, yet it is the Dulbecco's Modified Eagle Medium (DMEM; aberrant HDAC activity that is considered a Invitrogen) supplemented with 10% Fetal potential target for cancer therapy. Depending Bovine on the experimental system, HDACi has been maintained at 37ºC, 5% CO2. MRC9 cells were reported to induce tumor cell differentiation, grown and maintained in minimum essential apoptosis, and/or growth arrest, putatively via medium (MEM; Invitrogen) supplemented modulation of gene expression (Shabason et with 10% FBS, non-essential amino acids al., 2011). Biomed Res J 2015;2(1):105-119 Serum (FBS; Invitrogen) and 107 Schlaff et al. (NEAA), and sodium pyruvate (Invitrogen). Cell cycle analysis Lyophilized CUDC-101 (MW: 434.49) was The evaluation of the cell cycle phase purchased from Selleck chemicals and 3.43 distribution was performed using the BD mg of CUDC-101 was reconstituted in 1 mL of FACSCalibur. Treatment protocols were DMSO (solubility: 20 mg/mL) and stored at - identical to the clonogenic treatment regimen 20 ºC at a concentration of 10 mM. DMSO was and cells were seeded into 10 cm petri dishes. not shown to have an effect and subsequent Samples were stained with propidium iodide experiments were done with a media only (PI) and analyzed using flow cytometry. To control unless otherwise noted. Cultures were determine the activation of the G2 cell cycle irradiated using the Pantak source at a dose of checkpoint, mitotic cells were distinguished 2.27 Gy/min. from G2 cells as previously reported by Xu et al. (2002) utilizing the mouse monoclonal antibody (Cell Signaling) against phospho-H3 Clonogenic Assay Cultures were dissociated with 0.25% histone (S10) (6G3) followed by staining with trypsin EDTA (Invitrogen) to create a single a Alexa Fluor-488® F(ab`)2 fragment of goat cell suspension and a specified number of cells anti-mouse conjugated secondary antibody were seeded into each well of a six well tissue (Invitrogen) (Xu and Kastan, 2004; Xu et al., culture plate. After allowing cells time to 2002). In this assay the increase of M phase adhere (24 h) cultures were treated with reflects the abrogation of the G2 checkpoint. varying doses of radiation: 0, 2, 4, 6, and 8 Gy, The data represents the mean ± SEM of followed by CUDC-101 (0.5 µM for U251 and minimum of three independent experiments. 1.0 µM for MDA-MB-231). The drug was not removed for duration of the assay. Ten to Apoptotic Cell Death fourteen days after seeding, colonies were The BD Annexin V: FITC Apoptosis stained with crystal violet, and the number of Detection Kit (Catalog Number: 556547) was colonies (≥ 50 cells) were determined. performed Surviving fractions were calculated and instructions. In brief, cells were washed twice survival curves generated by normalizing for with cold PBS and resuspended in 1X binding the amount of CUDC-101-induced cell death. buffer. The data represents the mean ± standard error suspension was incubated for 15 minutes at of mean (SEM) of minimum two independent room temperature (25ºC) in the dark after experiments. adding 5 µL of FITC-Annexin V and PI. as per Subsequently, the 100 manufacturer's µL of cell Samples were brought to a final volume of 500 Biomed Res J 2015;2(1):105-119 CUDC-101 induced tumor cell radiosensitization 108 µL and run on the FACSCalibur capturing limiting criteria. For each condition 00 cells 10,000 events. were scored. The data represents the mean ± SEM Immunofluorescent staining for γH2AX of minimum three independent experiments. Immunofluorescent staining and counting of γH2AX nuclear foci was performed as Immunoblotting and antibodies previously described (Xu and Kastan, 2004; Cells were seeded onto 60-mm dishes, lysed in Xu et al., 2002). Cells were seeded on four radioimmunoprecipitation well chamber slides and treated with 2 Gy buffer (Sigma) containing phosphatase and irradiation followed by treatment with CUDC- protease inhibitors (Roche, Indianapolis, IN). 101. Slides were examined on a Leica upright Protein concentrations were quantified using a fluorescent were DC Protein Assay kit (Bio-Rad Laboratories, imported into ImageJ image software for Hercules, CA). Thirty micrograms of protein analysis. For each treatment condition γH2AX was resolved on 4% to 20% Tris-Glycine gels foci were counted in 50 cells. The data and transferred to nitrocellulose membranes represents the mean ± SEM of minimum three (Bio-Rad Laboratories) and probed with the independent experiments. indicated antibodies. Primary antibodies were: microscope. Images assay (RIPA) actin, CD9 (Millipore, Germany), phosphoMitotic Catastrophe /total EGFR, and acetyl-/total histone H3 (Cell The presence of fragmented nuclei was used to Signaling Technology, Danvers, MA) and define cells undergoing mitotic catastrophe. phospho-/total HER2 (Upstate Cell Signaling Cells were seeded on cover slips and treated Solutions, Lake Placid, NY). Blots with with 2 Gy irradiation followed by treatment phosphorylated targets were visualized with with nuclear Super-Signal West Femtoluminol substrate fragmentation cells were fixed with a 10% (Thermo Scientific, Rockfold IL) and ECL neutral buffered formalin solution and Prime luminol reagent (GE Health-care, incubated with α-tubulin followed by anti- Pittsburgh, PA) was used for total protein. mouse Alexa-555 and mounted with Prolong Actin was used as a loading control. CUDC-101. To visualize gold antifade reagent containing DAPI. Normal cells and cells undergoing mitotic Statistical Analysis catastrophe were manually counted as cells In vitro experiments were repeated minimum with nuclear fragmentation. For cells to be twice and Student's t-test was used for considered positive for mitotic catastrophe, statistical analyses. Data are presented as cells with greater than 2 lobes were used as the mean ± SEM. A α value of p < 0.05 was Biomed Res J 2015;2(1):105-119 Schlaff et al. 109 considered significant. Analyses were done in assay was performed initially to determine GraphPad version 6 (Prism). appropriate dosage with minimal toxic effects. For this study, cells were plated at clonogenic RESULTS density, allowed to attach overnight, and CUDC-101 can effectively target intended treated with 4 Gy irradiation followed ligands immediately by treatment with CUDC-101 To assess the effects of CUDC-101 on the using a dose range including the previously radiosensitivity of tumor cells, a cytotoxicity reported average IC50 values of U251 and Figure 1: CUDC-101 effectively targets the intended ligands. The U251 was radiated (0 or 4 Gy) and incubated with 0.1 or 0.5 µM of CUDC-101 for 24 h. (A) Levels of phosphorylated HER2 and EGFR, and acetylated histone H3 were analyzed via immunoblot analysis. (B) Results show a decrease in phosphorylated HER2 and total EGFR in the presence or absence of radiation at 0.5 µM; however no effect was seen on pHER2 and pEGFR at 0.1 µM. Furthermore, acetyl-H3 is maintained and increased in the presence of CUDC-101 with/without radiation at both concentrations tested, supporting the effect seen by clonogenic survival. Biomed Res J 2015;2(1):105-119 CUDC-101 induced tumor cell radiosensitization 110 Figure 2: CUDC-101 radiosensitivity is tumor cell specific. Cells were radiated with increasing doses of radiation (closed circles) or post radiation incubated with various concentrations of CUDC-101 for 10 to 14 days (open circles). Colony-forming ability was assessed and survival curves generated after normalizing for the cytotoxicity of CUDC-101. (A) U251 cells were given 0.5 µM CUDC-101 and (B) MB231 cells were given 1.0 µM. (C) Normal fibroblast cell line MRC9 was irradiated with increasing doses of radiation (closed circles) or 0.5 µM CUDC-101 (open circles) or 1.0 µM CUDC-101 (closed triangles) following irradiation. Survival curves show that the radiosensitive activity of CUDC-101 is tumor cell specific, whereas MRC9 cells were not affected. Dose enhancement factors (DEFs) were assessed at surviving fractions (SFs) 0.1 and 0.01 where applicable. Data represents three-independent experiments (A-B) and twoindependent experiments (C) with points representing mean, and error bars SEM. MDA-MB-231 cells. U251 and MDA-MB- molecule's intended RTK targets, CUDC-101 231 cells were kept in drug containing media was given alone or immediately following for duration of the assay (10 to 14 days). Cells irradiation (4 Gy) to U251 cells and RTK were then stained with crystal violet and inhibition was analyzed via immunoblot. assessed for inhibition of colony formation. Results confirmed that CUDC-101 targeted Dosing CUDC-101 at 0.5 μM and 1.0 μM the intended ligands (Fig. 1). CUDC-101 immediately post-radiation for the duration of treatment the entire assay was most effective in phosphorylated inhibiting colony formation while having dependently, irrespective to the addition of minimal single agent toxic effect on U251 and irradiation, while pEGFR levels were only MDA-MB-231 respectively (Data not shown). modulated at 0.5 µM and no further decrease These concentrations were used to further in ligands level with combination treatment investigate the radiosensitive effects of was CUDC-101. however, the levels of total EGFR decreased To confirm that CUDC-101 inhibited the Biomed Res J 2015;2(1):105-119 reduced observed the HER2 (Fig. activation (pHER2) 1A). of dose Interestingly dose dependently in both single agent and 111 Schlaff et al. combination treatment cohorts. Also, as approximate value for the degree of expected of an HDACi, treatment with cytotoxicity for the evaluation of CUDC-101 CUDC-101 increased levels of acetylated H3 in combination with radiation. As shown in in a dose dependent manner. The protein levels Fig. 2A and B, a dose enhancement factor are quantitated and normalized against a (DEF) of 1.42 in U251 and 1.37 in MDA-MB- loading control, actin (Fig. 1B). 231 was observed at a SF of 0.1. Accordingly, the potential for tumor- CUDC-101 treatment inhibits clonogenic specific survival tumor specifically radiosensitivity was determined. The normal The hallmark of radiosensitivity is reduced fibroblast cell line MRC9 was treated with clonogenicity. In both the tumor cell lines, both 0.5 μM and 1.0 μM using the same treatment with treatment schedule and colony formation was irradiation resulted CUDC-101 following actions of CUDC-101 on in assessed as previously mentioned. CUDC-101 radiosensitivity as assessed by colony forming treatment reduced the MRC9 SF to 0.79 ± 0.15 ability 10 to 14 days later. U251 cells treated and 0.87 ± 0.007 respectively; in contrast to with 0.5 μM CUDC-101 yielded a surviving the tumor cell lines, CUDC-101 had little fraction (SF) of 0.67 ± 0.12; while treatment of effect on the radiosensitivity (1.13 at 0.5 μM; MDA-MB-231 cells with 1.0 μM gave a SF of 1.25 at 1.0 μM) of MRC9 (Fig. 1C). Albeit 0.65 ± 0.07. These values indicate an some sensitivity was induced it was not in an increase Figure 3: Treatment with CUDC-101 plus radiation impairs the DNA damage repair response. U251 cells seeded in chamber slides were exposed to 2 Gy irradiation followed by 0.5 μM CUDC-101 and fixed at specified times for immunoflourescent analysis of nuclear γH2AX foci retention. Foci were evaluated in ≥ 50 nuclei per treatment per experiment. (A) Representative images obtained from media at 24 h (top left panel), 2 Gy irradiation at 24 h (top right panel), 0.5 μM CUDC-101 treatment after 24 h exposure (bottom left panel), and 0.5 μM CUDC-101 immediately following 2 Gy irradiation at 24 h exposure (bottom right panel). Significant retention of γH2AX foci occurs with drug alone and combination therapy after 24 h with the combinatory effect significantly increased over drug only. (B) Data represents three independent experiments. Columns represent the mean and error bars are the SEM. * p< 0.05 ** p< 0.001. Biomed Res J 2015;2(1):105-119 112 CUDC-101 induced tumor cell radiosensitization Figure 4: CUDC-101 increases the percentage of cells in more sensitive phases of the cell cycle. U251 cells were seeded in 10-cm petri dishes, stained with PI and pH3 to differentiate mitotic cells and analyzed by flow cytometry. (A) Representative histogram of cell cycle distribution of U251 cells treated with drug or irradiation alone or combination treatment from an independent experiment. (B) Representative dot plots of cell cycle distribution of U251 cells from an independent experiment, gating and analysis was done using FloJo analysis software. (C) Treatment with 0.5 µM CUDC101 in the absence of irradiation significantly increased the number of cells in M-phase; however this effect was not augmented by the addition of irradiation (2 Gy). Additionally an increase in the G2 phase was observed for both CUDC101 alone and in combination with irradiation but was not statistically significant. Data represents three independent experiments. Columns represent the mean and error bars are the SEM. *p<0.05; **p< 0.001. Figure 5: CUDC-101 increases mitotic catastrophe. U251 cells were grown on cover slips and were irradiated (2 Gy) and exposed to CUDC-101. At 24, 48 and 72 h after treatment cells were fixed for immunocytochemical analysis of mitotic catastrophe. Nuclear fragmentation (defined as the presence of two or more distinct lobes within a single cell) was evaluated in at least 150 cells per cohort. Representative fluorescent micrographs are of cells fixed at 72 h after treatment for (A) medium, (B) 0.5 µM CUDC-101, (C) 2 Gy irradiation and (D) 0.5 µM CUDC-101 following 2 Gy irradiation. (E) Graph shows percent mitotic catastrophe; *p< 0.05; ***p< 0.0001. Analysis was done using a two-way ANOVA with Bonferroni multiple comparisons post-test. Biomed Res J 2015;2(1):105-119 113 Schlaff et al. statistically significant. Since the effect seen in treated with combination radiation and 0.5 μM GBM was significant compared to drug alone CUDC-101 (14.40 ± 0.92; p = 0.0003). As and the DEF was greater than that seen in the expected at 24 h almost complete resolution of breast cancer cell line, we focused on this DNA DSBs was seen in cells that were given histology. Further experiments to investigate irradiation only, as the average number of foci the radiosensitization effects of CUDC-101 per cell returned to near baseline levels. The used U251 cells. significant inhibition of γH2AX resolution at 24 h suggests that CUDC-101 inhibits repair CUDC-101 treatment increased retention of DNA DSBs. of γH2AX foci The most lethal form of injury to cells is DNA CUDC-101 post irradiation redistributes damage. Thus drugs inhibiting or retarding GBM cells within cell cycle DNA repair have the potential of effective Progress through the cell cycle depends on radiosensitizers. Therefore, the rate of DNA regulated set of checkpoints, which become repair via resolution of γH2AX foci was activated in the event of DNA damage. It is measured. The γ-variant of the H2A family known that radiation has an effect on inducing was shown to be a biomarker of DNA double G2/M delay stranded delayed Therefore, the distribution of tumor cells resolution of the foci indicate inhibition of within the cell cycle was assessed by flow DNA repair mechanisms (Kuo and Yang, cytometry (Fig. 4A-B). As shown in Fig. 4C, at 2008). CUDC-101 was added following 24 h an increase of cells in G2 was observed, irradiation (2 Gy) and at 1, 6 and 24 h and U251 although the increase was not significant (25.8 nuclei were visualized via immunofluorescence ± 3.5% to 40.5 ± 7.0%; p = 0.134). Addition- for resolution. ally, at 24 h a significant increase in M phase Representative images at 24 h are shown in from 2.30 ± 0.24% to 6.70 ± 1.1% was Fig. 3A. At 1 h and 6 h a significant difference observed with CUDC-101 alone (p = 0.02). in the average number of foci per cell was not The addition of radiation to CUDC-101, observed (Fig. 3B). However, there was a however, did not augment this increase in M significant increase in foci between cells phase. In fact, a slight decrease in M phase in treated with drug only compared to no cells treated with combination treatment (8.37 treatment at 24 h in U251 (6.33 ± 0.73; p = ± 1.1% to 6.86 ± 0.08%) was observed, 0.003) was observed. Furthermore, at 24 h however this difference was not statistically there was a significant difference in the significant (p = 0.247). Based on these results, average number of foci per cell in those cells it may be possible that treatment with CUDC- breaks inhibition (DSBs) of and γH2AX (Hall and Giaccia, 2006). Biomed Res J 2015;2(1):105-119 CUDC-101 induced tumor cell radiosensitization 114 101 may push cells into M phase by having off- involves an target effects in altering the activity of critical catastrophe. Cells with nuclear fragmentation, regulators of this checkpoint (e.g. ATM and defined as the presence of 2 or more distinct Chk2) regardless of the amount of DNA nuclear lobes within a single cell, were damage, and may partially account for CUDC- classified 101-mediated enhancement in radiation- catastrophe. As shown in the representative induced cell killing. fluorescent micrograph in Fig. 5A, cells as enhancement going of mitotic through mitotic undergoing mitotic catastrophe could be CUDC-101 increases mitotic catastrophe as distinguished after treatment of irradiation (2 mode of cell death Gy), CUDC-101, and combination treatment. Lai et al. (2010) reported that treatment with There was a time dependent increase in the CUDC-101 number induced expression of of cells undergoing mitotic proapoptotic and antiproliferative proteins in catastrophe after the treatment with either breast and colon cancer cells lines. To radiation or CUDC-101 up to 72 h. In cells determine receiving the combination treatment, a whether the increase in radiosensitivity resulting from CUDC-101 significantly treatment was due to an enhancement of undergoing mitotic catastrophe was detected radiation-induced V at 48 and 72 h, 61.6 ± 11.1% and 70.3 ± 6.2% staining 24 h after treatment was measured. As respectively. Furthermore, this increase in expected for a solid tumor cell line, radiation mitotic catastrophe was greater than additive induced little apoptotic cell death; treatment as compared to irradiation and CUDC-101 with CUDC-101 yielded essentially identical alone. These data suggest that the CUDC-101 levels of apoptosis, and the combination of mediated radiosensitization is achieved by an irradiation (2 Gy) and CUDC-101 had no inhibition in DNA DSB repair resulting in an effect on the frequency of apoptotic cell death increase events, indicating that the CUDC-101- catastrophe. Additionally, the data supports mediated radiosensitization of U251 glioma the observations seen in cell cycle analysis cells does not involve enhanced susceptibility indicating that the increase in M phase seen to apoptosis. (Data not shown) with drug alone, maintained when combined apoptosis, Annexin in greater cells number undergoing of cells mitotic The apparent inhibition of DSB repair, with irradiation at 24 h, was indicative of an increase of cells in M phase, and no increase in increase in mitotic catastrophe at later time- radiation-induced apoptosis suggests that points. CUDC-101 induced Biomed Res J 2015;2(1):105-119 radiosensitization 115 Schlaff et al. DISCUSSION in 40–50% of tumors, often followed by gene GBM (WHO Grade IV) is the most common rearrangement malignant central nervous system tumor with independent, constitutively phosphorylated an incidence of 0.4 to 2.8 per year per 100,000 and cell surface localized receptor tyrosine persons, and typified by nuclear atypia, kinases (RTK) that enhances tumorgenicity mitosis, endothelial proliferation and necrosis (Heimberger et al., 2005; Lal et al., 2002; (Mineo et al., 2006). Despite aggressive Lopez-Gines et al., 2010; Sugawa et al., 1990; therapies, prognoses for GBM remains poor, Wikstrand et al., 1997). Furthermore, recent and average overall survival remains 12-16% preclinical and clinical studies showed that (Stupp et al., 2009). A majority of these EGFR may play a role in radioresistance patients will at some in a ligand- undergo through activation of downstream signaling radiotherapy typically combined with a cascades such as, PI3K/Akt/mTOR, and its radiosensitizing agent. Attempts to develop involvement clinically have (Palumbo et al., 2014). Targeting HER2 is a cytotoxic well established target for breast cancer chemotherapeutic agents (Camphausen et al., therapy and a negative prognostic factor for 2005). cancers of breast, lung and brain (Hiesiger et relevant traditionally point resulting radiosensitizers been limited to Recently, agents targeting HDAC have in regulating autophagy al., 1993; Tateishi et al., 1991). gained in popularity, as they are shown to In regards to GBM, however, HER2 is not enhance the radio response by relaxing the expressed in adult glial cells, but its expression chromatin, leaving it more susceptible to DNA has been shown to increase with the degree of damage, among other mechanisms. Lai et al. astrocytoma degeneration (2010) showed the potent inhibition of 2006). Taken with the fact that the major multiple oncogenic pathways with CUDC-101 action of HER2 is the heterodimerization of as currently HER2 with other tyrosine kinase family investigating the effects of this compound in a members like EGFR; and this HER2 dose escalation study for various cancers as a heterodimerization, produces a more potent single in RTK with higher ligand affinity and tyrosine combination with radiation and cisplatin for kinase activity, and a lower internalization and locally advanced head and neck cancer degradation rate, inhibiting HER2 may not (NCT01384799). the only decrease HER2 activity, but could also investigation to assess the radiosensitizing affect the activity of its dimerization partners potential of CUDC-101 in GBM. (EGFR), thus helping to explain the effects a monotherapy, agent and are (NCT01171924), We and extended In GBM, EGFR is genomically amplified (Mineo et al., observed here with CUDC-101. Limited Biomed Res J 2015;2(1):105-119 CUDC-101 induced tumor cell radiosensitization 116 evidence exists showing the role of HER2 in been shown to correlate with amount of DNA radio response; however, a study by Duru et al. DSBs (Kuo and Yang, 2008). When X-rays (2012) showed that a pro-survival network induce DNA damage γH2AX is recruited to initiated by HER2 was responsible for the damage site and repair proteins such as radioresistance in breast cancer stem cells. ATM or DNA-PK phosphorylate γH2AX, and Additionally, irradiation of breast cancer cell p53BP, MRN and BRCA1 are recruited to lines has been associated with increased complete the repair complex (Mah et al., expression of EGFR and HER2, which augments 2010). We have previously shown that the the response to HER2-targeted therapy with HDACi valproic acid induces retention in trasztuzumab (Wattenberg et al., 2014). γH2AX foci on the sites of DNA DSBs yet, the Interestingly, we also saw an increased sites of damage are being repaired as observed expression of HER2 and EGFR in U251 cells by comet assay (Camphausen et al., 2005). A treated with irradiation alone. similar effect may occur with CUDC-101. We show CUDC-101 inhibited HER2, Additionally, based on the increase of retained EGFR and HDAC in U251 and MDA-MB- foci at 24 h and cells in the M-phase of the cell 231 cell lines, which was accompanied by cycle, CUDC-101 may be inducing DNA enhancement in radiosensitivity, while no damage as a single agent, and therefore significant radiation-induced cell kill was seen causing an abrogation of the G2/M checkpoint. in a normal fibroblast cell line. Although Alternatively CUDC-101 may show indirect 0.1µM CUDC-101 effectively maintained Ac- effects on critical regulators of the checkpoint. H3, it did not affect HER2 and EGFR Therefore, this conflict between the DNA deactivation or reduce colony formation. 0.5 repair complex remaining on the DNA damage µM CUDC-101 maintained or increased Ac- sites after repair, and the cells still being H3 levels, decreased levels of pHER2 and total pushed into M-phase, may explain the EGFR, and achieved a significant DEF. Thus increase in mitotic catastrophe and the CUDC- the question arises, if some combinatorial 101-induced radiosensitization. permutation of these targets is essential for In order for a putative radiosensitizing inducing radiosensitization, or is it solely an agent to be effective clinically, the effects seen HDACi effect? Albeit an important question, in vitro must be replicated under in vivo tumor it was beyond the scope of this study, and xenograft models. Aside from defining tumor needs further investigation. radioresponsiveness, the ability of the agent to The apparent mechanism of CUDC-101- target its intended ligands is critical. The induced radiosensitization appears to involve radiosensitizing ability seen in vitro by an inhibition of DNA DSB repair. γH2AX has clonogenic survival with a DEF of 1.42 was Biomed Res J 2015;2(1):105-119 117 Schlaff et al. not replicated in vivo (DEF 1.2) due to a high The evaluation of the radiosensitizing degree of toxicity and substantial weight loss potential of CUDC-101, provides the basis for (data not shown). Various other agents have additional preclinical exploration of the shown promise in vitro, but their effects were radiosensitizing unable to be translated to in vivo models or investigation and understanding of the clinically, limited by the toxicity (Camphausen specific molecular mechanisms addressing the et al., 2005). Much of the biological necessity to target all three ligands to achieve consequences and mechanisms of action clinically explaining this phenomenon are unclear. warranted. potential. applicable Further sensitization, is Additionally, in the case of CUDC-101, whose targets are nuclear (HDAC) and surface ACKNOWLEDGEMENTS receptors (EGFR and HER2), CUDC-101 may This research was supported by the Intramural not effectively reach and inhibit the target(s) Research Program of the NIH, National responsible for enhancing radiosensitization Cancer Institute, National Institutes of Health. in sufficient number of tumor cells in vivo to The authors acknowledge NIH CCR Confocal achieve a significant response. It may be Microscropy Core Facility, Radiation Biology feasible to combine multiple drugs together as Branch and Dr. Deedee Smart for use of the single agents (i.e. lapatinib + erlotinib + equipment. vorinostat) with radiotherapy, as reported in various haematologic cancers. 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Biomed Res J 2015;2(1):105-119 Research Article Phenotypic and Functional Characterization of a Marrow-derived Stromal Cell Line, M210B4 and its Comparison with Primary Marrow Stromal Cells Shweta Singh, Suprita Ghode, Moirangthem Ranjita Devi, Lalita Limaye and Vaijayanti Kale* Stem Cell Laboratory, National Centre for Cell Science, NCCS Complex, University of Pune Campus, Ganeshkhind, Pune, India In vitro co-culture system consisting of bone marrow stromal cells (BMSCs) or mesenchymal stromal cell lines of marrow origin has provided important clues about the regulation of hematopoietic stem cells (HSCs) by their microenvironment or niche. In the current studies, we have compared phenotypic and functional characters of a marrow-derived mesenchymal stem cell line, M210B4, with BMSCs. We demonstrate that M210B4 resembles BMSCs in terms of phenotypic characters. Unlike the BMSCs, M210B4 differentiated only towards adipogenic lineage, and was refractory towards osteogenic differentiation. However, M210B4 cells exhibited a higher HSC-supportive ability as assessed by flow cytometry analyses of the output cells from co-cultures. We observed that M210B4 cells show a constitutively higher activation of p44/42 and p-38 MAPK pathways compared to BMSCs, contributing to their higher HSC-support in vitro. Overall, the results show that M210B4 forms a suitable in vitro system to study HSC regulation in vitro. INTRODUCTION Hematopoiesis takes place in bone marrow, a various cytokines and extra-cellular molecules complex micro-environment comprised of (ECMs). various cells like osteoblast, endothelial cells, emanating from these cells also contribute CXCL12-abundant reticular (CAR) cells, actively in the HSC fate decision (Blank et al., adipocytes, cells 2008; Eckfeldt et al., 2005). Mesenchymal (MSCs), pericytes, etc. (Calvi et al., 2003; stromal cells (MSCs) form a very important Ding et al., 2012; Kiel et al., 2005; Mendez- part of the HSC microenvironment. Although Ferrer et al., 2010; Omatsu et al., 2010; Park et these cells have been used as feeder layers for al., 2012; Sacchetti et al., 2007; Sugiyama et several years (Jang et al., 2006), their precise al., 2006; Zhang et al., 2003). Hematopoietic participation stem cells (HSCs) occur in close contact with documented recently (Jing et al., 2010; the cells of the microenvironment, which Mehrasa et al., 2014; Walenda et al., 2010). control the fate of the HSCs via secretion of Subsequent studies have demonstrated that mesenchymal stromal Various in signaling the HSC mechanisms niche was Key words: Mesenchymal stromal cells, M210B4, HSC. *Corresponding Author: Vaijayanti Kale, National Centre for Cell Science, NCCS complex, University of Pune Campus, Ganeshkhind, Pune, India. Email: vpkale@nccs.res.in, vaijayanti.kale@gmail.com Biomed Res J 2015;2(1):120-133 Singh et al. 121 MSCs actively participate in regulation of whether they express MSC-like phenotype and hematopoiesis and play an important role in support hematopoiesis with efficiency at par homing and engraftment of transplanted HSCs with BMSCs. In the present study, we have (Bensidhoum et al., 2004; Sohni et al., 2013). compared M210B4 cell line with the primary In vitro co-culture of HSCs with stromal feeder layers forms an excellent model to bone marrow-derived stromal cells (BMSCs) using phenotypic and functional parameters. study molecular mechanisms involved in the regulation of hematopoiesis in general, and MATERIALS AND METHODS HSC fate in particular. Murine stromal cells Cells were isolated and characterized by Tropel et The protocols used in animal experimentation al. (2004) and constituted an important in vitro were approved by the institutional animal tool to study stromal cell biology. To have a ethics committee (IAEC). The C57BL/6J constant supply of feeder cells, several stromal (CD45.2) cell lines were generated. Some of the clonal (Ptprc; stromal cell lines that have been established Laboratory, Bar Harbor, USA) were housed are PA6 (Piacibel et al., 1996), M210B4 and bred in our experimental animal facility (Sutherland et al., 1991), S17 (Winwman et (EAF). BMSCs were isolated from bone al., 1993), and MS5 (Tordjman et al., 1999). marrow of 6–8 weeks C57BL6/J mice by These cells have been successfully used as flushing the femurs with complete medium, feeder layers and possess hematopoiesis- constituting Iscove's modified Dulbecco's supportive ability in vitro. medium (IMDM) (HiMedia, Mumbai, India) and B6.SJL-PtprcaPepcb/BoyJ CD45.1) mice (The Jackson The M210B4 cell line is a clone derived supplemented with 20% mesenchymal stem from bone marrow stromal cells from cell FBS (Mesen-FBS; Stem Cell Technology, (C57BL/6J × C3H/HeJ) F1 mouse (Lemoine Vancouver, British Columbia, Canada) and et al., 1988), and supports hematopoiesis when plated in a petridish. After 8–10 days of used as feeder layer for long-term culture- incubation, with intermittent removal of non- initiating cell (LTC-IC) assay (Burroughs et al adherent cells and addition of fresh medium, 1994). Our group has used this cell line to the adherent cells were used for the study various aspects of hematopoiesis (Bajaj experiments (Anjos-Afonso et al., 2008). et al., 2011; Hinge et al., 2010). M210B4 cell line was purchased from ATCC In spite of its extensive use in LTC-IC and maintained in RPMI1640 (HiMedia, assays, these cells have not been critically Mumbai, India) supplemented with 10% FBS evaluated primary (GIBCO, Invitrogen, Carlsbad, California, marrow-derived stromal cells. It is not known USA). Lineage negative (lin-) cells were in comparison with Biomed Res J 2015;2(1):120-133 Characterization of a marrow-derived stromal cell line, M210B4 122 isolated from bone marrow mononuclear cells irradiated (8000 rads of gamma ray, Co60) (MNCs) of Ptprc mice by using biotin-labelled feeders for 7 days in either IMDM anti-mouse lineage antibody cocktail prepared supplemented with 10% MSC-FBS (MSC from Biotin Mouse Lineage panel (BD qualified Pharmingen, San Diego, California, USA) and California, USA) or Myelocult medium (Stem ® Dynabeads biotin binder (Invitrogen, Calrsbad, California, USA). Cell FBS, Invitrogen, technology, Columbia, Carlsbad, Vancouver, Canada). The British medium was supplemented with 25 ng/ml murine IL-6, 25 Differentiation towards adipocytes and ng/ml murine SCF and 10 ng/ml murine IL-3 osteoblasts (Peprotech, Rocky Hill, USA). We compared For adipocytic differentiation, BMSCs or the HSC-supportive ability of M210B4 and M210B4 cells were treated with standard BMSCs adipogenic conditions: differentiation medium under four A) different Non-irradiated culture BMSCs/ comprising insulin (4 μg/ml), 3-isobutyl, 1- M210B4 in (IMDM + 10% Mesen-FBS), methyl B) Irradiated BMSCs/M210B4 in (IMDM + xanthine (IBMX) (500 µM), dexamethasone (0.25 μM), indomethacin (200 10% Mesen FBS), C) Non-irradiated µM) and 2 µg/ml insulin for 15-18 days. The BMSCs/M210B4 in Myelocult medium and adipogenic differentiation was confirmed by D) Irradiated BMSCs/M210B4 in Myelocult staining lipid droplets with Oil Red O dye medium. After 7 days of co-culture, the cells osteoblastic were harvested and analyzed for LSK (lin- differentiation, BMSCs or M210B4 cells were Sca1+ c-Kit+) stem cell population using flow treated with β glycerophosphate (10 nmol/L), cytometry. The LSK population was further dexamethasone (100 nmol/L) and ascorbic analyzed as long-term HSCs (LT-HSCs) or acid (0.05 nmol/L) for 15–18 days. To confirm short-term HSCs (ST-HSCs) based on CD34 osteoblastic differentiation, cells were stained expression. The absolute numbers were with Alizarin Red S to detect calcium deposits calculated based on % of cells obtained on (Sila-Asna et al.,2007). flow cytometer and total yield, while the fold (Bajaj et al., 2011). For increase was calculated by comparing Co-culture assay absolute numbers of input population to that of BMSCs or M210B4 cells were seeded in the output population. collagen-coated (50 µg/ml) 24-well plate as feeder layer. After 24 h, 1 × 105 lin- cells Flow Cytometry isolated from mouse bone marrow were co- For the phenotypic characterization, BMSCs cultured and M210B4 cells were stained with APC- with either non-irradiated Biomed Res J 2015;2(1):120-133 or Singh et al. 123 conjugated anti-mouse CD44, PE-conjugated membranes (Biorad, California, USA). The anti-mouse CD73, APC-conjugated anti- blots were incubated with specific primary mouse CD90.1, PE-conjugated anti-mouse antibodies CD105, APC-conjugated anti-mouse CD106 (Thr202/Tyr204), anti-p38, and anti-p-p38 (eBioscience, San Diego, California, USA), (Thr180/Tyr182); and horseradish peroxidase- PE-conjugated anti-mouse Sca-1, FITC- conjugated conjugated anti-mouse CD45 and FITC- Signaling Technology, Danvers, Massachu- conjugated (BD settes, USA). The signals were detected using Pharmingen, San Diego, California, USA). Lumiglo reagent (Cell Signaling Technology, For LSK profiling, cells were stained with Danvers, Massachusettes, USA) and the APC-conjugated mouse lineage antibody signals were captured on X-Ray films. cocktail, anti-mouse PECy7-conjugated CD117(c-Kit), Sca-1 CD34 (BD PE-conjugated anti-p44/42, secondary anti-p-p44/42 antibodies (Cell anti-mouse anti-mouse Data were analyzed by Sigma Stat software California, USA) and FITC-conjugated anti- using one-way repeated measure analysis of mouse CD34 (eBioscience, San Diego, variance (One-Way RM ANOVA; Jandel California, USA). The isotype-stained cells Scientific Software, California, USA). The were used as controls. The stained cells were plots represent the values as mean ± standard acquired error of mean (SEM), and p value ≤ 0.05 was FACS Canto San Statistical analysis Diego, on Pharmingen, – II (Becton Dickinson, New Jersey, USA) and analyzed considered significant. using BD FACS-DIVA SOFTWARE version 5.0. RESULTS M210B4 cell line is comparable to BMSCs Western blots at phenotypic level Whole cell lysates were prepared using RIPA BMSCs are phenotypically characterized by lysis buffer supplemented with Phosphatase surface markers as Sca1+CD44+ CD73+ Inhibitor-1(PI-1), Phosphatase Inhibitor-2 (PI- CD90.1+ CD105+ CD106+ CD45- CD34- 2), Protease Inhibitor Cocktail (PIC), 1 mM (Chamberlain et al., 2007; Boxall SL et al., Phenyl Methyl Sulphonyl Fluoride (PMSF), 1 2012; Mabuchi et al., 2013). When M210B4 mM Sodium-orthovanadate and 1 mM cells Sodium Fluoride (NaF) (Sigma-Aldrich, St. phenotypic analyses using the standard Louis, Missouri, USA). The samples having markers, we observed that both BMSCs as equal protein concentration were separated by well as M210B4 are highly positive for CD44, 9% SDS-PAGE and transferred to PVDF CD106 and Sca-1; and moderately positive for and BMSCs were subjected to Biomed Res J 2015;2(1):120-133 Characterization of a marrow-derived stromal cell line, M210B4 124 Table 1: Percent and mean fluorescence intensity (MFI) of different surface markers in M210B4 and BMSCs. S. No. Surface Markers 1 CD44 2 CD73 3 CD90.1 4 5 CD105 CD106 6 Sca-1 7 CD34 8 CD45 M210B4 BMSCs a) % population 99.99 73.89 b) MFI 35445 1680 a) % population 8.92 25.84 b) MFI 216.43 178.41 a) % populati1on 16.17 28.73 b) MFI 253.06 173.34 a) % population 31.48 56.75 b) MFI 230.91 595.31 a) % population 99.96 93 b) MFI 4872 2432 a) % population 98.99 86.51 b) MFI 24088 5751 a) % population Nil Nil b) MFI Nil Nil a) % population Nil Nil b) MFI Nil Nil CD73, CD90.1 and CD105 (Fig. 1A). In terms differentiation potential. We observed that of percentages, more number of BMSCs M210B4 cells differentiated into adipocytes expressed CD73, CD90.1 and CD105. Both (Fig. 1B), and not towards the osteogenic cells were negative for CD34 and CD45. The lineage. The data showed that M210B4 data showed that M210B4 cells compare well efficiently differentiates towards adipogenic with BMSCs at phenotypic level, albeit with lineage, but not towards the osteoblastic one. minor numerical differences (Table 1). M210B4 cells expand long-term HSCs in M210B4 cell line differentiates towards vitro adipocytic lineage The results showed that in type 'A' (Non- The marrow-derived Mesenchymal stromal irradiated feeders in IMDM supplemented cells are expected to differentiate towards with 10% MSC-FBS) and type 'B' (irradiated osteoblastic, and adipocytic lineages under feeders in IMDM supplemented with 10% appropriate stimuli (Prockop DJ., 1997; MSC-FBS) co-cultures, the total number of Pittenger MF et al., 1999; Dominici et al., cells harvested was significantly low in 2006). However, when these cells are cultured M210B4 set (Fig. 2A-a, 2B-a). The absolute for long periods they lose their differentiation numbers of LT-HSCs (LSK CD34-) were capacity. So we investigated whether M210B4 significantly increased, whereas the absolute cells possess both adipogenic and osteogenic numbers of LSK HSC (Lin-Sca-1+ c-Kit+), and Biomed Res J 2015;2(1):120-133 Singh et al. 125 Figure 1: Characterization of murine bone marrow stromal cells and M210B4 cell-line. A) Flow cytometric analysis for the MSC surface markers expressed on BMSCs versus M210B4 cell-line; B) The panel illustrates images of adipocytes formed from M210B4 cells and BMSCs stained with Oil Red O. The panel shows that BMSCs differentiate towards the osteoblastic lineage as evident by Alizarin Red S staining; M210B4 cells do not show such differentiation. (Original magnification 100X). Biomed Res J 2015;2(1):120-133 126 Characterization of a marrow-derived stromal cell line, M210B4 Figure 2: Co-culture of murine hematopoietic stem cells with BMSCs versus M210B4 cell-line. Total numbers of hematopoietic cells obtained in various co-cultures are depicted (a). Quantitative data showing absolute number of HSCs (b) and fold change over input cells (c) with respect to total cells harvested, LSK, LT-HSC and ST-HSC, at 4 different conditions (2A) Non-irradiated BMSCs/M210B4 in (IMDM+10%MSC-FBS), (2B) Irradiated BMSCs/M210B4 in (IMDM+10%MSC-FBS), (2C) Non-irradiated BMSCs/M210B4 in Myelocult medium and (2D)Irradiated BMSCs/M210B4 in Myelocult medium. The data of one representative experiment are depicted and are represented as mean ± SEM. N=3. ST-HSC (LSK CD34+) were significantly (Fig. 2A-c and 2B-c). There was no significant decreased in the M210B4 set as compared to difference in M210B4 set versus BMSCs set in the BMSCs set (Fig. 2A-b and 2B-b; Table 2). type 'C' (non-irradiated feeders in Myelocult In terms of fold increase over input, the result medium) co-cultures with respect to LSK- was the same as that of the absolute numbers HSC, LT-HSC and ST-HSC populations in Biomed Res J 2015;2(1):120-133 Singh et al. 127 Table 2: The absolute numbers (mean ± SEM, n=3) and fold increase over input from one representative experiment have been tabulated. 3 Absolute numbers × 10 Culture condition LSK LT-HSC ST-HSC BMSCs M210B4 BMSCs M210B4 BMSCs M210B4 A 30.53±0.85 13.81±0.93 3.22±0.25 10.59±1.26 26.84±1.02 2.11±0.57 B 19.15±1.12 12.67±0.61 1.10±0.07 4.40±0.52 17.94±1.06 7.95±0.10 C 9.69±1.06 13.38±3.86 7.57±1.03 11.63±3.52 1.31±0.07 0.90±0.23 D 3.27±0.15 10.42±0.44 1.88±0.16 7.99±0.34 1.17±0.06 1.75±0.30 Fold increase over input Culture condition LSK LT-HSC ST-HSC BMSCs M210B4 BMSCs M210B4 BMSCs M210B4 A 10.60±0.29 4.80±0.32 1.62±0.12 5.33±0.63 26.62±1.01 2.10±0.56 B 6.65±0.39 4.40±0.21 0.55±0.03 2.22±0.26 17.80±1.05 7.88±0.09 C 3.36±0.37 4.65±1.34 3.81±0.52 5.85±1.77 1.29±0.07 0.89±0.23 D 1.14±0.05 3.62±0.15 0.95±0.08 4.02±0.17 1.16±0.06 1.74±0.29 terms of absolute numbers and fold increase A, B and D culture conditions used in the over input (Fig. 2C-b–c; Table 2), with the experiments, with respect to the primitive LT- exception of significantly lower total cells HSC population (Table 2). Under the culture harvested in M210B4 as compared to that of condition C, however, both BMSCs and BMSCs (Fig. 2C-a). Type 'D' co-cultures M210B4 gave comparable output of various (irradiated feeders in Myelocult medium) did HSC populations (Fig. 2C). not show any difference in the total cells harvested in M210B4 and BMSCs (Fig. 2D-a), Signaling pathways operative in M210B4 but the absolute numbers and fold increase vs. BMSCs over input with respect to LSK and LT-HSC Earlier data showed that M210B4 exhibits a were significantly higher in M210B4 (Fig. 2D- better HSC-supportive ability compared to the b–c; Table 2). The absolute number and fold BMSCs. To examine whether this was related increase over input of ST-HSC between to the difference in their signaling gamut, we M210B4 and BMSCs was non-significant. subjected the lysates of these cells to western Flow cytometry graphs for all the 4 sets of co- blot analyses to detect phosphorylation of p38 culture conditions (A–D) are depicted in and p44/42 MAPK. The four different culture supplementary Fig. S1. The data showed that conditions described above were used. M210B4 cells have a better HSC-supportive We observed that under most of the culture potential in vitro as compared to BMSCs under conditions the levels of phospho p-38 Biomed Res J 2015;2(1):120-133 Characterization of a marrow-derived stromal cell line, M210B4 128 Figure 3: Western blot analyses of phosphorylated forms of p38 and p44/42 in BMSCs versus M210B4 cell-line. Total cell lysate of 48 h cultured BMSCs and M210B4 at different conditions, as shown in the figure were subjected to Western blot analyses. The blots were probed with (i) anti p-p38 and total p38 and (ii) anti p-p44/42 and total p44/42 antibodies. Equal input of proteins was ensured by probing the blots with antibody to α-tubulin. The intensity of the bands was quantitated by densitometry using ImageJ software. (Thr180/Tyr182) were higher in M210B4 as regulation of HSC fate. The cell line models compared to the BMSCs (Fig.3A). The levels become especially useful, when one needs to of phospho p44/42 (Thr202/Tyr204) were use a genetic approach to over-express or higher in M210B4 under culture conditions A, silence any particular gene. Primary cells can B and D as compared to BMSCs (Fig. 3B). also be genetically modified, but need viral Under the culture condition C, the level of p- vectors to get sufficient numbers of modified p38 was high in M210B4, whereas the level of cells. Secondly, being primary cells, the p-p44/42 was comparable to BMSCs. modified cells wither off, necessitating their Collectively, the data suggests that reestablishment. Cell lines give a distinct increased levels of phospho p38 and phospho advantage of unlimited supply of cells, and p-44/42 in M210B4 may have conferred upon also allow use of simple plasmid-based system M210B4 a better HSC-supportive ability for gene manipulations. They also facilitate under culture conditions A, B and D. generation of independent clones showing stable expression. Several cell lines like PA6 (Piacibel et al., DISCUSSION In vitro co-culture system using non-irradiated 1996), M210B4 (Sutherland et al., 1991), S17 or irradiated feeder layers formulated with (Winwman et al., 1993), and MS-5 (Tordjman primary mesenchymal et al., 1991) have been established and stromal cells or cell lines, is a useful tool to successfully used in co-culture studies. Our understand group has mainly used M210B4 cell line in our marrow the derived stromal Biomed Res J 2015;2(1):120-133 cell-mediated Singh et al. 129 previous work (Hinge et al., 2010; Bajaj et al., differentiate towards chondrocytic, and also 2011). Presently, our group is involved in towards other lineages like neural, muscular, genetically modifying these cells using etc. plasmid vectors expressing various molecules We subjected both M210B4 and primary involved in regulation of hematopoiesis. stromal cells in co-culture studies. Since the When we initiated these studies, we noticed feeder cells can be used in non-irradiated or that though this cell line has been frequently irradiated form, we employed both the used in experiments (Sutherland et al., 1991), conditions and used two different kinds of its phenotype in comparison with BMSCs has media typically used in such experiments. The not its data showed that though under most of the hematopoiesis support in comparison with culture conditions the total output of BMSCs has not been examined. hematopoietic cells in M210B4 set was low, been established. Secondly, Therefore we compared phenotype of except in co-culture condition 'D', the fold M210B4 with BMSCs. We showed that these increase of LT-HSCs over the input was cells are phenotypically comparable to consistently high in M210B4 set, except under primary BMSCs. On examination of the condition 'C'. The data suggest that M210B4 differentiation and cells possess better ability to support the LT- adipocytic lineages, we observed that the HSCs. In culture condition 'C', the absolute M210B4 cells differentiated into adipocytes number and the fold increase over input for LT- (Fig. 1B), but were refractive for osteoblastic HSCs were higher than in the BMSCs set, but differentiation. BMSCs are most commonly the data did not reach significance. This could known to differentiate into osteoblastic, be related to milder activation of p44/42 adipocytic and chondrogenic lineages. We MAPK activation under this culture condition have shown such tri-lineage differentiation of (Fig. 3B). Adipocytes are considered as placental MSCs (Sharma et al 2012). negative Adipocytes and osteoblasts form important (Naveiras et al., 2009), but M210B4 cells in components of the HSC niche and thus the spite of being able to differentiate to differentiation of BMSCs towards these two adipocytes supported HSC proliferation. This lineages is relevant in the context of shows that differentiated adipocytes, but not hematopoiesis. Therefore, in the present study pre-adipocytes, may exert negative effect on we examined the ability of M210B4 to the HSCs. The overall low output of differentiate and hematopoietic cells thus appears to be osteoblastic lineages. However, it will be primarily due to low proliferation of interesting to see whether these cells committed progenitors. towards towards osteoblastic adipogenic modulators of hematopoiesis Biomed Res J 2015;2(1):120-133 Characterization of a marrow-derived stromal cell line, M210B4 130 The signaling mechanisms operative in the stromal cells are known to affect HSC fate. that alteration of p44/42 and p38 pathways in the stromal cells can affect HSC fate. Our data showed that constitutively activated In summary, our study shows that p44/42 and p38 pathways in M210B4 cells M210B4 cell line shows phenotypic similarity under all culture conditions except for the with the primary BMSCs and has higher HSC- levels of p-p44/42 under the culture condition supportive properties by the virtue of the 'C,' may be responsible for their better HSC signaling gamut present in them. This cell line support. Stromal cells regulate the HSC fate thus forms a suitable model to examine the via secretion of various cytokines and ECM stromal cell-mediated regulation of stem cell molecules (Scadden, 2006; Baraniak et al., fate. Similarly, this cell line is a suitable model 2010). Cell–cell interactions are also known to to study adipogenesis (Bajaj et al., 2011). play important role in this process. In our future experiments we propose to examine ACKNOWLEDGEMENTS whether show The authors acknowledge Department of differential ECM and adhesion molecule Biotechnology (DBT), Government of India, profile. New Delhi (Grant M210B4 and BMSCs BT/PR14036/MED/31/ Our group has shown that p44/42 and p38 101/2010), Director, NCCS, FACS core pathways are coupled and inversely regulated facility, and Council of Scientific and in primitive stem cells (Kale 2004; Kale et al., Industrial Research (CSIR) for fellowship to 2004; SS, SG and MRD). Kale autonomous 2005). 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