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 Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo 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 www.biotechnology-journal.com 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: [email protected] 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 730 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 www.biotecvisions.com 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™ 731 Biotechnology Journal Biotechnol. J. 2013, 8, 730–737 www.biotechnology-journal.com 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 732 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 www.biotecvisions.com 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 733 Biotechnology Journal Biotechnol. J. 2013, 8, 730–737 www.biotechnology-journal.com 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. 734 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Biotechnol. J. 2013, 8, 730–737 www.biotecvisions.com 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 735 Biotechnology Journal Biotechnol. J. 2013, 8, 730–737 www.biotechnology-journal.com 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 Biotechnol. J. 2013, 8, 730–737 www.biotecvisions.com 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. 6 References [1] Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J., Autophagy fights disease through cellular self-digestion. Nature 2008, 451, 1069–1075. [2] Aymard, E., Barruche, V., Naves, T., Bordes, S. et al., Autophagy in human keratinocytes: An early step of the differentiation? 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