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Universidade de São Paulo
Biblioteca Digital da Produção Intelectual - BDPI
Departamento de Parasitologia - ICB/BMP
Artigos e Materiais de Revistas Científicas - ICB/BMP
2013-06
Rapid screening of potential autophagic
inductor agents using mammalian cell lines
Biotechnology Journal, Weinheim, v.8, n.6, p.730-737, 2013
http://www.producao.usp.br/handle/BDPI/44607
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ISSN 1860-6768 · BJIOAM 8 (6) 633–752 (2013) · Vol. 8 · June 2013
Systems & Synthetic Biology ·
Nanobiotech · Medicine
6/2013
Biofuels
Bioreactor design
Antibody purification
Biochemical
engineering
sciences
www.biotechnology-journal.com
Special issue: Biochemical Engineering Sciences
This Special Issue is a collection of the latest research in biochemical engineering science presented
at the 9th ESBES Conference in Istanbul, Turkey, in 2012. The cover illustrates the development in
biochemical engineering science by showing symbols for several biochemical engineering sub-disciplines, such as process engineering, strain and drug design, and material science, linked by covalent
bonds in a hypothetical biological molecule.
Images: © JarnoM, © Amelie Olivier, © teracreonte, © ermess, © by-studio, © Sergey Nivens, all from Fotolia.com.
Biotechnology Journal – list of articles published in the June 2013 issue.
Editorial: ESBES – European Society of Biochemical
Engineering Sciences
Alois Jungbauer and Guilherme Ferreira
http://dx.doi.org/10.1002/biot.201300220
Review
Bioreactor design for clinical-grade expansion of stem cells
Francisco F. dos Santos, Pedro Z. Andrade, Cláudia
Lobato da Silva and Joaquim M.S. Cabral
http://dx.doi.org/10.1002/biot.201200373
Review
Host cell protein analysis in therapeutic protein
bioprocessing – methods and applications
Anne Luise Tscheliessnig, Julita Konrath, Ron Bates
and Alois Jungbauer
Supporting information see
http://dx.doi.org/10.1002/biot.201200018
Review
Functional monolithic platforms: Chromatographic tools
for antibody purification
Telma Barroso, Abid Hussain, Ana C. A. Roque
and Ana Aguiar-Ricardo
http://dx.doi.org/10.1002/biot.201200328
Review
Large-scale production of diesel-like biofuels – process design
as an inherent part of microorganism development
Maria C. Cuellar, Joseph J. Heijnen
and Luuk A.M. van der Wielen
http://dx.doi.org/10.1002/biot.201200319
Research Article
Acoustic detection of cell adhesion to a coated quartz crystal
microbalance – implications for studying the biocompatibility
of polymers
Ana-Carina Da-Silva, Sandra S. Soares
and Guilherme N. M. Ferreira
Research Article
Harnessing Candida tenuis and Pichia stipitis in whole-cell
bioreductions of o-chloroacetophenone: Stereoselectivity,
cell activity, in situ substrate supply and product removal
Christoph Gruber, Stefan Krahulec, Bernd Nidetzky
and Regina Kratzer
http://dx.doi.org/10.1002/biot.201200322
Research Article
Stimuli-Responsive magnetic nanoparticles for monoclonal
antibody purification
Luís Borlido, Leila Moura, Ana M. Azevedo, Ana C. A.
Roque, Maria R. Aires-Barros and José P. S. Farinha
http://dx.doi.org/10.1002/biot.201200329
Regular Articles
Research Article
Organic co-solvents affect activity, stability and
enantioselectivity of haloalkane dehalogenases
Veronika Stepankova, Jiri Damborsky
and Radka Chaloupkova
http://dx.doi.org/10.1002/biot.201200378
Technical Report
Rapid screening of potential autophagic inductor agents
using mammalian cell lines
Waleska K. Martins, Divinomar Severino,
Cleidiane Souza, Beatriz S. Stolf and Maurício S. Baptista
http://dx.doi.org/10.1002/biot.201200306
Research Article
Designing a fully automated multi-bioreactor plant for fast
DoE optimization of pharmaceutical protein production
Jens Fricke, Kristof Pohlmann, Nils A. Jonescheit,
Andree Ellert, Burkhard Joksch and Reiner Luttmann
http://dx.doi.org/10.1002/biot.201200190
http://dx.doi.org/10.1002/biot.201200320
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biotechnology
Journal
DOI 10.1002/biot.201200306
Biotechnol. J. 2013, 8, 730–737
Technical Report
Rapid screening of potential autophagic inductor agents
using mammalian cell lines
Waleska K. Martins1, Divinomar Severino,1, Cleidiane Souza1, Beatriz S. Stolf2 and Maurício S. Baptista1
1 Department
2 Department
of Biochemistry, University of São Paulo, São Paulo, Brazil
of Parasitology, University of São Paulo, São Paulo, Brazil
Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and
specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic
vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used
assays to determine cell viability – Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) – to predict
autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of
NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and
CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.
Received
Revised
Accepted
Accepted
article online
25 AUG 2012
12 DEC 2012
14 FEB 2013
19 FEB 2013
Keywords: Betulinic acid · Bioextracts · Colorimetric assay · Drug screening · Quantitation of autophagic cell death
1 Introduction
Autophagy or macroautophagy is a lysosomal degradative mechanism that participates in critical functions
including cellular homeostasis and energy production [1],
cell differentiation [2], and aging [3]. Under certain circumstances, autophagy can also lead to type II programmed cell death [4, 5].
During autophagy, intact organelles and/or parts of
the cytoplasm are involved by double-membrane vacu-
Correspondence: Dr. Waleska K. Martins, Instituto de Química (IQ),
Universidade de São Paulo, Av. Prof Lineu Prestes 748, sala 1262,
CEP 05508-000 São Paulo, Brazil
E-mail: wkerllenmartins@gmail.com
Abbreviations: AO, Acridine Orange; BA, betulinic acid; CQ, chloroquine;
CVS, Crystal Violet staining; HP, hydrogen peroxide; MTT, 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide; NR, Neutral Red; NRU, Neutral
Red uptake; STS, staurosporine; TEM, temsirolimus
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oles known as autophagosomes. In the normal autophagic flux, autophagosomes mature by fusing with lysosomes, thereby forming the so-called autolysosomes, in
which lysossomal hydrolases are activated and degrade
the luminal content [4, 5].
Recommended methods for monitoring autophagy in
higher eukaryotes have been discussed in recent reviews
[6, 7]. However, only laborious and/or expensive methods
such as electron microscopy, dye-staining of autophagic
vesicles, and LC3-II and LAMP2A immunoblotting or
immunoassaying are available for reliable autophagy
identification [5-7]. A high throughput screening of autophagy and/or autophagic cell death modulators is still
missing. Aiming to fulfill this technical gap, we present
here a convenient and fast method to quantify autophagic cell death by associating three colorimetric chemosensitive assays largely used in cell viability analysis through
a semi-automatic microplate scanning spectrophotometer. One of the techniques is the MTT reduction assay,
based on reduction by mitochondrial succinic dehydro-
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2013, 8, 730–737
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genase of 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide tetrazolium salt (MTT) to an insoluble purple formazan product [8]. The Crystal Violet staining
(CVS) assay is another simple and reproducible colorimetric assay of cytotoxicity based on the growth rate evaluation [9]. The Neutral Red uptake (NRU) assay is based
on the accumulation of the Neutral Red (NR) dye in the
lysosomes of viable cells [10].
In order to create a parameter sensitive and specific to
autophagic cell death we divided NRU survival rate by the
mean of the MTT and CVS survival rates obtaining AAU,
i.e. autophagic arbitrary units.
The proposed strategy allowed the quantitation of
lysossomal alterations associated with cell death after
treatment with the known autophagy inducers chloroquine (CQ) [11] and temsirolimus (TEM) [12], and the
cytotoxic drugs hydrogen peroxide (HP) [13] and staurosporine (STS) [13, 14]. Furthermore, we tested whether
a promising anticancer drug, pentacyclic triterpene
betulinic acid (BA) [15], could induce autophagy. Finally,
to evaluate the applicability of this strategy as a highthroughput screening technique for autophagy detection,
10 natural extracts of Brazilian plants were screened.
Our results indicate that this approach may be useful
for drug discovery associated to autophagic cell death in
a high throughput screening using mammalian cell lines
in a single microplate assay.
2.3 Determination of AV accumulation by
NR-staining and spectroscopy analysis
Cells were stained with 30 µg mL–1 of NR (Sigma) at 37°C
for 2 h and washed twice with PBS. NR was eluted with
an alcoholic-based 1.0% v/v acetic acid fixing solution for
10 min at room temperature and measured at 540 nm
using the microplate reader TECAN INFINITE 200M, with
a wavelength correction set at 800 nm for subtraction of
backgrounds. Cell survival rates were normalized to the
absorbance values of untreated cells.
2.4 Cell survival assays – MTT and CVS
Cell survival was estimated according to CVS and MTT [8]
assays carried out independently. Briefly, in each well
containing the cells we added 0.2 mL of medium containing MTT (Sigma) at 50 µg mL–1 and incubated at 37°C for
2 h. At the end of the incubation period, the medium with
MTT was removed and 0.1 mL dimethyl sulfoxide (DMSO;
Sigma) was added. The plate was shaken and absorbance
values were read at 550 nm.
For the CVS assay, NR-labeled wells were washed
twice with distilled water and stained with Crystal Violet
(CV; Sigma) at 0.02% w/v for 5 min at room temperature.
After washing with distilled water, CV was eluted by 50%
v/v ethanol-0.1 M sodium citrate, and absorbance was
read at 585 nm. For both assays cell survival rates were
normalized to the absorbance values of untreated cells.
2 Materials and methods
2.5 AAU calculation
2.1 Cell lines and cell culture
Human keratinocyte cell line HaCaT, human uterine sarcoma cell lines MES-SA and MES-SA/Dx5 cells were cultured in Dulbecco modified eagle medium (DMEM, Sigma) supplemented with 10% v/v fetal bovine serum (FBS;
Sigma), 100 U mL–1 of penicillin, and 100 pg mL–1 of streptomycin in a 37°C incubator at a moist atmosphere of 5%
carbon dioxide.
Exponentially growing HaCaT, MES-SA, or MESSA/Dx5 cells were seeded at 6 × 104 cells cm–2 in 96-wellmicrotiter culture dish (Corning®) for 24 h. After washing
with phosphate buffered saline (PBS), cells were treated
with BA (Sigma), CQ (Sigma), HP (Sigma), STS (Sigma), or
TEM (Sigma) in DMEM 1.0% v/v FBS in a dose-dependent manner for 24 h at 37°C. Untreated cells in DMEM
1.0% v/v FBS served as controls.
2.2 Bioextracts
We tested 10 hidro-glycoalcoholic (propylene glycol) at
0.56% v/v and ethanol at 0.1% v/v) bioextracts furnished by
FarmaService BioExtract (São Paulo, Brazil) diluted at 1.0%
v/v in DMEM 1.0% v/v FSB. The same hidro-glycoalcoholic
solution diluted at 1.0% v/v served as control.
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
To calculate AAU, the NRU survival rate was normalized
to the mean of the MTT and CVS survival rates according
to the formula:


xa
AAU = 

 [ x b + x c ] / 2 
where xa, xb, and xc were respectively the survival rates
measured by NRU, CVS, and MTT assays.
This strategy is recommended for assaying cell survival after at least 24 or 48 h post-treatment; otherwise,
there can be important differences in survival rates
between CVS and MTT assays.
2.6 AO and NR staining of live cells
For live-cell imaging experiments [16], the lysosomotropic dye Acridine Orange (AO hemi(zinc chloride)) salt, Sigma) was added to a final concentration of 1.0 µg mL–1 for
10 min at 37°C. For NR live-cell imaging experiments,
cells were stained with NR to a final concentration of
30 µg mL–1 for 10 min at 37°C. After washing twice in PBS,
live-cells were visualized using an inverted microscope
for transmitted light and epifluorescence (Zeiss™
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Axiovert 200, Germany) equipped with an C-APOCHROMAT 40×/1.20 W Corr M27 objective (Zeiss™) and
imaged using Image J Software (National Institutes of
Health, Bethesda). Dye fluorescence of AO-stained cell
was detected by using the filter set 09 (Zeiss™) that provides an excitation band pass (BP) of 450-490 nm with
emission long pass (LP) of 515 nm.
2.7 LC3-II and LAMP2A immunoassaying
The LC3-II immunoassaying was performed according to
manufacturer’s instruction. Briefly, after fixation and
blocking cells were incubated overnight at 4°C with primary rabbit monoclonal anti-LC3-II (Cell Signaling Technology®) and primary mouse monoclonal anti-LAMP2A
(ABCAM®) antibodies diluted 1:200 and 1:100, respectively in staining buffer. Next, the stained cells were incubated for 2 h at room temperature with secondary antibodies from Molecular Probes® (Alexa 488-goat fluorochrome-conjugated anti-rabbit IgG and Alexa 633-goat
fluorochrome-conjugated anti-mouse IgG) diluted at
1:500 in blocking buffer. Cover slips were mounted onto
microscope slides using the mounting media ProLong¨
Gold antifade reagent with DAPI (Molecular Probes®).
Slides were visualized using a confocal microscope
(Zeiss™ Axiovert 200 LSM 510 Laser and Confocor Modules, Germany) equipped with a Plan-APOCHROMAT
63X/1.40 oil DIC M27 objective (Zeiss™) and imaged
using Image J Software (National Institutes of Health,
Bethesda). The spatial overlap between LC3-II and
LAMP2A was measured by a plugin “Colocalization
analysis” at Image J [17].
2.8 Statistics
Comparative statistical analysis was used to characterize
related samples. In case of a Gaussian distribution, parametric paired Student’s t-test was applied. Otherwise, the
non-parametric Wilcoxon test was used. The analysis of
correlation was done using Spearman’s (non-parametric
test) or Pearson’s coefficient (parametric test). The results
were obtained from at least three independent experiments expressed as mean values ±IC95%. p-values lower
than 0.05 were considered significant.
3 Results
3.1 Analysis of autophagy induction after treatment
of HaCaT cells with chloroquine
In an attempt to identify late autophagic vacuoles or
autolysosomes formation associated with autophagy
induction we analyzed acidotropic-lysosomotropic dye
AO labeling in HaCaT cells treated with CQ. AO stains
cell cytoplasm and nucleolus in bright green fluorescence
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and stains late autophagic vacuoles in bright red color
[7, 16]. AO staining revealed acidic perinuclear vesicles
suggestive of lysosomes (white arrow) and late autophagic vacuoles (gray arrow) in untreated and CQ treated cells,
respectively (Fig. 1A). CQ treatment induced similar cell
staining patterns of autolysosomes for AO (Fig. 1A) and
the lysosomotropic NR (Fig. 1B). To confirm whether the
stained structures were autolysosomes, immunoassays
were performed for LC3-II and LAMP2A. As can be
observed in Fig. 1C, colocalization of LC3-II and LAMP2A
(white arrow and Mander’s overlap coefficient r = 0.85)
indicated that the vacuoles stained with AO and NR are
indeed autolysosomes and that CQ at 60 µM induced
autophagy in HaCaT cells.
3.2 In vitro quantification of autophagy
We characterized the concentration-dependent cytotoxicity of CQ in the mammalian cell line HaCaT using the colorimetric cell viability assays CVS, MTT and NRU. As can
be observed in Fig. 1D, 48 h after treatment with CQ at
60 µM for 24 h, CVS and MTT assays indicate similar survival rates of 42% and 40%, respectively, while NRU indicated a significant discrepancy rate of 68%.
NR incorporates in lysosomes of viable cells. However,
autophagic vacuole accumulation led to an increase in NR
incorporation in autolysosomes. NR survival rates were
overestimated and thus, were weakly correlated with CQ
treatment, as stated by ρ’s correlation coefficients (−0.33,
p = 10–6). In contrast, using CVS and MTT assays we
noticed a higher correlation between cell survival and CQ
increment (ρ = −0.91 and −0.87, respectively, p = 10–6). As
represented in Fig. 1E, the concentration-dependence of
CQ cytotoxicity ascribed to AAU indicates its association
with autophagic cell death (ρ = 0.94, p = 10–6). The scatter-plot of AAU and cell survival measured by CVS shown
in Fig. 1F and the corresponding Spearman’s test analysis (ρ = −0.87, p = 10–6) indicate a negative and significant
correlation between AAU and cell survival.
Next, correlation analyses were made to evaluate the
sensitivity of AAU to quantify autophagy. To suppress
autophagy induced by CQ, HaCaT cells were pre-treated
with Bafilomicyn A1 (BAF) at a non-cytotoxic concentration (1.0 nM) for 24 h at 37°C, followed by dose-dependent
(20–80 µM) CQ treatment for 24 h. BAF induces defective
lysossomal acidification that can impair their fusion with
autophagosomes [18, 19]. After BAF treatment, the
autophagic cell death induced by CQ at 80 µM was significantly diminished (84%–57%, p < 0.001) as measured
by MTT reduction assay. AAU levels and cell survival
measured by CVS assay showed a higher and significant
Pearson’s correlation coefficient in untreated compared to
BAF-treated HaCaT cells (Fig. 1G).
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2013, 8, 730–737
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Figure 1. CQ induces autophagy in HaCaT cells. Autolysosomes accumulation in HaCaT cells treated with 60 µM CQ for 24 h and stained with (A) acridine
orange or (B) NR 48 h after treatment. C, control (untreated) cells; CQ, chloroquine-treated cells original magnification 520×. (C) Immunoassays for LC3-II
and LAMP2A 48 h after-treatment. Original magnification 1260×. (D) Survival rates 48 h after treatment with CQ (10-80 µM) estimated by CVS, MTT reduction, and NRU. (E) Histogram showing linear correlation between AAU levels and CQ concentrations. (F) Scatter-plots showing Spearman’s ρ correlation
coefficient (dotted line) between AAU levels and cell survival evaluated by CVS. (G) Scatter-plots correlating AAU and cell survival measured by CVS assay
after CQ treatment in BAF pre-treated (1.0 ηM for 24 h; green markers) and untreated (blue markers) cells. All images represent random fields of a slide
from two replicates of two independent experiments and scale bars indicates 20 µm. The results were obtained from at least three independent experiments and are expressed as mean values ± IC95%.
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Validation of AAU strategy to quantify autophagy. Experiments performed 48 h after treatment with different agents for 24 h. (A) Scatter-plots
correlating AAU and cell survival for MES-SA and MES-SA/Dx5 cells after treatment with CQ (10–80 µM). (B) Scatter-plots correlating AAU and cell survival
for HaCaT cells after treatment with different concentrations of HP, STS and TEM. (C) LC3-II immunoassaying in HaCaT cells after treatment with HP
(1.0 mM), STS (10 ηM), or TEM (15 µM). C, control (untreated) cells. Images represent random fields of a slide from two replicates with original magnification 630×. Scale bar indicates 20 µm. LC3-II staining is indicated by white arrows. The results were obtained from at least three independent experiments.
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2013, 8, 730–737
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3.3 Strategy validation
4 Discussion
3.3.1 Different cell lines
Autophagy or macroautophagy is a lysossomal degradative mechanism involved in protein and organelle catabolism to generate small macromolecules that are essential
for cell survival. Autophagy is activated by either conventional ATG5-dependent [4, 5, 22] or alternative ATG5independent [23, 24] systems. Despite some differences,
the last stages of both systems depend on lysosomes to
form autolysosomes [24]. Therefore, autolysosomes can
be considered a downstream biomarker of the autophagy
pathway, and assays that measure their accumulation
could be useful to identify autophagy induction independent of the system.
Quantifying autophagy is difficult, since this cell
mechanism is a dynamic process involving formation and
processing of protein biomarkers such as LC3-II. This limitation reflects the challenge of measuring autophagy
induction in a high throughput platform for identification
of new promising antitumor agents.
In an attempt to fulfill this technical gap, we presented here an approach to quantify autophagic cell death in
terms of a numeric variable named AAU, developed using
CQ. CQ is a weak base that concentrates in acidic vesicles like lysosomes and triggers apoptotic and non-apoptotic death pathways [11]. It has recently been shown that
treatment of gliomas [11], epithelial [25], and neuroblastoma cells [26] with CQ induced accumulation of autophagic vacuoles measured by LC3-II, which may result in
autophagic cell death. This autophagic cell death may
result from the accumulation of autophagosomes due to
the failure of lysosomes to successfully degrade these
autophagosomes and their contents [25]. In other situations, CQ may lead to accumulation of late autophagic
vacuoles (autolysosomes) due to the failure of lysosomes
to successfully degrade their contents [11, 26].
In our model, CQ induced autophagic flux with accumulation of autolysosomes, as shown by colocalization of
LC3-II (autophagosome marker) with LAMP2A (lysossomal marker; white arrow and Mander’s overlap coefficient r = 0.85). AAU was capable of quantifying the accumulation of CQ induced autolysosomes. In fact, we
observed a significant and strong correlation between
AAU levels and autophagic cell death. Quantifying AAU
allowed a rapid screening of potential autophagic deathinductor agents using mammalian cell lines B16-F10,
HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a single
microplate.
We also observed cytoplasmic accumulation of
autolysosomes in HaCaT cells in response to STS and
TEM. TEM induced autophagic cell death according to
both AAU levels and LC3-II immunoassays. Although STS
is well-known as an apoptosis inductor, evidence other
than ours also suggested that it induces autophagy [27].
These findings reinforced that AAU is directly associated
with autophagic cell death and may be applied to high
To evaluate the flexibility of our strategy, we used the
human cell lines originating from uterine sarcoma
MES-SA and MES-SA/Dx5. The AAU correlation with
autophagic cell death induced by CQ in MES-SA and
MES-SA/Dx5 had similar Pearson’s coefficients (r = −0.9,
p < 10–15; Fig. 2A), also comparable to HaCaT cells
(Fig. 1F). The AAU association with autophagy in B16F10 and HeLa cells was also similar (data not shown).
3.3.2 Different treatments
To evaluate both sensitivity and specificity of our strategy
in quantitating autophagy, we treated HaCaT cells with
cytotoxic drugs well-known to induce apoptosis (HP and
STS) [13, 14] or autophagy (TEM) [12]. After 24 h of treatment AAU levels were correlated with cell survival assayed
by the CVS protocol. As displayed in Fig. 2B, HP treatment
did not induce significant correlation between AAU levels
and cell survival and thus was not associated with
autophagic cell death. Contrarily, STS and TEM treatments
showed a significant and strong correlation between AAU
levels and cell survival (Fig. 2B), indicating that autophagic
cell death was induced by these drugs. These findings are
in agreement with autophagic LC3-II labeling under the
same treatment conditions (Fig. 2C).
3.4 Application of AAU to evaluate autophagy
induced by drugs and plant extracts
To challenge the ability of our strategy in quantifying
autophagy, we treated HaCaT cells with the lupane-type
triterpene BA, tested as a therapeutic anticancer drug for
dysplastic nevi [20]. It induces apoptosis in several cell
lines [15], and its glycosylated derivative form B10 leads
to lysossomal cell death, converting autophagy into a
detrimental process [21]. Forty-eight hours after a 24 h
treatment with BA, HaCaT cells showed a significant linear dose-dependent autophagy induction (Fig. 3A),
which was significantly correlated with decrease of cell
survival (Fig. 3B). These findings are in agreement with
autophagic LC3-II labeling under the same treatment
conditions (Fig. 3C), indicating that not only apoptosis
but also autophagy is induced by BA.
We then applied our strategy to high-throughput cell
viability platform usually employed in industry. We treated HaCaT cells for 24 h with 10 different natural extracts,
evaluated the cell survival by CVS, MTT reduction, and
NRU protocols, and determined AAU levels (Fig. 3D). As
can be seen in Fig. 3E, extracts of eucalyptus, guarana,
pomegranate, and rosemary at cytotoxic concentrations
(at least 25% of cell death) showed increase in mean AAU
levels associated with cell death. The autophagic cell
death detected by the AAU strategy was successfully
confirmed by LC3-II immunoassays (Fig. 3F).
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. Application of AAU to evaluate autophagy induced by drugs and plant extracts in HaCaT cells. Experiments performed 48 h after treatment with
different agents for 24 h. (A) Histogram showing linear correlation between AAU levels and BA concentrations. (B) Scatter-plots correlating AAU and cell
survival after treatment with BA. (C) LC3-II immunoassaying after treatment with 20 µM BA. Original magnification 1260×. Histograms showing cell survival rates estimated by CVS, MTT reduction, and NRU (D) and AAU levels (E) after treatment with 10 different natural extracts at 1.0% (v/v). (F) LC3-II
immunoassaying 48 h after treatment with four potential autophagic cell death inducers for 24 h. Original magnification 1260×. All images represent random fields of a slide from three independent experiments and scale bar indicates 20 µm. The results were obtained from at least three independent experiments and are expressed as mean values ± IC95%.
throughput screening of unknown compounds or bioextracts.
Interestingly, our strategy was capable of indicating
pentacyclic triterpene BA as a new inductor of autophagy.
BA is a multitarget agent responsible for inducing apoptosis, displaying antiangiogenic, anti-inflammatory as
736
well as antioxidant effects and enhancing cell differentiation in tumor cells [15].
As already discussed, there are many techniques
being employed for autophagy detection, but there is no
universal assay for conventional and alternative autophagy that can be widely applied to experimental
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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research. No individual assay is guaranteed to be the
most appropriate in every situation, and the use of multiple assays to verify an autophagic response is highly recommended [6, 7]. The AAU strategy proposed here associates three different and simple assays, and can be
applied in large scale for identification of both conventional and alternative autophagic cell death.
5 Concluding remarks
The strategy established here is more convenient than
others usually applied to measure autophagic cell death in
vitro. The main advantage is that AAU allows both qualitative and quantitative analyses of autophagic cell death
in a high throughput screening using a single microplate
assay. The qualitative evaluation can be made through
morphological analysis of NR stained autolysosomes
using optical microscopy before the elution step.
Our strategy identifies both conventional and alternative autophagic cell death, since it is based on autolysosome accumulation that is independent of its biological
type of membrane autolysosome accumulation. In addition, it may help to identify new promising autophagic
cell death inducers by convenient and fast colorimetric
chemosensitive assays.
The authors are grateful to Ana C. Viotto and Edson Alves
for technical assistance; Adriana Y. Matsukuma and
Wilton J. R. Lima for helping in confocal microscopy. This
work was supported by CAPES PNPD/FINEP grant (number 02533/09-0), Brazil and by FarmaService Bioextract,
São Paulo, Brazil, grant 1874-FUSP.
The authors declare no conflict of interest.
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