Mol Biol Rep (2014) 41:85–94 DOI 10.1007/s11033-013-2840-8
Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells Vinod Vijayakurup • Carmela Spatafora • Corrado Tringali • Padmakrishnan Chorakkode Jayakrishnan Priya Srinivas • Srinivas Gopala
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Received: 15 December 2012 / Accepted: 26 October 2013 / Published online: 5 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBLinduced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-
V. Vijayakurup P. C. Jayakrishnan S. Gopala (&) Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Science and Technology, Thiruvanathapuram 695011, India e-mail: [email protected] C. Spatafora C. Tringali (&) Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy e-mail: [email protected] P. Srinivas Integrated Cancer Research Program V, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, India
induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells. Keywords Cancer
Autophagy Lignan Caffeic acid
Introduction Autophagy is a catabolic process that degrades cellular components through lysosomal machinery. During autophagy, cytoplasmic constituents such as exhausted out organelles or damaged proteins are sequestered into double membrane vesicles called autophagosomes and are subsequently delivered to lysosomes for degradation and recycling [1]. Although it is generally considered a mechanism to maintain cellular homeostasis, the role of induced autophagy in tumor cells in response to chemotherapeutic drugs remains a topic of controversy [2]. It can act as a pro-survival or death mechanism in tumor cells depending on the cellular context and the mode and intensity of chemotherapeutic insults [3–5]. Autophagy, as a part of survival responses in tumor cells, is capable of removing proteins and organelles that are critically
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Materials and methods Reagents
Fig. 1 Chemical structure of PCBL
damaged because of the stress induced by chemotherapeutic agents [3]. On the other hand, under persistent autophagic stimulation, uncontrolled autophagy can result in cell death by digesting essential cytoplasmic components in tumor cells [4]. Though there are numerous reports to substantiate the role of autophagy in both cell survival and death, autophagy induced in tumor cells in response to drug-induced stress may not be an essential cell death regulatory mechanism. The possibility that such induced autophagy is a ‘bystander’ with no active role in regulating cell death has been mentioned in recent reviews on autophagy [5, 6]. Such unpredictable roles of autophagy in tumor cell death make it an interesting research topic, particularly when studying autophagy induced during drug treatment. Recently, the mechanisms underlying the cell death inducing effects of the novel semi-synthetic compound bis(2-phenylethyl)-6,9,10-trihydroxybenzo[kl]xanthene1,2-dicarboxylate (or, more shortly, Phenethyl Caffeate Benzo[kl]xanthene Lignan, PCBL) has reported by us [7]. The structure of PCBL is shown in Fig. 1. PCBL is a molecule derived by the dimerization of the natural product Caffeic Acid Phenethyl Ester (CAPE, a honeybee propolis component). It belongs to the group of benzoxanthene lignans, which are rarely encountered in nature. They have consequently been studied only recently, once their biomimetic synthesis at a sufficient quantity became practically successful using Mn-mediated oxidative coupling of caffeic acid esters [8]. Among different benzoxanthene lignans obtained through such chemical processes, PCBL is the most promising antitumor agent because of its DNA interacting capacity, antiproliferative activities [9], antiangiogenic properties [10], and its cell death inducing effects in tumor cells [7]. In the current study, we have extended these studies by analyzing the role of autophagy in PCBL-induced cell death.
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3-(4-5-Dimethylthiazol-2-yl)-2-5-diphenyl-tetrazolium bromide (MTT), phenylmethanesulfonyl fluoride (PMSF), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), B7 supplement, L-glutamine, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), trypan blue, 3-methyladenine (3-MA), bafilomycin A1, and antibodies for b-actin and LC3 II were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer for protein extraction, antibodies against Atg5, Atg12, phospho-p70 S6 kinase, and secondary anti rabbit antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies against p62 and Beclin 1 were purchased from Abcam (Cambridge, MA, USA). A West Pico Chemiluminiscence Detection kit used for the development of western blots and a DyLightÒ 488 conjugated secondary antibody for immunoflourescence were purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). A Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). PCBL was prepared through oxidative coupling of Caffeic Acid Phenethyl Ester (CAPE) using Mn(OAc)3 as the oxidative agent, following previously reported procedures [8]. Cell culture WiDr colon adenocarcinoma cells were the kind gift of Professor William R. Wilson, Auckland Cancer Society Research Centre, University of Auckland, New Zealand. The cells were grown as monolayer cultures in DMEM, containing 10 % FBS and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) in a humidified atmosphere of 5 % CO2 at 37 °C. Human glioma tissues were obtained from patients undergoing surgical treatment at Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvanthapuram, India. Informed consent was obtained from patients as approved by the institutional ethical committee. The biopsy specimens were transported in Hanks Balanced Salt Solution (HBSS). Tumor tissues were washed with HBSS and minced to small fragments, which were then dissociated into single cells with papain treatment. The dissociated tumor cells were washed, resuspended and cultured in DMEM: F-12 supplemented with 10 % FBS, B 27 supplement, 2 mM of L-glutamine, 20 ng/ml of bFGF, 20 ng/ml of EGF, and 100 U/ml of antibiotics penicillin and streptomycin. The cells were incubated at 37 °C in 5 % CO2 and 95 % air. For all experiments, DMEM containing 2.5 % FBS was used or the medium is specified. Glioma cells after first passage were used for experiments.
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Cell viability assay using MTT Cell viability was measured by MTT assay as described elsewhere [11]. WiDr cells were plated at a density of 1 9 104 cells per well in 96 well plates and incubated for 24 h. After incubation, WiDr cells were treated with different concentrations of PCBL alone or in combination with bafilomycin A1 or 3-MA. During combination treatments, 10 mM 3-MA or 10 nM bafilomycin A1 were pretreated for 1 h followed by PCBL treatment for 18 h. Following treatment, MTT dissolved in the culture media was added to each well (final concentration 1 mg/ml) and incubated at 37° C for 2 h. Acidified isopropanol was added to dissolve the MTT crystals and the optical density was measured at 570 nm (with 630 nm as reference wavelength) using a microplate reader (BioTek Instruments, Winooski, VT, USA). Western blot analysis For western blotting, cells were lysed in cell lysis buffer supplemented with a protease inhibitor cocktail. Lysates were separated by 10 or 15 % SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (pH 7.8) containing 0.5 % Tween 20 and 5 % skim milk to reduce non-specific binding. The blocked membrane was probed with primary antibody against the target protein followed by HRP-conjugated secondary antibody and the bands were visualized using West Pico Chemiluminescence Detection Kit as per the instructions of the manufacturer. The bands obtained were exposed to X-ray films and documented using an image analysis system (BioRad Laboratories, Hercules, CA, USA). Immunoflourescence microscopy For immunocytochemistry, WiDr cells were plated on a 24 well plate (5 9 104 cells per well) and incubated for 24 h. Following incubation, cells were treated with different concentrations of PCBL for 8 h and fixed with 4 % paraformaldehyde for 10 min. The fixed cells were blocked with PBS containing 3 % Bovine Serum Albumin (BSA) and 0.3 % Triton X-100 (PBST) for 1 h. The cells were incubated with LC3 II antibody (1:600 dilution) overnight at 4 °C. After washing, the cells were incubated with DyLight 488-conjugated secondary antibody (1:800 dilution) at room temperature and visualized using a fluorescent microscope equipped with appropriate excitation and emission filters (excitation/emission at 490/520 nm). Acridine orange staining Autophagic induction is characterized by increased acidic autophagosomes, which can be detected by acridine orange
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staining [12]. WiDr cells were seeded in 60 mm culture dishes at a 70 % confluence and treated with PCBL (25 lM) for 8 h. The cells were then rinsed twice with PBS and incubated with acridine orange at a final concentration of 1 mg/ml for 5 min. These cells were washed again with PBS to remove the excess stain and observed under a fluorescent microscope. Trypan blue exclusion assay For assessment of cell death, WiDr Cells were plated on a 24 well plate (5 9 104 cells per well), and incubated for 24 h. The cells were pre-treated with bafilomycin A1 (10 nM) for 1 h followed by co-treatment with or without 25 lM of PCBL for 12 or 24 h. After treatment, cells were trypsinized and resuspended in PBS. Equal amount of cell suspension was mixed with 0.4 % trypan blue solution and incubated for 3 min. Stained (dead) and unstained (viable) cells were counted in a hemocytometer and a quantitative measure of viability was expressed as the percentage of viable cells out of the total cell count. Caspase assay Caspase assay was performed using Caspase-Glo 3/7 Assay kit per the manufacturer’s instructions. Briefly, 3 9 104 glioma cells were seeded into a 96 well plate and incubated for 24 h and treated with or without 25 lM of PCBL for 12 h. After treatment, 100 ll of caspase reagent from the kit was added to the cells without removing the medium. The contents of the wells were gently shaken for 2 h and the luminescence emitted was measured using a luminometer (Berthold Technologies GmbH & Co. KG, Bad wildbad, Germany). Statistical analysis All statistical calculations were carried out using GraphPad Prism software. All values are expressed as the mean ± standard deviation (SD). The differences among the mean values were analyzed with one-way ANOVA followed by Tukey’s post hoc t test analysis.
Results PCBL induces autophagy in WiDr cells PCBL-treated WiDr cells exhibited vacuolated structures (Fig. 2a), a morphological feature of autophagy. Autophagy is a process characterized by increased acidic vesicular organelles (AVOs), which can be detected by acridine orange, a stain that accumulates in acid vesicles and
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88 Fig. 2 PCBL increases the level of autophagosomes in WiDr cells. a Vacuolated structures, probably autophagosomes, observed in PCBL-treated WiDr cells were documented using a phase contrast microscope. b WiDr cells treated with or without PCBL were stained with acridine orange and observed under a fluorescence microscope. Cells treated with PCBL show acridine orangestained acidic vesicles, an indication of increased levels of autophagosomes. c WiDr cells were treated with different concentrations of PCBL for 8 h, fixed, and immunostained with LC3 II antibody. A punctuated distribution of LC3 II was observed in PCBL-treated WiDr cells. This suggests that PCBL can induce autophagy. (Color figure online)
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Fig. 3 PCBL increases autophagic flux. a and b WiDr cells were treated with PCBL for the indicated time periods and at the indicated concentrations and were analyzed for LC3 II expression by western blotting. WiDr cells were treated with PCBL (25 lM) for different time periods or for 12 h with different concentrations of PCBL. Results show time- and dose-dependent increase of LC3 II in PCBLtreated cells c WiDr cells treated with bafilomycin A1 for 4 h were analyzed for LC3 II expression by western blotting. Bafilomycin A1 blocks fusion of autophagosomes with lysosomes and induces accumulation of LC3 II. Results show that the saturated concentration needed for blocking autophagosome-lysosome fusion was 10 nM and higher concentrations did not yield further accumulation of LC3 II. d The Protein levels were expressed as fold changes over control after normalising to b-actin. e WiDr cells were treated with bafilomycin A1, PCBL, or PCBL?bafilomycin A1. f The PCBL treatment was applied over 8 h and bafilomycin A1 co-treatment was applied only
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during the last 4 h of PCBL treatment. Increased expression of LC3 II was observed in cells co-treated with PCBL and bafilomycin A1 compared to cells treated with PCBL or bafilomycin A1 alone. g The protein levels were expressed as fold changes over control after normalising to b-actin. All of the above experiment were repeated thrice. *** P value \0.001, **P value \0.01, ### P value \0.001. Symbol asterisk (*) represents statistical significance between control and treatment groups where as hash (#) represents statistical significance between different treatment groups). h WiDr cells treated with bafilomycin A1 for 4 h were analyzed for accumulation of p62 by western blotting. Bafilomycin A1 treatment increased accumulation of p62 in WiDr cells. i WiDr cells treated with PCBL for 8 h and 12 h were analyzed for p62 expression by western blotting. PCBL treatment decreased the expression of p62 in WiDr cells
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fluoresces in bright red. Vital staining of WiDr cells with acridine orange showed an increase in AVO in the cells exposed to 25 lM PCBL (Fig. 2b). The capacity of PCBL to induce autophagy was further examined by analyzing the intracellular distribution of LC3 II (autophagy marker) upon PCBL treatment by immunofluorescence. A punctuate distribution pattern of LC3 II, an indication of increased autophagy, was observed in WiDr cells treated with different concentrations of PCBL (25, 37.5 and 50 lM) (Fig. 2c). Autophagic induction in PCBL-treated WiDr cells were further demonstrated by analyzing LC3 II expression using western blotting. WiDr cells were treated with a concentration of 25 lM of PCBL for different time periods (4, 8 and 12 h) or for 12 h with different concentrations of PCBL (25, 37.5 and 50 lM). Our results show time- and dose-dependent increase of LC3 II in PCBL-treated cells (Fig. 3a, b). The autophagic flux during PCBL treatment, which is an accurate indication of autophagic activity, was also analyzed in this study using bafilomycin A1. Bafilomycin A1 blocks the degradation of autophagosome cargo in lysosomes, leading to the accumulation of autophagosome components including LC3 II. Western blot analysis of bafilomycin-A1-treated cells shows that the effects of bafilomycin A1 (in terms of LC3 II accumulation and autophagic inhibition in WiDr cells) saturated at a 10 nM concentration (Fig. 3c, d), and this concentration was used for further experiments analyzing autophagic flux. Treatment of PCBL (25 lM) for 4 h followed by its co-treatment with bafilomycin A1 for another 4 h in WiDr cells shows increased accumulation of LC3-II compared to cells treated with PCBL or bafilomycin A1 alone (Fig. 3e–g). Such surplus accumulation of LC3 II induced by PCBL in the presence of bafilomycin A1 demonstrates enhanced autophagic flux in PCBL-treated WiDr cells. Furthermore, we analyzed the expression dynamics of p62 (autophagic substrate protein) in WiDr cells. Inhibition of autophagy by bafilomycin A1 resulted in p62 accumulation confirms that a considerable amount of p62 is degraded via autophagy in WiDr cells (Fig. 3h). Since p62 degradation is enhanced by increased autophagic flux, its expression should remain low in autophagy-induced cells. As expected, PCBL treatment reduced the expression of p62 in WiDr cells (Fig. 3i). Taken together, these observations confirm that PCBL induces autophagy. PCBL-induced autophagy is class III PI3-kinase dependent To obtain information regarding the role of class III PI3kinase in PCBL-induced autophagy, we used 3-methyladenine (3-MA), which is a known inhibitor of class III PI3kinase. When administered in combination with 3-MA,
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PCBL failed to induce LC3 II in WiDr cells (Fig. 4a). Moreover, PCBL-induced autophagy in WiDr cells was associated with increased expression of Beclin 1, an autophagy related protein associated with the class III PI3kinase complex (Fig. 4b). Both of these results indicate a positive involvement of class III PI3-kinase in PCBLinduced autophagy. PCBL treatment did not alter p70 S6k phosphorylation To study the role of mTOR signaling in PCBL-induced autophagy, we analyzed phosphorylation of p70 S6 kinase. Active mTOR can phosphorylate its substrate p70 S6 kinase at ser 279 and thr 376 positions. Phosphorylation at these sites, an indication of mTOR activity, was analyzed by Western blotting. We found unaltered phosphorylation of p70 S6 kinase at ser 279 or thr 376 in PCBL-treated WiDr cells (Fig. 5a, b), indicating that PCBL does not alter mTOR activity. These results further indicate that mTOR signaling may not play a critical role in regulating PCBLinduced autophagy. PCBL treatment did not induce Atg5 and Atg12 expression Atg5 and Atg12 proteins in their conjugated form (Atg5– Atg12), having a molecular weight of about 55 kDa, are required for the recruitment of LC3 II to autophagosomes. Western blot analysis indicated that PCBL treatment did not increase the expression of Atg5 or Atg12 (conjugated form, 55 kDa) in WiDr cells (Fig. 6a, b). This result also shows that Atg5–Atg12 conjugation did not increase following PCBL treatment. PCBL induces autophagy and apoptosis in primary glioma cells The autophagic and apoptotic effects of PCBL are not cell line specific. Our finding of decreased pro-caspase 7
Fig. 4 PCBL-induced autophagy is class III PI3-kinase dependent. a WiDr cells were pre-treated with 3-MA for 1 h and subsequently exposed to PCBL (25 lM) for 8 h and subjected to western blotting. PCBL in combination with 3-MA did not induce LC3 II expression in WiDr cells. b WiDr cells were treated with PCBL for the indicated time periods and were analyzed for Beclin 1 expression by western blotting. PCBL-treated WiDr cells showed increased expression of Beclin 1
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Fig. 5 PCBL had no effect on p70 S6 kinase phosphorylation. WiDr cells were treated with PCBL for different time periods and the phosphorylation of the mTOR substrate p70 S6 kinase was analyzed
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by western blotting. There was no decrease in phosphorylation at thr 389 or ser 371 residues of p70 S6 kinase (a and b)
Fig. 6 PCBL treatment did not increase Atg5 and Atg12 expression. WiDr cells treated with 25 lM PCBL for different time periods were analyzed for the expression of Atg5 (a) or Atg12 (b). Expression of Atg5 and Atg12 remain uninduced in PCBL-treated cells
expression and increased caspase 3/7 activity in glioma cells treated with PCBL for 12 h demonstrates that PCBL can induce apoptosis (Fig. 7a, b). PCBL-treated (25 lM) glioma cells exhibited increased bright red fluorescent structures when stained with acridine orange (Fig. 7c). This suggests the presence of increased AVO in PCBL-treated glioma cells. PCBL-treated glioma cells also showed increased LC3 II accumulation and decreased p62 expression (Fig. 7d), a pattern expected in autophagy induced cells. Taken together, these observations confirm the capacity of PCBL to induce autophagy along with apoptosis in glioma cells. Cell death induced by PCBL was unaltered by inhibition of autophagy The role of autophagy in PCBL-induced cell death was analyzed by inhibiting autophagy in WiDr cells. Inhibition of autophagy with bafilomycin A1 and 3-MA did not alter the viability reduction or cell death induction caused by PCBL in WiDr cells (Fig. 8a, b). The capacity of PCBL to induce apoptosis was also not altered by inhibiting autophagy. The pattern of cleaved PARP expression in WiDr cells co-treated with bafilomycin A1 and PCBL was similar to that of WiDr cells treated with PCBL alone (Fig. 8c). Taken together, these results demonstrate that PCBL-induced cell death is independent of its capacity to induce autophagy.
Fig. 7 PCBL induced apoptosis and autophagy in primary glioma cells. a Glioma cells treated with PCBL for 12 h was analyzed for pro-caspase 7 by western blotting. PCBL treatment decreased procaspase 7 expression in glioma cells. b Glioma cells treated with PCBL (25 lM) for 12 h were analyzed for caspase activity using a caspase-Glo 3/7 assay kit. PCBL-treated cells exhibited increased luminescence, which was graphically represented as caspase 3/7 activity. c Glioma cells were treated with PCBL for 12 h and stained with acridine orange. Increased acridine orange staining of intracellular vesicles was observed in PCBL-treated glioma cells. d PCBLtreated glioma cells were analyzed for the expression of p62 and LC3 II by western blotting. PCBL-treated (25 lM) glioma cells showed increased LC3 II and decreased p62 expression
Discussion We recently reported the effects of PCBL on DNA damage and apoptosis in tumor cells, specifically WiDr cells [7]. Autophagy has generally been considered a pathway that has the potential to regulate the course of drug-induced cell death. Hence, it is appropriate to investigate the capacity of a novel synthetic compound to induce autophagy while studying its antitumor properties. Since this investigation pertains to the role of autophagy in PCBL-induced cell
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Fig. 8 Autophagic inhibition fails to alter PCBL-induced cell death. a WiDr cells were treated with different concentrations of PCBL (0, 12.5, 25, 37.5, or 50 lM) in combination with 3-methyladenine (5 mM) or bafilomycin A1 (10 nM) for 18 h and analyzed for cell viability by MTT assay. 3-MA and bafilomycin A1 were applied for 1 h before the addition of PCBL. The antitumor efficacy of PCBL was not altered in cells treated with bafilomycin A1 or 3-methyladenine. ns indicates a lack of statistical significance. b WiDr cells were treated with PCBL (25 lM), bafilomycin A1 (10 nM), or the combination of both for 12 or 24 h and were analyzed for cell
viability by trypan blue assay. WiDr cells were pre-treated with bafilomycin A1 for 1 h before the addition of PCBL, when treated in combination. c WiDr cells treated with 25 lM PCBL and/or 10 nM bafilomycin A1 for 12 h were analyzed for cleaved PARP levels by western blotting. Bafilomycin A1 was pre-treated for 1 h before the addition of PCBL for the inhibition of autophagy. The expression of cleaved PARP was increased in PCBL-treated cells but was not further altered because of bafilomycin A1 treatment. ***P value \0.005, ns non-significant
death, we used PCBL at concentrations and treatment regimens known to induce cell death in WiDr cells. During autophagy, autophagosomes fuse with lysosomes forming autophagolysosomes, and its components, including LC3 II, of the autophagosome membrane become subsequently degraded in the lysosomes [13]. Though increased acridine orange fluorescence and LC3 II expression provide evidence of increased autophagosomes in PCBL-treated cells, these findings cannot be accepted as definitive evidence of increased autophagy because an increased levels of autophagosomes or LC3 II in the cytosol at a particular time can either be because of increased autophagy or reduced clearance of
autophagosomes (blockage of autophagy) [14]. Hence, instead of attempting to take a snap shot of autophagosomes, assaying its increased turnover (autophagic flux) may be a more reliable indicator of autophagy in response to a particular treatment [15]. The guidelines provided by Rubinsztein et al., to analyze autophagic flux in response to drug treatment were used in our study [16]. An increased level of LC3 II or autophagosomes when autophagosome degradation is blocked by bafilomycin A1 reflects the basal rate of autophagosome formation (autophagy flux) in cells. The additional accumulation of autophagosomes and LC3 II following treatment with the test compound in the presence of bafilomycin A1 indicates a true autophagosomal
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increase because of the influence of the test compound. If a compound is inducing autophagy, in the presence of bafilomycin A1, it should induce a surplus accumulation of autophagosomes and LC3 II when compared to treatment with either bafilomycin A1 or the compound alone. We observed such additional accumulation of PCBL-induced LC3 II in the presence of bafilomycin A1, suggesting that PCBL induces autophagy. We also analyzed expression of p62, another protein marker of autophagy, to provide further evidence regarding PCBL-induced autophagy. The protein p62 serves as a link between LC3 and the cargo substrates that typically degrade in autophagolysosomes. During the process of cargo protein targeting, p62 itself gets degraded in autophagolysosomes, reducing its expression during increased autophagy [17]. Accumulation of p62 in bafilomycin-A1-treated cells suggests that a sufficient amount of p62 was being targeted and degraded in autophagolysosmes during autophagy in WiDr cells, as reported in other cell types [18]. Reduced expression of p62 thus suggests increased protein degradation through autophagy in PCBL-treated WiDr cells. Signaling mediated by mTOR and class III PI3-kinase has been reported to be involved in regulating autophagy. Signaling by mTOR can provide a gauge of cellular nutrient stress and can regulate autophagy accordingly. Autophagy, once activated, can compensate for nutrient deficits by degrading and recycling cellular components for energy needs. Under normal nutrient conditions, mTOR suppresses autophagy by phosphorylating Atg13 and thereby blocking its interaction with ULK, a process required for the initiation of autophagy [19, 20]. Signaling by mTOR is generally considered to be a potent regulator of autophagy, but drugs that can induce autophagy independent of the mTOR pathway has also been reported frequently [21]. PCBL failed to reduce mTOR activity, suggesting that autophagy induced by PCBL was not regulated through the mTOR pathway. It is interesting to note that drugs that induce autophagy independent of the mTOR pathway possess therapeutic benefits against neurodegenerative disorders such as Parkinson’s and Huntington’s disease. Most of these drugs can induce autophagy by reducing intracellular inositol or inositol 1,4,5-trisphosphate levels [22]. Another well established positive regulator of autophagy is class III PI3-kinase, a molecular complex present in mammals, whose function is to produce phosphatidylinositol-3-phosphate from phosphatidyl group [19]. Phosphoinositol-3-phosphate formed by the activation of class III PI3-kinase is supposed to help the assembly of autophagosomes. Autophagy dependent on class III PI3kinase is termed canonical. The need to specify autophagy as ‘canonical’ arises from reports of the existence of autophagy that does not require the activity of class III PI3kinase for its action [23]. Such forms of autophagy, termed
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non canonical, do not depend on Beclin 1 and are insensitive to 3-MA, a class III PI3-kinase inhibitor, as reported with resveratrol-induced autophagy [24]. Autophagy induced by PCBL is class III PI3-kinase dependent, as PCBL-induced autophagy is 3-MA sensitive and associated with increased expression of Beclin 1. This provides evidence for the canonical nature of PCBL-induced autophagy. Atg5–Atg12 conjugative protein is responsible for the conjugation between LC3 protein and phosphatidyl ethanolamine in autophagosome membranes and thus plays an important role in the assembly of autophagosomes [19]. The expression of Atg5 and Atg12 proteins in their conjugative form in PCBL-treated cells prompted us to agree with the view of Klionsky et al. [13] that increased Atg5– Atg12 conjugation is not necessary to induce autophagy. Our observations of Atg5 and Atg12 expression also supports the argument cautioning against the use of autophagy related proteins other than LC3 II and p62 in assaying autophagy. Because of the overt tendency to use the term ‘autophagic cell death’ to address the increased autophagy associated with cell death, recent reviews stressed the need to demarcate clearly between ‘cell death with autophagy’ and ‘cell death by autophagy’. The term ‘cell death with autophagy’ is proposed to address a passive form of autophagy induced in a dying cell with no role in regulating cell death. Similarly ‘cell death by autophagy’ can be used as a term to describe cell death where autophagy acts as a prominent cell death mechanism [5, 6]. Comparisons of cell death induced by PCBL in normal and autophagy inhibited WiDr cells suggests that autophagy induced by PCBL is ‘cell death with autophagy’. Our previous report demonstrated that apoptosis was the main mode of cell death induced by PCBL [7]. Given that PCBL induces autophagy along with apoptosis, ‘apoptosis with autophagy as a bystander’ is probably the apt terminology for describing such cell death. Indeed, autophagy is not a specialized mechanism for cell death. It can even be a prosurvival pathway. Such pro-survival effects of autophagy are capable of providing resistance against the cell death induced by classic chemotherapeutic agents, thereby reducing their effectiveness. The neutral modes of autophagy induced in WiDr cells by PCBL also imply that the activated autophagy mounts no resistance to PCBLinduced cell death. Considering the perplexing, yet overwhelming reports about the role of autophagy in cell death induced by antitumor agents, it is worthwhile to gather information regarding the role of autophagy in cell death induced by a novel compound with promising antitumor potential. In summary, we demonstrated that PCBL-induced cell death was associated with canonical, mTOR-independent autophagy. However, autophagy associated with PCBL has
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only a bystander role in terms of WiDr cell death. From our observations, autophagy may not possess a critical death regulatory role in PCBL-induced cell death. However, this report, in general perspective, raises concerns against the canonical dogma that couples autophagy associated with cell death as a prominent cell death regulator. Moreover, this is the first work demonstrating autophagy induced by PCBL.
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Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells Vinod Vijayakurup • Carmela Spatafora • Corrado Tringali • Padmakrishnan Chorakkode Jayakrishnan Priya Srinivas • Srinivas Gopala
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Received: 15 December 2012 / Accepted: 26 October 2013 / Published online: 5 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBLinduced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-
V. Vijayakurup P. C. Jayakrishnan S. Gopala (&) Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Science and Technology, Thiruvanathapuram 695011, India e-mail: [email protected] C. Spatafora C. Tringali (&) Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy e-mail: [email protected] P. Srinivas Integrated Cancer Research Program V, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, India
induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells. Keywords Cancer
Autophagy Lignan Caffeic acid
Introduction Autophagy is a catabolic process that degrades cellular components through lysosomal machinery. During autophagy, cytoplasmic constituents such as exhausted out organelles or damaged proteins are sequestered into double membrane vesicles called autophagosomes and are subsequently delivered to lysosomes for degradation and recycling [1]. Although it is generally considered a mechanism to maintain cellular homeostasis, the role of induced autophagy in tumor cells in response to chemotherapeutic drugs remains a topic of controversy [2]. It can act as a pro-survival or death mechanism in tumor cells depending on the cellular context and the mode and intensity of chemotherapeutic insults [3–5]. Autophagy, as a part of survival responses in tumor cells, is capable of removing proteins and organelles that are critically
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Materials and methods Reagents
Fig. 1 Chemical structure of PCBL
damaged because of the stress induced by chemotherapeutic agents [3]. On the other hand, under persistent autophagic stimulation, uncontrolled autophagy can result in cell death by digesting essential cytoplasmic components in tumor cells [4]. Though there are numerous reports to substantiate the role of autophagy in both cell survival and death, autophagy induced in tumor cells in response to drug-induced stress may not be an essential cell death regulatory mechanism. The possibility that such induced autophagy is a ‘bystander’ with no active role in regulating cell death has been mentioned in recent reviews on autophagy [5, 6]. Such unpredictable roles of autophagy in tumor cell death make it an interesting research topic, particularly when studying autophagy induced during drug treatment. Recently, the mechanisms underlying the cell death inducing effects of the novel semi-synthetic compound bis(2-phenylethyl)-6,9,10-trihydroxybenzo[kl]xanthene1,2-dicarboxylate (or, more shortly, Phenethyl Caffeate Benzo[kl]xanthene Lignan, PCBL) has reported by us [7]. The structure of PCBL is shown in Fig. 1. PCBL is a molecule derived by the dimerization of the natural product Caffeic Acid Phenethyl Ester (CAPE, a honeybee propolis component). It belongs to the group of benzoxanthene lignans, which are rarely encountered in nature. They have consequently been studied only recently, once their biomimetic synthesis at a sufficient quantity became practically successful using Mn-mediated oxidative coupling of caffeic acid esters [8]. Among different benzoxanthene lignans obtained through such chemical processes, PCBL is the most promising antitumor agent because of its DNA interacting capacity, antiproliferative activities [9], antiangiogenic properties [10], and its cell death inducing effects in tumor cells [7]. In the current study, we have extended these studies by analyzing the role of autophagy in PCBL-induced cell death.
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3-(4-5-Dimethylthiazol-2-yl)-2-5-diphenyl-tetrazolium bromide (MTT), phenylmethanesulfonyl fluoride (PMSF), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), B7 supplement, L-glutamine, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), trypan blue, 3-methyladenine (3-MA), bafilomycin A1, and antibodies for b-actin and LC3 II were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer for protein extraction, antibodies against Atg5, Atg12, phospho-p70 S6 kinase, and secondary anti rabbit antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies against p62 and Beclin 1 were purchased from Abcam (Cambridge, MA, USA). A West Pico Chemiluminiscence Detection kit used for the development of western blots and a DyLightÒ 488 conjugated secondary antibody for immunoflourescence were purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). A Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). PCBL was prepared through oxidative coupling of Caffeic Acid Phenethyl Ester (CAPE) using Mn(OAc)3 as the oxidative agent, following previously reported procedures [8]. Cell culture WiDr colon adenocarcinoma cells were the kind gift of Professor William R. Wilson, Auckland Cancer Society Research Centre, University of Auckland, New Zealand. The cells were grown as monolayer cultures in DMEM, containing 10 % FBS and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) in a humidified atmosphere of 5 % CO2 at 37 °C. Human glioma tissues were obtained from patients undergoing surgical treatment at Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvanthapuram, India. Informed consent was obtained from patients as approved by the institutional ethical committee. The biopsy specimens were transported in Hanks Balanced Salt Solution (HBSS). Tumor tissues were washed with HBSS and minced to small fragments, which were then dissociated into single cells with papain treatment. The dissociated tumor cells were washed, resuspended and cultured in DMEM: F-12 supplemented with 10 % FBS, B 27 supplement, 2 mM of L-glutamine, 20 ng/ml of bFGF, 20 ng/ml of EGF, and 100 U/ml of antibiotics penicillin and streptomycin. The cells were incubated at 37 °C in 5 % CO2 and 95 % air. For all experiments, DMEM containing 2.5 % FBS was used or the medium is specified. Glioma cells after first passage were used for experiments.
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Cell viability assay using MTT Cell viability was measured by MTT assay as described elsewhere [11]. WiDr cells were plated at a density of 1 9 104 cells per well in 96 well plates and incubated for 24 h. After incubation, WiDr cells were treated with different concentrations of PCBL alone or in combination with bafilomycin A1 or 3-MA. During combination treatments, 10 mM 3-MA or 10 nM bafilomycin A1 were pretreated for 1 h followed by PCBL treatment for 18 h. Following treatment, MTT dissolved in the culture media was added to each well (final concentration 1 mg/ml) and incubated at 37° C for 2 h. Acidified isopropanol was added to dissolve the MTT crystals and the optical density was measured at 570 nm (with 630 nm as reference wavelength) using a microplate reader (BioTek Instruments, Winooski, VT, USA). Western blot analysis For western blotting, cells were lysed in cell lysis buffer supplemented with a protease inhibitor cocktail. Lysates were separated by 10 or 15 % SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (pH 7.8) containing 0.5 % Tween 20 and 5 % skim milk to reduce non-specific binding. The blocked membrane was probed with primary antibody against the target protein followed by HRP-conjugated secondary antibody and the bands were visualized using West Pico Chemiluminescence Detection Kit as per the instructions of the manufacturer. The bands obtained were exposed to X-ray films and documented using an image analysis system (BioRad Laboratories, Hercules, CA, USA). Immunoflourescence microscopy For immunocytochemistry, WiDr cells were plated on a 24 well plate (5 9 104 cells per well) and incubated for 24 h. Following incubation, cells were treated with different concentrations of PCBL for 8 h and fixed with 4 % paraformaldehyde for 10 min. The fixed cells were blocked with PBS containing 3 % Bovine Serum Albumin (BSA) and 0.3 % Triton X-100 (PBST) for 1 h. The cells were incubated with LC3 II antibody (1:600 dilution) overnight at 4 °C. After washing, the cells were incubated with DyLight 488-conjugated secondary antibody (1:800 dilution) at room temperature and visualized using a fluorescent microscope equipped with appropriate excitation and emission filters (excitation/emission at 490/520 nm). Acridine orange staining Autophagic induction is characterized by increased acidic autophagosomes, which can be detected by acridine orange
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staining [12]. WiDr cells were seeded in 60 mm culture dishes at a 70 % confluence and treated with PCBL (25 lM) for 8 h. The cells were then rinsed twice with PBS and incubated with acridine orange at a final concentration of 1 mg/ml for 5 min. These cells were washed again with PBS to remove the excess stain and observed under a fluorescent microscope. Trypan blue exclusion assay For assessment of cell death, WiDr Cells were plated on a 24 well plate (5 9 104 cells per well), and incubated for 24 h. The cells were pre-treated with bafilomycin A1 (10 nM) for 1 h followed by co-treatment with or without 25 lM of PCBL for 12 or 24 h. After treatment, cells were trypsinized and resuspended in PBS. Equal amount of cell suspension was mixed with 0.4 % trypan blue solution and incubated for 3 min. Stained (dead) and unstained (viable) cells were counted in a hemocytometer and a quantitative measure of viability was expressed as the percentage of viable cells out of the total cell count. Caspase assay Caspase assay was performed using Caspase-Glo 3/7 Assay kit per the manufacturer’s instructions. Briefly, 3 9 104 glioma cells were seeded into a 96 well plate and incubated for 24 h and treated with or without 25 lM of PCBL for 12 h. After treatment, 100 ll of caspase reagent from the kit was added to the cells without removing the medium. The contents of the wells were gently shaken for 2 h and the luminescence emitted was measured using a luminometer (Berthold Technologies GmbH & Co. KG, Bad wildbad, Germany). Statistical analysis All statistical calculations were carried out using GraphPad Prism software. All values are expressed as the mean ± standard deviation (SD). The differences among the mean values were analyzed with one-way ANOVA followed by Tukey’s post hoc t test analysis.
Results PCBL induces autophagy in WiDr cells PCBL-treated WiDr cells exhibited vacuolated structures (Fig. 2a), a morphological feature of autophagy. Autophagy is a process characterized by increased acidic vesicular organelles (AVOs), which can be detected by acridine orange, a stain that accumulates in acid vesicles and
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88 Fig. 2 PCBL increases the level of autophagosomes in WiDr cells. a Vacuolated structures, probably autophagosomes, observed in PCBL-treated WiDr cells were documented using a phase contrast microscope. b WiDr cells treated with or without PCBL were stained with acridine orange and observed under a fluorescence microscope. Cells treated with PCBL show acridine orangestained acidic vesicles, an indication of increased levels of autophagosomes. c WiDr cells were treated with different concentrations of PCBL for 8 h, fixed, and immunostained with LC3 II antibody. A punctuated distribution of LC3 II was observed in PCBL-treated WiDr cells. This suggests that PCBL can induce autophagy. (Color figure online)
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Fig. 3 PCBL increases autophagic flux. a and b WiDr cells were treated with PCBL for the indicated time periods and at the indicated concentrations and were analyzed for LC3 II expression by western blotting. WiDr cells were treated with PCBL (25 lM) for different time periods or for 12 h with different concentrations of PCBL. Results show time- and dose-dependent increase of LC3 II in PCBLtreated cells c WiDr cells treated with bafilomycin A1 for 4 h were analyzed for LC3 II expression by western blotting. Bafilomycin A1 blocks fusion of autophagosomes with lysosomes and induces accumulation of LC3 II. Results show that the saturated concentration needed for blocking autophagosome-lysosome fusion was 10 nM and higher concentrations did not yield further accumulation of LC3 II. d The Protein levels were expressed as fold changes over control after normalising to b-actin. e WiDr cells were treated with bafilomycin A1, PCBL, or PCBL?bafilomycin A1. f The PCBL treatment was applied over 8 h and bafilomycin A1 co-treatment was applied only
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during the last 4 h of PCBL treatment. Increased expression of LC3 II was observed in cells co-treated with PCBL and bafilomycin A1 compared to cells treated with PCBL or bafilomycin A1 alone. g The protein levels were expressed as fold changes over control after normalising to b-actin. All of the above experiment were repeated thrice. *** P value \0.001, **P value \0.01, ### P value \0.001. Symbol asterisk (*) represents statistical significance between control and treatment groups where as hash (#) represents statistical significance between different treatment groups). h WiDr cells treated with bafilomycin A1 for 4 h were analyzed for accumulation of p62 by western blotting. Bafilomycin A1 treatment increased accumulation of p62 in WiDr cells. i WiDr cells treated with PCBL for 8 h and 12 h were analyzed for p62 expression by western blotting. PCBL treatment decreased the expression of p62 in WiDr cells
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fluoresces in bright red. Vital staining of WiDr cells with acridine orange showed an increase in AVO in the cells exposed to 25 lM PCBL (Fig. 2b). The capacity of PCBL to induce autophagy was further examined by analyzing the intracellular distribution of LC3 II (autophagy marker) upon PCBL treatment by immunofluorescence. A punctuate distribution pattern of LC3 II, an indication of increased autophagy, was observed in WiDr cells treated with different concentrations of PCBL (25, 37.5 and 50 lM) (Fig. 2c). Autophagic induction in PCBL-treated WiDr cells were further demonstrated by analyzing LC3 II expression using western blotting. WiDr cells were treated with a concentration of 25 lM of PCBL for different time periods (4, 8 and 12 h) or for 12 h with different concentrations of PCBL (25, 37.5 and 50 lM). Our results show time- and dose-dependent increase of LC3 II in PCBL-treated cells (Fig. 3a, b). The autophagic flux during PCBL treatment, which is an accurate indication of autophagic activity, was also analyzed in this study using bafilomycin A1. Bafilomycin A1 blocks the degradation of autophagosome cargo in lysosomes, leading to the accumulation of autophagosome components including LC3 II. Western blot analysis of bafilomycin-A1-treated cells shows that the effects of bafilomycin A1 (in terms of LC3 II accumulation and autophagic inhibition in WiDr cells) saturated at a 10 nM concentration (Fig. 3c, d), and this concentration was used for further experiments analyzing autophagic flux. Treatment of PCBL (25 lM) for 4 h followed by its co-treatment with bafilomycin A1 for another 4 h in WiDr cells shows increased accumulation of LC3-II compared to cells treated with PCBL or bafilomycin A1 alone (Fig. 3e–g). Such surplus accumulation of LC3 II induced by PCBL in the presence of bafilomycin A1 demonstrates enhanced autophagic flux in PCBL-treated WiDr cells. Furthermore, we analyzed the expression dynamics of p62 (autophagic substrate protein) in WiDr cells. Inhibition of autophagy by bafilomycin A1 resulted in p62 accumulation confirms that a considerable amount of p62 is degraded via autophagy in WiDr cells (Fig. 3h). Since p62 degradation is enhanced by increased autophagic flux, its expression should remain low in autophagy-induced cells. As expected, PCBL treatment reduced the expression of p62 in WiDr cells (Fig. 3i). Taken together, these observations confirm that PCBL induces autophagy. PCBL-induced autophagy is class III PI3-kinase dependent To obtain information regarding the role of class III PI3kinase in PCBL-induced autophagy, we used 3-methyladenine (3-MA), which is a known inhibitor of class III PI3kinase. When administered in combination with 3-MA,
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PCBL failed to induce LC3 II in WiDr cells (Fig. 4a). Moreover, PCBL-induced autophagy in WiDr cells was associated with increased expression of Beclin 1, an autophagy related protein associated with the class III PI3kinase complex (Fig. 4b). Both of these results indicate a positive involvement of class III PI3-kinase in PCBLinduced autophagy. PCBL treatment did not alter p70 S6k phosphorylation To study the role of mTOR signaling in PCBL-induced autophagy, we analyzed phosphorylation of p70 S6 kinase. Active mTOR can phosphorylate its substrate p70 S6 kinase at ser 279 and thr 376 positions. Phosphorylation at these sites, an indication of mTOR activity, was analyzed by Western blotting. We found unaltered phosphorylation of p70 S6 kinase at ser 279 or thr 376 in PCBL-treated WiDr cells (Fig. 5a, b), indicating that PCBL does not alter mTOR activity. These results further indicate that mTOR signaling may not play a critical role in regulating PCBLinduced autophagy. PCBL treatment did not induce Atg5 and Atg12 expression Atg5 and Atg12 proteins in their conjugated form (Atg5– Atg12), having a molecular weight of about 55 kDa, are required for the recruitment of LC3 II to autophagosomes. Western blot analysis indicated that PCBL treatment did not increase the expression of Atg5 or Atg12 (conjugated form, 55 kDa) in WiDr cells (Fig. 6a, b). This result also shows that Atg5–Atg12 conjugation did not increase following PCBL treatment. PCBL induces autophagy and apoptosis in primary glioma cells The autophagic and apoptotic effects of PCBL are not cell line specific. Our finding of decreased pro-caspase 7
Fig. 4 PCBL-induced autophagy is class III PI3-kinase dependent. a WiDr cells were pre-treated with 3-MA for 1 h and subsequently exposed to PCBL (25 lM) for 8 h and subjected to western blotting. PCBL in combination with 3-MA did not induce LC3 II expression in WiDr cells. b WiDr cells were treated with PCBL for the indicated time periods and were analyzed for Beclin 1 expression by western blotting. PCBL-treated WiDr cells showed increased expression of Beclin 1
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Fig. 5 PCBL had no effect on p70 S6 kinase phosphorylation. WiDr cells were treated with PCBL for different time periods and the phosphorylation of the mTOR substrate p70 S6 kinase was analyzed
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by western blotting. There was no decrease in phosphorylation at thr 389 or ser 371 residues of p70 S6 kinase (a and b)
Fig. 6 PCBL treatment did not increase Atg5 and Atg12 expression. WiDr cells treated with 25 lM PCBL for different time periods were analyzed for the expression of Atg5 (a) or Atg12 (b). Expression of Atg5 and Atg12 remain uninduced in PCBL-treated cells
expression and increased caspase 3/7 activity in glioma cells treated with PCBL for 12 h demonstrates that PCBL can induce apoptosis (Fig. 7a, b). PCBL-treated (25 lM) glioma cells exhibited increased bright red fluorescent structures when stained with acridine orange (Fig. 7c). This suggests the presence of increased AVO in PCBL-treated glioma cells. PCBL-treated glioma cells also showed increased LC3 II accumulation and decreased p62 expression (Fig. 7d), a pattern expected in autophagy induced cells. Taken together, these observations confirm the capacity of PCBL to induce autophagy along with apoptosis in glioma cells. Cell death induced by PCBL was unaltered by inhibition of autophagy The role of autophagy in PCBL-induced cell death was analyzed by inhibiting autophagy in WiDr cells. Inhibition of autophagy with bafilomycin A1 and 3-MA did not alter the viability reduction or cell death induction caused by PCBL in WiDr cells (Fig. 8a, b). The capacity of PCBL to induce apoptosis was also not altered by inhibiting autophagy. The pattern of cleaved PARP expression in WiDr cells co-treated with bafilomycin A1 and PCBL was similar to that of WiDr cells treated with PCBL alone (Fig. 8c). Taken together, these results demonstrate that PCBL-induced cell death is independent of its capacity to induce autophagy.
Fig. 7 PCBL induced apoptosis and autophagy in primary glioma cells. a Glioma cells treated with PCBL for 12 h was analyzed for pro-caspase 7 by western blotting. PCBL treatment decreased procaspase 7 expression in glioma cells. b Glioma cells treated with PCBL (25 lM) for 12 h were analyzed for caspase activity using a caspase-Glo 3/7 assay kit. PCBL-treated cells exhibited increased luminescence, which was graphically represented as caspase 3/7 activity. c Glioma cells were treated with PCBL for 12 h and stained with acridine orange. Increased acridine orange staining of intracellular vesicles was observed in PCBL-treated glioma cells. d PCBLtreated glioma cells were analyzed for the expression of p62 and LC3 II by western blotting. PCBL-treated (25 lM) glioma cells showed increased LC3 II and decreased p62 expression
Discussion We recently reported the effects of PCBL on DNA damage and apoptosis in tumor cells, specifically WiDr cells [7]. Autophagy has generally been considered a pathway that has the potential to regulate the course of drug-induced cell death. Hence, it is appropriate to investigate the capacity of a novel synthetic compound to induce autophagy while studying its antitumor properties. Since this investigation pertains to the role of autophagy in PCBL-induced cell
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Fig. 8 Autophagic inhibition fails to alter PCBL-induced cell death. a WiDr cells were treated with different concentrations of PCBL (0, 12.5, 25, 37.5, or 50 lM) in combination with 3-methyladenine (5 mM) or bafilomycin A1 (10 nM) for 18 h and analyzed for cell viability by MTT assay. 3-MA and bafilomycin A1 were applied for 1 h before the addition of PCBL. The antitumor efficacy of PCBL was not altered in cells treated with bafilomycin A1 or 3-methyladenine. ns indicates a lack of statistical significance. b WiDr cells were treated with PCBL (25 lM), bafilomycin A1 (10 nM), or the combination of both for 12 or 24 h and were analyzed for cell
viability by trypan blue assay. WiDr cells were pre-treated with bafilomycin A1 for 1 h before the addition of PCBL, when treated in combination. c WiDr cells treated with 25 lM PCBL and/or 10 nM bafilomycin A1 for 12 h were analyzed for cleaved PARP levels by western blotting. Bafilomycin A1 was pre-treated for 1 h before the addition of PCBL for the inhibition of autophagy. The expression of cleaved PARP was increased in PCBL-treated cells but was not further altered because of bafilomycin A1 treatment. ***P value \0.005, ns non-significant
death, we used PCBL at concentrations and treatment regimens known to induce cell death in WiDr cells. During autophagy, autophagosomes fuse with lysosomes forming autophagolysosomes, and its components, including LC3 II, of the autophagosome membrane become subsequently degraded in the lysosomes [13]. Though increased acridine orange fluorescence and LC3 II expression provide evidence of increased autophagosomes in PCBL-treated cells, these findings cannot be accepted as definitive evidence of increased autophagy because an increased levels of autophagosomes or LC3 II in the cytosol at a particular time can either be because of increased autophagy or reduced clearance of
autophagosomes (blockage of autophagy) [14]. Hence, instead of attempting to take a snap shot of autophagosomes, assaying its increased turnover (autophagic flux) may be a more reliable indicator of autophagy in response to a particular treatment [15]. The guidelines provided by Rubinsztein et al., to analyze autophagic flux in response to drug treatment were used in our study [16]. An increased level of LC3 II or autophagosomes when autophagosome degradation is blocked by bafilomycin A1 reflects the basal rate of autophagosome formation (autophagy flux) in cells. The additional accumulation of autophagosomes and LC3 II following treatment with the test compound in the presence of bafilomycin A1 indicates a true autophagosomal
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increase because of the influence of the test compound. If a compound is inducing autophagy, in the presence of bafilomycin A1, it should induce a surplus accumulation of autophagosomes and LC3 II when compared to treatment with either bafilomycin A1 or the compound alone. We observed such additional accumulation of PCBL-induced LC3 II in the presence of bafilomycin A1, suggesting that PCBL induces autophagy. We also analyzed expression of p62, another protein marker of autophagy, to provide further evidence regarding PCBL-induced autophagy. The protein p62 serves as a link between LC3 and the cargo substrates that typically degrade in autophagolysosomes. During the process of cargo protein targeting, p62 itself gets degraded in autophagolysosomes, reducing its expression during increased autophagy [17]. Accumulation of p62 in bafilomycin-A1-treated cells suggests that a sufficient amount of p62 was being targeted and degraded in autophagolysosmes during autophagy in WiDr cells, as reported in other cell types [18]. Reduced expression of p62 thus suggests increased protein degradation through autophagy in PCBL-treated WiDr cells. Signaling mediated by mTOR and class III PI3-kinase has been reported to be involved in regulating autophagy. Signaling by mTOR can provide a gauge of cellular nutrient stress and can regulate autophagy accordingly. Autophagy, once activated, can compensate for nutrient deficits by degrading and recycling cellular components for energy needs. Under normal nutrient conditions, mTOR suppresses autophagy by phosphorylating Atg13 and thereby blocking its interaction with ULK, a process required for the initiation of autophagy [19, 20]. Signaling by mTOR is generally considered to be a potent regulator of autophagy, but drugs that can induce autophagy independent of the mTOR pathway has also been reported frequently [21]. PCBL failed to reduce mTOR activity, suggesting that autophagy induced by PCBL was not regulated through the mTOR pathway. It is interesting to note that drugs that induce autophagy independent of the mTOR pathway possess therapeutic benefits against neurodegenerative disorders such as Parkinson’s and Huntington’s disease. Most of these drugs can induce autophagy by reducing intracellular inositol or inositol 1,4,5-trisphosphate levels [22]. Another well established positive regulator of autophagy is class III PI3-kinase, a molecular complex present in mammals, whose function is to produce phosphatidylinositol-3-phosphate from phosphatidyl group [19]. Phosphoinositol-3-phosphate formed by the activation of class III PI3-kinase is supposed to help the assembly of autophagosomes. Autophagy dependent on class III PI3kinase is termed canonical. The need to specify autophagy as ‘canonical’ arises from reports of the existence of autophagy that does not require the activity of class III PI3kinase for its action [23]. Such forms of autophagy, termed
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non canonical, do not depend on Beclin 1 and are insensitive to 3-MA, a class III PI3-kinase inhibitor, as reported with resveratrol-induced autophagy [24]. Autophagy induced by PCBL is class III PI3-kinase dependent, as PCBL-induced autophagy is 3-MA sensitive and associated with increased expression of Beclin 1. This provides evidence for the canonical nature of PCBL-induced autophagy. Atg5–Atg12 conjugative protein is responsible for the conjugation between LC3 protein and phosphatidyl ethanolamine in autophagosome membranes and thus plays an important role in the assembly of autophagosomes [19]. The expression of Atg5 and Atg12 proteins in their conjugative form in PCBL-treated cells prompted us to agree with the view of Klionsky et al. [13] that increased Atg5– Atg12 conjugation is not necessary to induce autophagy. Our observations of Atg5 and Atg12 expression also supports the argument cautioning against the use of autophagy related proteins other than LC3 II and p62 in assaying autophagy. Because of the overt tendency to use the term ‘autophagic cell death’ to address the increased autophagy associated with cell death, recent reviews stressed the need to demarcate clearly between ‘cell death with autophagy’ and ‘cell death by autophagy’. The term ‘cell death with autophagy’ is proposed to address a passive form of autophagy induced in a dying cell with no role in regulating cell death. Similarly ‘cell death by autophagy’ can be used as a term to describe cell death where autophagy acts as a prominent cell death mechanism [5, 6]. Comparisons of cell death induced by PCBL in normal and autophagy inhibited WiDr cells suggests that autophagy induced by PCBL is ‘cell death with autophagy’. Our previous report demonstrated that apoptosis was the main mode of cell death induced by PCBL [7]. Given that PCBL induces autophagy along with apoptosis, ‘apoptosis with autophagy as a bystander’ is probably the apt terminology for describing such cell death. Indeed, autophagy is not a specialized mechanism for cell death. It can even be a prosurvival pathway. Such pro-survival effects of autophagy are capable of providing resistance against the cell death induced by classic chemotherapeutic agents, thereby reducing their effectiveness. The neutral modes of autophagy induced in WiDr cells by PCBL also imply that the activated autophagy mounts no resistance to PCBLinduced cell death. Considering the perplexing, yet overwhelming reports about the role of autophagy in cell death induced by antitumor agents, it is worthwhile to gather information regarding the role of autophagy in cell death induced by a novel compound with promising antitumor potential. In summary, we demonstrated that PCBL-induced cell death was associated with canonical, mTOR-independent autophagy. However, autophagy associated with PCBL has
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only a bystander role in terms of WiDr cell death. From our observations, autophagy may not possess a critical death regulatory role in PCBL-induced cell death. However, this report, in general perspective, raises concerns against the canonical dogma that couples autophagy associated with cell death as a prominent cell death regulator. Moreover, this is the first work demonstrating autophagy induced by PCBL.
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