Oral Diseases (2014) doi:10.1111/odi.12305 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
Obatoclax induces Beclin 1- and ATG5-dependent apoptosis and autophagy in adenoid cystic carcinoma cells L-Z Liang1, B Ma2, Y-J Liang3, H-C Liu3, T-H Zhang4, G-S Zheng3, Y-X Su3,5,*, G-Q Liao3,* 1
Department of Oral and Maxillofacial Surgery, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai; 2Department of Stomatology, Shanxi Academy of Medical Sciences, Shanxi Dayi Hospital, Taiyuan; 3Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-Sen University, Guangzhou; 4Department of Stomatology, Affiliated Zhongshan Hospital, Sun Yat-sen University, Zhongshan; 5Discipline of Oral & Maxillofacial Surgery, Faculty of Dentistry, the University of Hong Kong, Hong Kong, China
OBJECTIVES: Adenoid cystic carcinoma (ACC) is one of the most common salivary gland cancers. The prognosis of adenoid cystic carcinoma is poor for its high frequency of distant metastases and insensitivity to chemotherapy or molecular therapies. This study investigated the effect of Obatoclax on adenoid cystic carcinoma cells and its cytotoxic mechanism. METHODS: Western blot, transmission electron microscopy, and pEGFP-LC3 plasmids transfection were carried out to detect autophagy in ACC cells treated with Obatoclax. 3-MA and RNA interference against Beclin 1 and ATG5 were used to inhibit autophagy. Then we used Western blot and Hochest 33342 staining for apoptosis assessment. Finally, cell viability was assessed by MTT assay. RESULTS: We found that Obatoclax induced cytoprotective autophagy which depended on ATG5 and partly on Beclin 1 in adenoid cystic carcinoma cells. Furthermore, pharmacologically inhibiting Obatoclax-induced autophagy promoted apoptosis. Downregulation of Beclin 1 or ATG5 attenuated the cytotoxicity of Obatoclax by suppressing both autophagy and apoptosis. Finally, when apoptosis was pharmacologically inhibited, autophagic cell death was initiated in adenoid cystic carcinoma cells treated with Obatoclax. CONCLUSION: In summary, Beclin 1 and ATG5 play important roles in regulating both Obatoclax-induced autophagy and apoptosis in adenoid cystic carcinoma.
Correspondence: Gui-Qing Liao, Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, 56 West Lingyuan Road, Guangdong, Guangzhou 510055, China. Tel: (86) 20 83862531, Fax: (86) 20 83822807, E-mail: [email protected] and Yu-Xiong Su, Division of Oral and Maxillofacial Surgery, Faculty of Dentistry, the University of Hong Kong, 34 Hospital Road, Hong Kong, China. Tel: (852) 28590267, Fax: (852) 28575570, E-mail: richsu@ hku.hk *These authors contributed equally to this work. Received 25 September 2014; revised 10 November 2014; accepted 22 November 2014
Oral Diseases (2014) doi: 10.1111/odi.12305 Keywords: adenoid autophagy; beclin 1
cystic
carcinoma;
apoptosis;
ATG5;
Introduction Adenoid cystic carcinoma accounts for 7.5–10.0% of salivary gland malignancies and about 1% of all head and neck malignancies (Barrett and Speight, 2009). Currently, surgery plus postoperative radiotherapy is the treatment of choice for adenoid cystic carcinoma. However, distant metastases to lung, bone, and soft tissues still occur among approximately 40–60% of patients, which is the major reason for treatment failure (Adams and Cory, 2007). Chemotherapy is essential to prevent and treat distant metastases, but adenoid cystic carcinoma is not sensitive to chemotherapy or molecular therapies (Dodd and Slevin, 2006). Most of these therapies kill tumor cells by inducing apoptosis. However, high expression of anti-apoptosis molecular such as Bcl-2 and Mcl-1 was found in ACC tissues (Norberg-Spaak et al, 2000), which may be a reason for the resistance to chemotherapy. Better understanding of the cell death pathway of ACC is needed. Autophagy is an evolutionarily conserved process in which cytoplasm and cellular organelles are degraded in lysosomes for amino acid and energy recycling (Chen and Karantza-Wadsworth, 2009). Autophagy genes, such as Beclin 1, will be activated and autophagic process will start to produce ATP when the cells are under starvation or other stress conditions. In contrast to survival, excessive autophagy can lead to cell death in a nonapoptotic manner without activating caspases, which is called type II programmed cell death (Ouyang et al, 2012). It is known that Bcl-2 and Beclin 1 complex regulates both autophagy and apoptosis (Marquez and Xu, 2012).
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Obatoclax, also named GX15-070, is a pan-Bcl-2 inhibitor, which disrupts the interaction between Bcl-2 and BH3-only proteins, and activates mitochondrial apoptosis. Moreover, studies found that Obatoclax releases Beclin 1 from the combination with Bcl-2 and induces autophagy or even autophagic cell death (Oltersdorf et al, 2005; Konopleva et al, 2008; Paik et al, 2011). Our previous studies (Liang et al, 2012; Ma et al, 2013) found that high Beclin 1 expression is associated with favorable prognosis in patients with salivary gland ACC, and inhibition of autophagy enhances cisplatin cytotoxicity in ACC cells. But the mechanism of Beclin 1 and Bcl-2 in regulating cell death in ACC cells is still unknown. Therefore, the aim of this study was to investigate the effect of Obatoclax on ACC cells and its cytotoxic mechanism.
Materials and methods Chemicals and antibodies Obatoclax (GX15-070) was purchased from Selleck Chemicals (Houston, TX, USA). 3-Methyladenine (3-MA) was obtained from Sigma-Aldrich (Shanghai, China). Chloroquine was from MP Biomedicals Inc. (Illkirch, France). Antibodies against LC3B and cleaved caspase-3 were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Beclin 1 and anti-GAPDH antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ATG5 antibody was from Abcam (Cambridge, UK). Goat anti-rabbit antibody was purchased from Kangweishiji (Beijing, China). pEGFP-LC3 plasmid (Plasmid 21073) was provided by Addgene Inc. (Cambridge, MA, USA). All experiments were performed at least in triplicate.
Cell lines and culture Human salivary gland adenoid cystic carcinoma cell line ACC-M was obtained from the Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine. The ACC-M cell line was maintained in RPMI1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 U ml 1 penicillin/streptomycin (Gibco) at 37°C under 5% CO2. This study was approved by the Ethics Committees at Guanghua School of Stomatology, Sun Yat-Sen University. Written informed consent was obtained from each subject in this study. To establish ACC-M cell line constitutively expressing green fluorescent protein (GFP)-microtubule-associated protein-1 light chain 3 (LC3) (ACCM/GFP-LC3), pEGFP-LC3 plasmids (4 lg; Addgene, Inc., Cambridge, MA, USA) were transfected into ACC-M using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions. Cells were selected with G418 (800 lg ml 1) (MP Biomedicals Inc.) 48 h after transfection for 2 weeks, then maintained in the presence of G418 (400 lg ml 1). Autophagy was then measured by fluorescence microscopic counting of cells with GFP-LC3 puncta.
RNA interference The siRNA sequences targeting Beclin 1 (5’CAGTTTGGCACAATCAATA3’), ATG5 (5’GGAAUAUCCU GCAGAAGAA3’), and a non-targeting control siRNA (5’GCGGAGAGGCUUAGGUGUA3’) were synthesized. ACC-M cells were transfected with 80 nM siRNA, respectively, using Lipofectamine 2000 as a transfection agent.
Cell viability assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma-Aldrich) was used to detect the number of viable cells. Briefly, after various treatments, 20 ll MTT (5 mg ml 1) was added to each well in 96-well plates and then incubated for 4 h at 37°C. MTTformazan crystals were then dissolved in 150 ll of DMSO per well. Finally, absorbance was read in a microplate reader (Infinite 200, Tecan, Switzerland) at 490 nm and 50% inhibition of cell growth (IC50) was evaluated. Oral Diseases
Apoptosis assessment Nuclear condensation was assessed by staining with Hochest 33342. Cells were seeded in 24-well plates and treated with Obatoclax and/or 3-MA for 24 h. Then nuclear material was stained with 1 lg ml 1 Hochest 33342 at 37°C for 10 min. Cell images were obtained using a Zeiss microscope.
Western blot analysis Western blot analysis was performed as described previously (Liang et al, 2011). The antibody dilutions were as follows: anti-LC3 1:500, anti-Beclin 1 1:500, anti-ATG5 1:1000, anti-cleaved caspase-3 1:250, anti-GAPDH 1:1000.
Transmission electron microscopy Autophagosomes in ACC-M cells were detected by transmission electron microscopy. Cells were trypsinized and fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 4 h at 37°C. Samples were fixed again in 1% osmium tetroxide after washed in cacodylate buffer (4–6 h), then dehydrated in ethanol, embedded in resin, and cut into ultrathin sections. The specimens were examined with a transmission electron microscope (Phillips CM-10: Phillips, Eindhoven, The Netherlands).
Results Obatoclax induced autophagic flux in ACC-M cells When autophagy is activated, LC3-I is lipidated to yield LC3-II, which localizes to autophagosomal membranes. The conversion from LC3-I to LC3-II indicates autophagy. Western blot results showed that Obatoclax induced timedependent and concentration-dependent accumulation of LC3-II in ACC-M cells (Figure 1a). Furthermore, after Obatoclax treatment, GFP-LC3 transfected ACC-M cells displayed punctate green fluorescence (Figure 1b), indicating autophagosome formation, while untreated cells displayed diffuse staining. The average number of GFP-LC3 dots were 0.3, 12.6, 13.1, 17.5 for each cell treated with 0, 0.625, 1.25, 2.5 lM Obatoclax for 2 h; and 1.2, 14.4, 16.2, 26.3 for each cell treated with 5 lM Obatoclax for 0, 0.5, 1, 2, and 4 h. As shown in Figure 1c, increased autophagic structures could be found under transmission electron microscopy in Obatoclax-treated cells compared with control cells. Because the amount of LC3-II at a certain time point does not reflect autophagic flux, delivery of autophagic substrates to lysosomes for degradation should also be measured. We analyzed LC3-II expression in cells treated with chloroquine (CQ), which inhibits the fusion of autophagosomes and lysosomes. As shown in Figure 1d, the accumulation of LC3-II enhanced in the presence of both CQ and Obatoclax compared with Obatoclax alone. This result indicated that Obatoclax activated autophagic flux in ACC-M cells. Obatoclax induced Beclin 1- and ATG5-dependent autophagy As both Beclin 1 and ATG5 are key molecules in the autophagic process, we thus examined the roles of Beclin 1 and ATG5 in Obatoclax-induced autophagy. Western blot revealed the protein level of Beclin 1 increased in Obatoclax-treated ACC-M cells in a time-dependent manner, but only a transient increase of ATG5 (32 kDa) expression was observed at 12 h (Figure 2a). We knocked down Beclin 1 and ATG5, respectively, by siRNA in ACC-M cells (Figure 2b) and found that Obatoclax-induced LC3 lipidation
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The mechanism of cytoprotective effect of Obatoclaxinduced autophagy is complex. Obatoclax can effectively release BAX or BAK from Bcl-2 and Mcl-1, then BAX and BAK travel to the outer membrane of mitochondria and release pro-apoptosis molecules (Konopleva et al, 2008). Autophagy can maintain genomic integrity by removing damaged organelles such as mitochondria, unfolded proteins, and reactive oxygen species (Paglin et al, 2001; Ito et al, 2005; Karantza-Wadsworth et al, 2007; Mathew et al, 2007). Accordingly, inhibition of autophagy in breast, prostate, and colon cancer cells enhanced radiotherapyinduced apoptotic cell death (Paglin et al, 2001; Ito et al, 2005). Furthermore, autophagy provides energy and nutrients for cells survival by degrading cellular organelles and macromolecules. These might be the prosurvival mechanism of Obatoclax-induced autophagy. Interestingly, we found that siRNA against essential autophagy genes (Beclin 1 or ATG5) suppressed Obatoclax-mediated cell death in ACC-M cells. Similar results could be found in acute lymphoblastic leukemia cells Oral Diseases
Figure 4 Obatoclax induced caspaseindependent cell death. (a–b) ACC-M cells transfected with control, Beclin 1-siRNA, or ATG5-siRNA were treated with 5 lM Obatoclax for 48 h. (a) Western blot results suggested that suppressing Beclin 1 or ATG5 expression inhibited Obatoclax-induced caspase-3 cleavage. (b) The MTT results suggested that suppressing Beclin 1 or ATG5 expression decreased the toxicity of Obatoclax in ACC-M cells for 48 h. (c) ACC-M cells were pretreated with 50 lM Z-VAD-fmk for 2 h, and then 5 lM Obatoclax was added in culture for 24 h. Western blot results showed that Z-VAD-fmk promoted Obatoclax-induced LC3 conversion. (d) ACC-M cells transfected with control, Beclin 1, or ATG5-siRNA pretreated with 50 lM Z-VAD-fmk for 2 h were incubated in 5 lM Obatoclax for 48 h. MTT results suggested that suppressing Beclin 1 or ATG5 expression reduced the toxicity of Obatoclax when apoptosis was inhibited. (* P < 0.05).
(Heidari et al, 2010) and non-small-cell lung cancer cells (McCoy et al, 2010). These results seemed to be conflicting with the cytoprotective role of autophagy mentioned above. It could be explained by the dual role of Beclin 1 and ATG5 played in autophagy and apoptosis. Unlike 3MA or CQ, siRNA against Beclin 1 or ATG5 decreased the caspase-3 cleavage in Obatoclax-treated ACC cells, which suggested that knocking down ATG5 or Beclin 1 might reduce the level of apoptosis. ATG5 can be cleaved by calpains, and its N-terminal product translocates to mitochondria, where it interacts with Bcl-XL and promotes cytochrome c release, caspase activation and apoptosis (Eisenberg-Lerner et al, 2009). Beclin 1 can also be cleaved by caspases and the C-terminal can amplify mitochondrial-mediated apoptosis (Wirawan et al, 2010). Accordingly, knockdown of Beclin 1 or ATG5 inhibits not only autophagy but also apoptosis. Finally, inhibiting apoptosis can promote autophagy. Previous study reported that inhibiting apoptosis by Z-VAD-fmk promoted autophagy induced by enterovirus
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Figure 2 Obatoclax induced Beclin 1- and ATG5-dependent autophagy. (a) The protein level of Beclin 1 increased in 5 lM Obatoclaxtreated ACC-M cells in a time-dependent manner. And a transient upregulation of ATG5 was observed when ACC-M cells were treated with Obatoclax for 12 h, then returned to the original level. (b) Western blot results showed that RNA interference effectively suppressed Beclin 1 and ATG5 expression in ACC-M cells at 48 h. (c) Suppressing Beclin 1 expression inhibited LC3 conversion in ACC-M cells which were treated with 5 lM Obatoclax for 12 h, but the conversion recovered at 24 and 48 h. However, knocking down ATG5 expression sustainably inhibited LC3 conversion. (* P < 0.05).
(Figure 4d). This indicated that cytotoxicity of Obatoclax depended on autophagic cell death when apoptosis was inhibited.
Discussion In the present study, we found that Obatoclax induced autophagy, which depended on ATG5 and Beclin 1, played a cytoprotective role in adenoid cystic carcinoma cells. Knockdown of Beclin 1 or ATG5 reduced the cytotoxicity of Obatoclax by suppressing both autophagy and apoptosis. When apoptosis was pharmacologically Oral Diseases
inhibited, Obatoclax induced autophagic cell death in ACC cells. These suggested that Beclin 1 and ATG5 played an important role in regulating Obatoclax-induced autophagy and apoptosis. Preclinical studies have demonstrated the anti-tumor effect of Obatoclax in various hematologic tumor cells including AML, CML, ALL, lymphomas and myelomas (Konopleva et al, 2008), and solid tumors including nonsmall cell lung cancer, prostate, colon, and cervical cancers (Oltersdorf et al, 2005). As a BH3-mimetic, Obatoclax was designed to counteract anti-apoptotic proteins such as Bcl-2. And it has been proved to induce
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Figure 3 Pharmacologically inhibiting Obatoclax-induced autophagy promoted apoptosis. (a) ACC-M cells were pretreated with 5 mM 3-MA for 1 h, then 5 lM Obatoclax was added into the culture for 24 h. 3-MA effectively inhibited the conversion of LC3 induced by Obatoclax. Obatoclax or 3-MA did not induce the cleavage of caspase-3, but 3-MA and Obatoclax significantly promoted caspase-3 cleavage. (b) ACC-M cells stably expressing GFPLC3 displayed punctate green fluorescence after incubating in 5 lM Obatoclax for 2 h. But the same concentration of Obatoclax failed to cause fluorescence aggregation by pretreating with 3-MA (5 mM) for 1 h. RNAi against Beclin 1- or ATG5-suppressed fluorescence aggregation induced by Obatoclax. (9400) (c) MTT assay showed that the cell inhibitory rate was significantly higher when ACC-M cells were coculture with 3-MA and Obatoclax for 48 h than Obatoclax alone. Similar result was found when ACC-M cells were treated with CQ and Obatoclax. (d) Hoechst 33342 staining displayed apoptosis in ACC-M cells. Incubating in Obatoclax or 3-MA for 24 h did not induce significant apoptosis. However, increased karyopyknosis was observed when cells were treated with 3-MA and Obatoclax for 24 h. (9200).
mitochondrial apoptosis depending on Bcl-2 associated x protein/Bcl-2 antagonist killer (BAX/BAK) (McCoy et al, 2010). Consistent with our results, recent studies showed that Obatoclax also induced autophagy (Pan et al, 2010). It is hypothesized that Obatoclax releases Beclin 1 from the binding of Bcl-2 which initiates autophagy (Maiuri et al, 2007). We also found that Obatoclax increased the expression of Beclin 1 and transiently increased ATG5 expression. High expression of these two proteins may contribute to the activation of autophagy. The fact that Obatoclax upregulates Beclin 1 expression was also found in various kinds of tumor cells (Pan et al, 2010). In terms of ATG5, We believe that when autophagy is activated ATG5 turns into ATG5-12 complex (Simonsen and Tooze, 2009). Accordingly, after a transient upregulation, ATG5 expression returns to the original level. Both Beclin 1 and ATG5 play crucial roles in autophagy. Beclin 1, originally discovered as a Bcl-2-interacting protein (Liang et al, 1998), acts as an obligatory activator of class III phosphatidylinositol 3-kinase (PI3K), which initiates autophagic pathway. It has been suggested that releasing Beclin 1 from Bcl-2 may induce a toxic form of autophagy in some cancer cells (Pattingre and Levine, 2006). ATG5 is one of conjugation proteins which take part in the elongation of the autophagosome membrane (Heidari et al, 2010). Moreover, ATG5–Atg12 complex directly regulates LC3 lipidation as a key enzyme
(Simonsen and Tooze, 2009). In our study, Western blot indicated that suppressing ATG5 expression using RNA interference continuously inhibited LC3 conversion. In contrast, suppressing Beclin 1 only decreased LC3 conversion within 12 h. Some studies demonstrated that Obatoclax induced ATG5, but not Beclin 1-dependent autophagy, suggesting a non-canonical pathway of autophagy (Heidari et al, 2010; Grishchuk et al, 2011). Our data suggested that Obatoclax-induced autophagy depended on Beclin 1 at the early stage, and when Beclin 1 was knocked down, Beclin 1 independent pathway might be activated. The relationship between Obatoclax-induced autophagy and apoptosis varies with different cell types, stimulus, and environments. Eisenberg-Lerner et al concluded that autophagy may act as a partner, antagonist, or enabler to apoptosis (Eisenberg-Lerner et al, 2009). In this study, we found that Obatoclax-induced autophagy attenuated apoptosis. Our data indicated that Obatoclax itself did not induce massive apoptosis in ACC-M cells, but increasing level of apoptosis was observed while autophagy was inhibited by 3-MA. Our result was consistent with previous study that Obatoclax induced cytoprotective autophagy in the esophagus cancer and osteosarcoma cancer cell lines (Simonsen and Tooze, 2009). However, Obatoclax killed pancreatic cancer cells via the autophagy pathway (Martin et al, 2009). These inconsistent results suggest that Obatoclax-induced autophagy played complicated roles in different cancer cell types. Oral Diseases
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The mechanism of cytoprotective effect of Obatoclaxinduced autophagy is complex. Obatoclax can effectively release BAX or BAK from Bcl-2 and Mcl-1, then BAX and BAK travel to the outer membrane of mitochondria and release pro-apoptosis molecules (Konopleva et al, 2008). Autophagy can maintain genomic integrity by removing damaged organelles such as mitochondria, unfolded proteins, and reactive oxygen species (Paglin et al, 2001; Ito et al, 2005; Karantza-Wadsworth et al, 2007; Mathew et al, 2007). Accordingly, inhibition of autophagy in breast, prostate, and colon cancer cells enhanced radiotherapyinduced apoptotic cell death (Paglin et al, 2001; Ito et al, 2005). Furthermore, autophagy provides energy and nutrients for cells survival by degrading cellular organelles and macromolecules. These might be the prosurvival mechanism of Obatoclax-induced autophagy. Interestingly, we found that siRNA against essential autophagy genes (Beclin 1 or ATG5) suppressed Obatoclax-mediated cell death in ACC-M cells. Similar results could be found in acute lymphoblastic leukemia cells Oral Diseases
Figure 4 Obatoclax induced caspaseindependent cell death. (a–b) ACC-M cells transfected with control, Beclin 1-siRNA, or ATG5-siRNA were treated with 5 lM Obatoclax for 48 h. (a) Western blot results suggested that suppressing Beclin 1 or ATG5 expression inhibited Obatoclax-induced caspase-3 cleavage. (b) The MTT results suggested that suppressing Beclin 1 or ATG5 expression decreased the toxicity of Obatoclax in ACC-M cells for 48 h. (c) ACC-M cells were pretreated with 50 lM Z-VAD-fmk for 2 h, and then 5 lM Obatoclax was added in culture for 24 h. Western blot results showed that Z-VAD-fmk promoted Obatoclax-induced LC3 conversion. (d) ACC-M cells transfected with control, Beclin 1, or ATG5-siRNA pretreated with 50 lM Z-VAD-fmk for 2 h were incubated in 5 lM Obatoclax for 48 h. MTT results suggested that suppressing Beclin 1 or ATG5 expression reduced the toxicity of Obatoclax when apoptosis was inhibited. (* P < 0.05).
(Heidari et al, 2010) and non-small-cell lung cancer cells (McCoy et al, 2010). These results seemed to be conflicting with the cytoprotective role of autophagy mentioned above. It could be explained by the dual role of Beclin 1 and ATG5 played in autophagy and apoptosis. Unlike 3MA or CQ, siRNA against Beclin 1 or ATG5 decreased the caspase-3 cleavage in Obatoclax-treated ACC cells, which suggested that knocking down ATG5 or Beclin 1 might reduce the level of apoptosis. ATG5 can be cleaved by calpains, and its N-terminal product translocates to mitochondria, where it interacts with Bcl-XL and promotes cytochrome c release, caspase activation and apoptosis (Eisenberg-Lerner et al, 2009). Beclin 1 can also be cleaved by caspases and the C-terminal can amplify mitochondrial-mediated apoptosis (Wirawan et al, 2010). Accordingly, knockdown of Beclin 1 or ATG5 inhibits not only autophagy but also apoptosis. Finally, inhibiting apoptosis can promote autophagy. Previous study reported that inhibiting apoptosis by Z-VAD-fmk promoted autophagy induced by enterovirus
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infection in human rhabdomyosarcoma (RD-A) cells (Xi et al, 2013). Furthermore, their data indicated that downregulating ATG5 inhibited apoptosis through caspase-3, reversely when caspase-3 was inhibited autophagy was promoted by activating ATG5. In the present study, when apoptosis was inhibited by Z-VAD-fmk, Obatoclaxinduced autophagy was potentiated. More interestingly, downregulation of Beclin 1 or ATG5 still saved cells from Obatoclax killing when apoptosis was inhibited. This result suggested that cytotoxicity of Obatoclax depended on autophagic cell death when apoptosis was inhibited. But how could autophagy be cytoprotective and cytotoxic in one cell line? Recent study showed that Obatoclaxinduced autophagy killed rituximab-resistant lymphoma cell line with low to absent levels of BAX/BAK. However, Obatoclax did not induce cytotoxic autophagy in the same cell line with high BAX/BAK expression (Heidari et al, 2010), which indicated that Obatoclax-mediated autophagy may play different roles in cells depending on their apoptotic threshold. Z-VAD-fmk increases the apoptotic threshold of ACC-M cells and leads to a toxic autophagy. Further studies on regulating pathways of apoptosis and autophagy will be needed for a better understanding of the underlying mechanisms. Certain limitations in this study should be taken into account. This study was performed based on one ACC cell line and may not reflect processes in the intact body. We are going to confirm our results in other cell lines and mice. In conclusion, Obatoclax induced cytoprotective autophagy, which was Beclin 1 and ATG5 dependent, in ACC-M cells. When autophagy was inhibited, apoptotic cell death was promoted. And while apoptosis was inhibited, autophagy may lead to cell death. Beclin 1 and ATG5 played important roles in regulating Obatoclaxinduced autophagy and apoptosis. Acknowledgements The project was supported by grants from National Natural Science Foundation of China (No. 81371160, 81172566, 81302367) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2013-1792).
Conflict of interest statement The authors declare that there is no conflict of interest.
Author contributions LZ Liang drafted manuscript and analyzed data. B Ma, YJ Liang, and HC Liu analyzed data. TH Zhang and GS Zheng provided technical support. YX Su and GQ Liao designed study and revised the article.
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ORIGINAL ARTICLE
Obatoclax induces Beclin 1- and ATG5-dependent apoptosis and autophagy in adenoid cystic carcinoma cells L-Z Liang1, B Ma2, Y-J Liang3, H-C Liu3, T-H Zhang4, G-S Zheng3, Y-X Su3,5,*, G-Q Liao3,* 1
Department of Oral and Maxillofacial Surgery, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai; 2Department of Stomatology, Shanxi Academy of Medical Sciences, Shanxi Dayi Hospital, Taiyuan; 3Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-Sen University, Guangzhou; 4Department of Stomatology, Affiliated Zhongshan Hospital, Sun Yat-sen University, Zhongshan; 5Discipline of Oral & Maxillofacial Surgery, Faculty of Dentistry, the University of Hong Kong, Hong Kong, China
OBJECTIVES: Adenoid cystic carcinoma (ACC) is one of the most common salivary gland cancers. The prognosis of adenoid cystic carcinoma is poor for its high frequency of distant metastases and insensitivity to chemotherapy or molecular therapies. This study investigated the effect of Obatoclax on adenoid cystic carcinoma cells and its cytotoxic mechanism. METHODS: Western blot, transmission electron microscopy, and pEGFP-LC3 plasmids transfection were carried out to detect autophagy in ACC cells treated with Obatoclax. 3-MA and RNA interference against Beclin 1 and ATG5 were used to inhibit autophagy. Then we used Western blot and Hochest 33342 staining for apoptosis assessment. Finally, cell viability was assessed by MTT assay. RESULTS: We found that Obatoclax induced cytoprotective autophagy which depended on ATG5 and partly on Beclin 1 in adenoid cystic carcinoma cells. Furthermore, pharmacologically inhibiting Obatoclax-induced autophagy promoted apoptosis. Downregulation of Beclin 1 or ATG5 attenuated the cytotoxicity of Obatoclax by suppressing both autophagy and apoptosis. Finally, when apoptosis was pharmacologically inhibited, autophagic cell death was initiated in adenoid cystic carcinoma cells treated with Obatoclax. CONCLUSION: In summary, Beclin 1 and ATG5 play important roles in regulating both Obatoclax-induced autophagy and apoptosis in adenoid cystic carcinoma.
Correspondence: Gui-Qing Liao, Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, 56 West Lingyuan Road, Guangdong, Guangzhou 510055, China. Tel: (86) 20 83862531, Fax: (86) 20 83822807, E-mail: [email protected] and Yu-Xiong Su, Division of Oral and Maxillofacial Surgery, Faculty of Dentistry, the University of Hong Kong, 34 Hospital Road, Hong Kong, China. Tel: (852) 28590267, Fax: (852) 28575570, E-mail: richsu@ hku.hk *These authors contributed equally to this work. Received 25 September 2014; revised 10 November 2014; accepted 22 November 2014
Oral Diseases (2014) doi: 10.1111/odi.12305 Keywords: adenoid autophagy; beclin 1
cystic
carcinoma;
apoptosis;
ATG5;
Introduction Adenoid cystic carcinoma accounts for 7.5–10.0% of salivary gland malignancies and about 1% of all head and neck malignancies (Barrett and Speight, 2009). Currently, surgery plus postoperative radiotherapy is the treatment of choice for adenoid cystic carcinoma. However, distant metastases to lung, bone, and soft tissues still occur among approximately 40–60% of patients, which is the major reason for treatment failure (Adams and Cory, 2007). Chemotherapy is essential to prevent and treat distant metastases, but adenoid cystic carcinoma is not sensitive to chemotherapy or molecular therapies (Dodd and Slevin, 2006). Most of these therapies kill tumor cells by inducing apoptosis. However, high expression of anti-apoptosis molecular such as Bcl-2 and Mcl-1 was found in ACC tissues (Norberg-Spaak et al, 2000), which may be a reason for the resistance to chemotherapy. Better understanding of the cell death pathway of ACC is needed. Autophagy is an evolutionarily conserved process in which cytoplasm and cellular organelles are degraded in lysosomes for amino acid and energy recycling (Chen and Karantza-Wadsworth, 2009). Autophagy genes, such as Beclin 1, will be activated and autophagic process will start to produce ATP when the cells are under starvation or other stress conditions. In contrast to survival, excessive autophagy can lead to cell death in a nonapoptotic manner without activating caspases, which is called type II programmed cell death (Ouyang et al, 2012). It is known that Bcl-2 and Beclin 1 complex regulates both autophagy and apoptosis (Marquez and Xu, 2012).
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Obatoclax, also named GX15-070, is a pan-Bcl-2 inhibitor, which disrupts the interaction between Bcl-2 and BH3-only proteins, and activates mitochondrial apoptosis. Moreover, studies found that Obatoclax releases Beclin 1 from the combination with Bcl-2 and induces autophagy or even autophagic cell death (Oltersdorf et al, 2005; Konopleva et al, 2008; Paik et al, 2011). Our previous studies (Liang et al, 2012; Ma et al, 2013) found that high Beclin 1 expression is associated with favorable prognosis in patients with salivary gland ACC, and inhibition of autophagy enhances cisplatin cytotoxicity in ACC cells. But the mechanism of Beclin 1 and Bcl-2 in regulating cell death in ACC cells is still unknown. Therefore, the aim of this study was to investigate the effect of Obatoclax on ACC cells and its cytotoxic mechanism.
Materials and methods Chemicals and antibodies Obatoclax (GX15-070) was purchased from Selleck Chemicals (Houston, TX, USA). 3-Methyladenine (3-MA) was obtained from Sigma-Aldrich (Shanghai, China). Chloroquine was from MP Biomedicals Inc. (Illkirch, France). Antibodies against LC3B and cleaved caspase-3 were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Beclin 1 and anti-GAPDH antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ATG5 antibody was from Abcam (Cambridge, UK). Goat anti-rabbit antibody was purchased from Kangweishiji (Beijing, China). pEGFP-LC3 plasmid (Plasmid 21073) was provided by Addgene Inc. (Cambridge, MA, USA). All experiments were performed at least in triplicate.
Cell lines and culture Human salivary gland adenoid cystic carcinoma cell line ACC-M was obtained from the Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine. The ACC-M cell line was maintained in RPMI1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 U ml 1 penicillin/streptomycin (Gibco) at 37°C under 5% CO2. This study was approved by the Ethics Committees at Guanghua School of Stomatology, Sun Yat-Sen University. Written informed consent was obtained from each subject in this study. To establish ACC-M cell line constitutively expressing green fluorescent protein (GFP)-microtubule-associated protein-1 light chain 3 (LC3) (ACCM/GFP-LC3), pEGFP-LC3 plasmids (4 lg; Addgene, Inc., Cambridge, MA, USA) were transfected into ACC-M using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions. Cells were selected with G418 (800 lg ml 1) (MP Biomedicals Inc.) 48 h after transfection for 2 weeks, then maintained in the presence of G418 (400 lg ml 1). Autophagy was then measured by fluorescence microscopic counting of cells with GFP-LC3 puncta.
RNA interference The siRNA sequences targeting Beclin 1 (5’CAGTTTGGCACAATCAATA3’), ATG5 (5’GGAAUAUCCU GCAGAAGAA3’), and a non-targeting control siRNA (5’GCGGAGAGGCUUAGGUGUA3’) were synthesized. ACC-M cells were transfected with 80 nM siRNA, respectively, using Lipofectamine 2000 as a transfection agent.
Cell viability assay MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma-Aldrich) was used to detect the number of viable cells. Briefly, after various treatments, 20 ll MTT (5 mg ml 1) was added to each well in 96-well plates and then incubated for 4 h at 37°C. MTTformazan crystals were then dissolved in 150 ll of DMSO per well. Finally, absorbance was read in a microplate reader (Infinite 200, Tecan, Switzerland) at 490 nm and 50% inhibition of cell growth (IC50) was evaluated. Oral Diseases
Apoptosis assessment Nuclear condensation was assessed by staining with Hochest 33342. Cells were seeded in 24-well plates and treated with Obatoclax and/or 3-MA for 24 h. Then nuclear material was stained with 1 lg ml 1 Hochest 33342 at 37°C for 10 min. Cell images were obtained using a Zeiss microscope.
Western blot analysis Western blot analysis was performed as described previously (Liang et al, 2011). The antibody dilutions were as follows: anti-LC3 1:500, anti-Beclin 1 1:500, anti-ATG5 1:1000, anti-cleaved caspase-3 1:250, anti-GAPDH 1:1000.
Transmission electron microscopy Autophagosomes in ACC-M cells were detected by transmission electron microscopy. Cells were trypsinized and fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 4 h at 37°C. Samples were fixed again in 1% osmium tetroxide after washed in cacodylate buffer (4–6 h), then dehydrated in ethanol, embedded in resin, and cut into ultrathin sections. The specimens were examined with a transmission electron microscope (Phillips CM-10: Phillips, Eindhoven, The Netherlands).
Results Obatoclax induced autophagic flux in ACC-M cells When autophagy is activated, LC3-I is lipidated to yield LC3-II, which localizes to autophagosomal membranes. The conversion from LC3-I to LC3-II indicates autophagy. Western blot results showed that Obatoclax induced timedependent and concentration-dependent accumulation of LC3-II in ACC-M cells (Figure 1a). Furthermore, after Obatoclax treatment, GFP-LC3 transfected ACC-M cells displayed punctate green fluorescence (Figure 1b), indicating autophagosome formation, while untreated cells displayed diffuse staining. The average number of GFP-LC3 dots were 0.3, 12.6, 13.1, 17.5 for each cell treated with 0, 0.625, 1.25, 2.5 lM Obatoclax for 2 h; and 1.2, 14.4, 16.2, 26.3 for each cell treated with 5 lM Obatoclax for 0, 0.5, 1, 2, and 4 h. As shown in Figure 1c, increased autophagic structures could be found under transmission electron microscopy in Obatoclax-treated cells compared with control cells. Because the amount of LC3-II at a certain time point does not reflect autophagic flux, delivery of autophagic substrates to lysosomes for degradation should also be measured. We analyzed LC3-II expression in cells treated with chloroquine (CQ), which inhibits the fusion of autophagosomes and lysosomes. As shown in Figure 1d, the accumulation of LC3-II enhanced in the presence of both CQ and Obatoclax compared with Obatoclax alone. This result indicated that Obatoclax activated autophagic flux in ACC-M cells. Obatoclax induced Beclin 1- and ATG5-dependent autophagy As both Beclin 1 and ATG5 are key molecules in the autophagic process, we thus examined the roles of Beclin 1 and ATG5 in Obatoclax-induced autophagy. Western blot revealed the protein level of Beclin 1 increased in Obatoclax-treated ACC-M cells in a time-dependent manner, but only a transient increase of ATG5 (32 kDa) expression was observed at 12 h (Figure 2a). We knocked down Beclin 1 and ATG5, respectively, by siRNA in ACC-M cells (Figure 2b) and found that Obatoclax-induced LC3 lipidation
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The mechanism of cytoprotective effect of Obatoclaxinduced autophagy is complex. Obatoclax can effectively release BAX or BAK from Bcl-2 and Mcl-1, then BAX and BAK travel to the outer membrane of mitochondria and release pro-apoptosis molecules (Konopleva et al, 2008). Autophagy can maintain genomic integrity by removing damaged organelles such as mitochondria, unfolded proteins, and reactive oxygen species (Paglin et al, 2001; Ito et al, 2005; Karantza-Wadsworth et al, 2007; Mathew et al, 2007). Accordingly, inhibition of autophagy in breast, prostate, and colon cancer cells enhanced radiotherapyinduced apoptotic cell death (Paglin et al, 2001; Ito et al, 2005). Furthermore, autophagy provides energy and nutrients for cells survival by degrading cellular organelles and macromolecules. These might be the prosurvival mechanism of Obatoclax-induced autophagy. Interestingly, we found that siRNA against essential autophagy genes (Beclin 1 or ATG5) suppressed Obatoclax-mediated cell death in ACC-M cells. Similar results could be found in acute lymphoblastic leukemia cells Oral Diseases
Figure 4 Obatoclax induced caspaseindependent cell death. (a–b) ACC-M cells transfected with control, Beclin 1-siRNA, or ATG5-siRNA were treated with 5 lM Obatoclax for 48 h. (a) Western blot results suggested that suppressing Beclin 1 or ATG5 expression inhibited Obatoclax-induced caspase-3 cleavage. (b) The MTT results suggested that suppressing Beclin 1 or ATG5 expression decreased the toxicity of Obatoclax in ACC-M cells for 48 h. (c) ACC-M cells were pretreated with 50 lM Z-VAD-fmk for 2 h, and then 5 lM Obatoclax was added in culture for 24 h. Western blot results showed that Z-VAD-fmk promoted Obatoclax-induced LC3 conversion. (d) ACC-M cells transfected with control, Beclin 1, or ATG5-siRNA pretreated with 50 lM Z-VAD-fmk for 2 h were incubated in 5 lM Obatoclax for 48 h. MTT results suggested that suppressing Beclin 1 or ATG5 expression reduced the toxicity of Obatoclax when apoptosis was inhibited. (* P < 0.05).
(Heidari et al, 2010) and non-small-cell lung cancer cells (McCoy et al, 2010). These results seemed to be conflicting with the cytoprotective role of autophagy mentioned above. It could be explained by the dual role of Beclin 1 and ATG5 played in autophagy and apoptosis. Unlike 3MA or CQ, siRNA against Beclin 1 or ATG5 decreased the caspase-3 cleavage in Obatoclax-treated ACC cells, which suggested that knocking down ATG5 or Beclin 1 might reduce the level of apoptosis. ATG5 can be cleaved by calpains, and its N-terminal product translocates to mitochondria, where it interacts with Bcl-XL and promotes cytochrome c release, caspase activation and apoptosis (Eisenberg-Lerner et al, 2009). Beclin 1 can also be cleaved by caspases and the C-terminal can amplify mitochondrial-mediated apoptosis (Wirawan et al, 2010). Accordingly, knockdown of Beclin 1 or ATG5 inhibits not only autophagy but also apoptosis. Finally, inhibiting apoptosis can promote autophagy. Previous study reported that inhibiting apoptosis by Z-VAD-fmk promoted autophagy induced by enterovirus
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Figure 2 Obatoclax induced Beclin 1- and ATG5-dependent autophagy. (a) The protein level of Beclin 1 increased in 5 lM Obatoclaxtreated ACC-M cells in a time-dependent manner. And a transient upregulation of ATG5 was observed when ACC-M cells were treated with Obatoclax for 12 h, then returned to the original level. (b) Western blot results showed that RNA interference effectively suppressed Beclin 1 and ATG5 expression in ACC-M cells at 48 h. (c) Suppressing Beclin 1 expression inhibited LC3 conversion in ACC-M cells which were treated with 5 lM Obatoclax for 12 h, but the conversion recovered at 24 and 48 h. However, knocking down ATG5 expression sustainably inhibited LC3 conversion. (* P < 0.05).
(Figure 4d). This indicated that cytotoxicity of Obatoclax depended on autophagic cell death when apoptosis was inhibited.
Discussion In the present study, we found that Obatoclax induced autophagy, which depended on ATG5 and Beclin 1, played a cytoprotective role in adenoid cystic carcinoma cells. Knockdown of Beclin 1 or ATG5 reduced the cytotoxicity of Obatoclax by suppressing both autophagy and apoptosis. When apoptosis was pharmacologically Oral Diseases
inhibited, Obatoclax induced autophagic cell death in ACC cells. These suggested that Beclin 1 and ATG5 played an important role in regulating Obatoclax-induced autophagy and apoptosis. Preclinical studies have demonstrated the anti-tumor effect of Obatoclax in various hematologic tumor cells including AML, CML, ALL, lymphomas and myelomas (Konopleva et al, 2008), and solid tumors including nonsmall cell lung cancer, prostate, colon, and cervical cancers (Oltersdorf et al, 2005). As a BH3-mimetic, Obatoclax was designed to counteract anti-apoptotic proteins such as Bcl-2. And it has been proved to induce
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Figure 3 Pharmacologically inhibiting Obatoclax-induced autophagy promoted apoptosis. (a) ACC-M cells were pretreated with 5 mM 3-MA for 1 h, then 5 lM Obatoclax was added into the culture for 24 h. 3-MA effectively inhibited the conversion of LC3 induced by Obatoclax. Obatoclax or 3-MA did not induce the cleavage of caspase-3, but 3-MA and Obatoclax significantly promoted caspase-3 cleavage. (b) ACC-M cells stably expressing GFPLC3 displayed punctate green fluorescence after incubating in 5 lM Obatoclax for 2 h. But the same concentration of Obatoclax failed to cause fluorescence aggregation by pretreating with 3-MA (5 mM) for 1 h. RNAi against Beclin 1- or ATG5-suppressed fluorescence aggregation induced by Obatoclax. (9400) (c) MTT assay showed that the cell inhibitory rate was significantly higher when ACC-M cells were coculture with 3-MA and Obatoclax for 48 h than Obatoclax alone. Similar result was found when ACC-M cells were treated with CQ and Obatoclax. (d) Hoechst 33342 staining displayed apoptosis in ACC-M cells. Incubating in Obatoclax or 3-MA for 24 h did not induce significant apoptosis. However, increased karyopyknosis was observed when cells were treated with 3-MA and Obatoclax for 24 h. (9200).
mitochondrial apoptosis depending on Bcl-2 associated x protein/Bcl-2 antagonist killer (BAX/BAK) (McCoy et al, 2010). Consistent with our results, recent studies showed that Obatoclax also induced autophagy (Pan et al, 2010). It is hypothesized that Obatoclax releases Beclin 1 from the binding of Bcl-2 which initiates autophagy (Maiuri et al, 2007). We also found that Obatoclax increased the expression of Beclin 1 and transiently increased ATG5 expression. High expression of these two proteins may contribute to the activation of autophagy. The fact that Obatoclax upregulates Beclin 1 expression was also found in various kinds of tumor cells (Pan et al, 2010). In terms of ATG5, We believe that when autophagy is activated ATG5 turns into ATG5-12 complex (Simonsen and Tooze, 2009). Accordingly, after a transient upregulation, ATG5 expression returns to the original level. Both Beclin 1 and ATG5 play crucial roles in autophagy. Beclin 1, originally discovered as a Bcl-2-interacting protein (Liang et al, 1998), acts as an obligatory activator of class III phosphatidylinositol 3-kinase (PI3K), which initiates autophagic pathway. It has been suggested that releasing Beclin 1 from Bcl-2 may induce a toxic form of autophagy in some cancer cells (Pattingre and Levine, 2006). ATG5 is one of conjugation proteins which take part in the elongation of the autophagosome membrane (Heidari et al, 2010). Moreover, ATG5–Atg12 complex directly regulates LC3 lipidation as a key enzyme
(Simonsen and Tooze, 2009). In our study, Western blot indicated that suppressing ATG5 expression using RNA interference continuously inhibited LC3 conversion. In contrast, suppressing Beclin 1 only decreased LC3 conversion within 12 h. Some studies demonstrated that Obatoclax induced ATG5, but not Beclin 1-dependent autophagy, suggesting a non-canonical pathway of autophagy (Heidari et al, 2010; Grishchuk et al, 2011). Our data suggested that Obatoclax-induced autophagy depended on Beclin 1 at the early stage, and when Beclin 1 was knocked down, Beclin 1 independent pathway might be activated. The relationship between Obatoclax-induced autophagy and apoptosis varies with different cell types, stimulus, and environments. Eisenberg-Lerner et al concluded that autophagy may act as a partner, antagonist, or enabler to apoptosis (Eisenberg-Lerner et al, 2009). In this study, we found that Obatoclax-induced autophagy attenuated apoptosis. Our data indicated that Obatoclax itself did not induce massive apoptosis in ACC-M cells, but increasing level of apoptosis was observed while autophagy was inhibited by 3-MA. Our result was consistent with previous study that Obatoclax induced cytoprotective autophagy in the esophagus cancer and osteosarcoma cancer cell lines (Simonsen and Tooze, 2009). However, Obatoclax killed pancreatic cancer cells via the autophagy pathway (Martin et al, 2009). These inconsistent results suggest that Obatoclax-induced autophagy played complicated roles in different cancer cell types. Oral Diseases
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The mechanism of cytoprotective effect of Obatoclaxinduced autophagy is complex. Obatoclax can effectively release BAX or BAK from Bcl-2 and Mcl-1, then BAX and BAK travel to the outer membrane of mitochondria and release pro-apoptosis molecules (Konopleva et al, 2008). Autophagy can maintain genomic integrity by removing damaged organelles such as mitochondria, unfolded proteins, and reactive oxygen species (Paglin et al, 2001; Ito et al, 2005; Karantza-Wadsworth et al, 2007; Mathew et al, 2007). Accordingly, inhibition of autophagy in breast, prostate, and colon cancer cells enhanced radiotherapyinduced apoptotic cell death (Paglin et al, 2001; Ito et al, 2005). Furthermore, autophagy provides energy and nutrients for cells survival by degrading cellular organelles and macromolecules. These might be the prosurvival mechanism of Obatoclax-induced autophagy. Interestingly, we found that siRNA against essential autophagy genes (Beclin 1 or ATG5) suppressed Obatoclax-mediated cell death in ACC-M cells. Similar results could be found in acute lymphoblastic leukemia cells Oral Diseases
Figure 4 Obatoclax induced caspaseindependent cell death. (a–b) ACC-M cells transfected with control, Beclin 1-siRNA, or ATG5-siRNA were treated with 5 lM Obatoclax for 48 h. (a) Western blot results suggested that suppressing Beclin 1 or ATG5 expression inhibited Obatoclax-induced caspase-3 cleavage. (b) The MTT results suggested that suppressing Beclin 1 or ATG5 expression decreased the toxicity of Obatoclax in ACC-M cells for 48 h. (c) ACC-M cells were pretreated with 50 lM Z-VAD-fmk for 2 h, and then 5 lM Obatoclax was added in culture for 24 h. Western blot results showed that Z-VAD-fmk promoted Obatoclax-induced LC3 conversion. (d) ACC-M cells transfected with control, Beclin 1, or ATG5-siRNA pretreated with 50 lM Z-VAD-fmk for 2 h were incubated in 5 lM Obatoclax for 48 h. MTT results suggested that suppressing Beclin 1 or ATG5 expression reduced the toxicity of Obatoclax when apoptosis was inhibited. (* P < 0.05).
(Heidari et al, 2010) and non-small-cell lung cancer cells (McCoy et al, 2010). These results seemed to be conflicting with the cytoprotective role of autophagy mentioned above. It could be explained by the dual role of Beclin 1 and ATG5 played in autophagy and apoptosis. Unlike 3MA or CQ, siRNA against Beclin 1 or ATG5 decreased the caspase-3 cleavage in Obatoclax-treated ACC cells, which suggested that knocking down ATG5 or Beclin 1 might reduce the level of apoptosis. ATG5 can be cleaved by calpains, and its N-terminal product translocates to mitochondria, where it interacts with Bcl-XL and promotes cytochrome c release, caspase activation and apoptosis (Eisenberg-Lerner et al, 2009). Beclin 1 can also be cleaved by caspases and the C-terminal can amplify mitochondrial-mediated apoptosis (Wirawan et al, 2010). Accordingly, knockdown of Beclin 1 or ATG5 inhibits not only autophagy but also apoptosis. Finally, inhibiting apoptosis can promote autophagy. Previous study reported that inhibiting apoptosis by Z-VAD-fmk promoted autophagy induced by enterovirus
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infection in human rhabdomyosarcoma (RD-A) cells (Xi et al, 2013). Furthermore, their data indicated that downregulating ATG5 inhibited apoptosis through caspase-3, reversely when caspase-3 was inhibited autophagy was promoted by activating ATG5. In the present study, when apoptosis was inhibited by Z-VAD-fmk, Obatoclaxinduced autophagy was potentiated. More interestingly, downregulation of Beclin 1 or ATG5 still saved cells from Obatoclax killing when apoptosis was inhibited. This result suggested that cytotoxicity of Obatoclax depended on autophagic cell death when apoptosis was inhibited. But how could autophagy be cytoprotective and cytotoxic in one cell line? Recent study showed that Obatoclaxinduced autophagy killed rituximab-resistant lymphoma cell line with low to absent levels of BAX/BAK. However, Obatoclax did not induce cytotoxic autophagy in the same cell line with high BAX/BAK expression (Heidari et al, 2010), which indicated that Obatoclax-mediated autophagy may play different roles in cells depending on their apoptotic threshold. Z-VAD-fmk increases the apoptotic threshold of ACC-M cells and leads to a toxic autophagy. Further studies on regulating pathways of apoptosis and autophagy will be needed for a better understanding of the underlying mechanisms. Certain limitations in this study should be taken into account. This study was performed based on one ACC cell line and may not reflect processes in the intact body. We are going to confirm our results in other cell lines and mice. In conclusion, Obatoclax induced cytoprotective autophagy, which was Beclin 1 and ATG5 dependent, in ACC-M cells. When autophagy was inhibited, apoptotic cell death was promoted. And while apoptosis was inhibited, autophagy may lead to cell death. Beclin 1 and ATG5 played important roles in regulating Obatoclaxinduced autophagy and apoptosis. Acknowledgements The project was supported by grants from National Natural Science Foundation of China (No. 81371160, 81172566, 81302367) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2013-1792).
Conflict of interest statement The authors declare that there is no conflict of interest.
Author contributions LZ Liang drafted manuscript and analyzed data. B Ma, YJ Liang, and HC Liu analyzed data. TH Zhang and GS Zheng provided technical support. YX Su and GQ Liao designed study and revised the article.
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