Human Melanoma Cells under Endoplasmic Reticulum Stress Are More Susceptible to Apoptosis Induced by the BH3 Mimetic Obatoclax 1

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1 Volume 11 Number 9 September 2009 pp Human Melanoma Cells under Endoplasmic Reticulum Stress Are More Susceptible to Apoptosis Induced by the BH3 Mimetic Obatoclax 1 Chen Chen Jiang 2, David Wroblewski 2, Fan Yang, Peter Hersey and Xu Dong Zhang Immunology and Oncology Unit, Room 443, Calvary Mater Newcastle Hospital, NSW, Australia Abstract Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma. Neoplasia (2009) 11, Introduction Melanoma continues to increase in incidence in many parts of the world, but there is currently no curative treatment once the disease has spread beyond the primary site because of the absence of effective systemic therapies. This is believed to be largely due to the resistance of melanoma cells to induction of apoptosis by available chemotherapeutic drugs and biologic reagents [1,2]. Deregulated expression of Bcl-2 family proteins, either as consequences of genetic alterations or resulting from environmental stimulations, is known to play a central role in the resistance of melanoma to apoptosis [1,2]. A number of cellular stress conditions, such as nutrient deprivation, hypoxia, alterations in glycosylation status, and disturbances of calcium flux, lead to accumulation and aggregation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) lumen and cause so-called ER stress [3 5]. The ER responds to stress conditions by activating a range of stress-response signaling pathways, which is referred to as the unfolded protein response (UPR) [3 5]. The UPR is fundamentally a cytoprotective response, but excessive or prolonged activation of the UPR can result in apoptosis. This involves many of the same molecules that have important roles in other apoptotic cascades [6 9]. Among Abbreviations: ER, endoplasmic reticulum; UPR, unfolded protein response; MAb, monoclonal antibody; Ab, antibody; TM, tunicamycin; TG, thapsigargin; ΔΨ m,mitochondrial membrane potential; sirna, small RNA interference Address all correspondence to: Dr. Xu Dong Zhang or Dr. Peter Hersey, Room 443, David Maddison Clinical Sciences Bldg, corner King and Watt streets, Newcastle, NSW 2300, Australia. Xu.Zhang@newcastle.edu.au, Peter.Hersey@newcastle.edu.au 1 This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council, Australia. X.D. Zhang is a Cancer Institute NSW Fellow. 2 These authors contributed equally to this work. Received 27 April 2009; Revised 24 May 2009; Accepted 26 May 2009 Copyright 2009 Neoplasia Press, Inc. All rights reserved /09/$25.00 DOI /neo.09692

2 946 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. Neoplasia Vol. 11, No. 9, 2009 them, Bcl-2 family proteins seem to be critical because ER stress induced apoptosis can be inhibited by the overexpression of Bcl-2 or its antiapoptotic homologs, suggesting that activation of proapoptotic proteins of this family is important in initiating ER stress mediated apoptotic signaling [7,8]. In support of this, Bax- and Bak-deficient cells are resistant to ER stress induced apoptosis [7]. A number of BH3-only proteins, including PUMA, Noxa, Bim, and BIK, have been shown to be upregulated/activated [10 15], whereas Bcl-2 and Mcl-1 have been reported to be downregulated [16,17], thus contributing to the induction of apoptosis by ER stress in various types of cells. Past studies have shown that melanoma cells have largely adapted to ER stress and are relatively resistant to ER stress induced apoptosis [18 20]. In contrast to down-regulation of Bcl-2 and Mcl-1 by ER stress in many other cell types [7,15], these antiapoptotic proteins are upregulated by the UPR in melanoma cells on ER stress [19]. Upregulation of Mcl-1 is of particular importance, in that it plays an important role in antagonizing PUMA and Noxa, which are also increased in melanoma cell cells submitted to ER stress [19]. Conversely, Bcl-2 does not seem to be essential in protection of melanoma cells against ER stress induced apoptosis because it cannot rescue melanoma cells deficient in Mcl-1 from apoptosis induced by ER stress [19]. Upregulation of Mcl-1 therefore seems to be an important adaptive mechanism for melanoma cells to survive ER stress conditions. Small-molecule mimics of BH3-only proteins are emerging as promising anticancer agents by inhibiting antiapoptotic Bcl-2 family proteins [21 25]. Obatoclax, also known as GX (Gemin X Biotechnologies, Inc, Montreal, Quebec, Canada), was identified by chemical library screening to bind the hydrophobic groove of antiapoptotic Bcl-2 family proteins and antagonize their function [23,25]. In contrast to another BH3 mimetic, ABT737 (Abbott Laboratories, Abbott Park, IL), which disables Bcl-2 and Bcl-X L but does not inhibit Mcl-1 [21,22], obatoclax can efficiently neutralize Mcl-1 [23,25]. It has been reported that obatoclax can potently kill some types of cancer cells, such as myeloma cells, as a single agent [26], and enhance apoptosis induced by various apoptotic stimuli such as tumor necrosis factor related apoptosis inducing ligand and the proteasome inhibitor bortezomib [23,24]. We have examined the apoptosis-inducing potential of obatoclax in melanoma cells submitted to acute ER stress. In this report, we show that although obatoclax at nanomolar concentrations is relatively ineffective in induction of apoptosis in melanoma cells, it potently overcomes resistance of melanoma cells to apoptosis induced by ER stress, and this is primarily due to the inhibition of Mcl-1. We demonstrate that obatoclax triggers further increases in the levels of Noxa in melanoma cells undergoing acute ER stress, which in turn cooperates with obatoclax to release Bak from Mcl-1, thus activating the mitochondrial apoptotic pathway. These results identify obatoclax as a potent agent that targets a major adaptive mechanism to ER stress in melanoma and suggest that combinations of obatoclax and agents that induce ER stress may be a useful strategy in the treatment of melanoma. Materials and Methods Cell Lines Human melanoma cell lines Mel-RM, MM200, IgR3, Mel-CV, Sk-Mel-28, and ME4405 have been described previously [19,27]. They were cultured in Dulbecco s modified Eagle medium containing 5% fetal calf serum (Commonwealth Serum Laboratories, Melbourne, Australia). Melanocytes were kindly provided by Dr. P. Parsons (Queensland Institute of Medical Research, Brisbane, Australia) and cultured in medium supplied by Clonetics (Edward Kellar, Victoria, Australia). Fresh Melanoma Isolates Isolation of melanoma cells from fresh surgical specimens was carried out as described previously [28,29]. Antibodies, Recombinant Proteins, and Other Reagents Tunicamycin (TM) and thapsigargin (TG) were purchased from Sigma Chemical, Co (Castle Hill, Australia). They were dissolved in dimethyl sulfoxide and made up in stock solutions of 1 mm. Obatoclax (GX15-070) was kindly supplied by Gemin X Biotechnologies, Inc. ABT737 was kindly provided by Abbott Laboratories. Obatoclax and ABT737 were dissolved in dimethyl sulfoxide and made up in stock solutions of 5 mm. The mouse monoclonal antibodies (MAbs) against Bcl-2, Bcl-X L, and Mcl-1 and the rabbit polyclonal antibodies (Abs) against Bak and Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The MAb against Noxa and the polyclonal Ab against Bim were purchased from Imgenex (San Diego, CA). The rabbit polyclonal Ab against PUMA and the mouse MAb against cleaved form of poly(adp-ribose) polymerase (PARP) were from Cell Signaling Technology (Beverly, MA). The mouse MAb against cytochrome c was from Pharmingen (Bioclone, Marrickville, Australia). The rabbit polyclonal Ab against caspase-3 was from Stressgen (Victoria, BC, Canada). Isotype control Abs used were the ID4.5 (mouse immunoglobulin G [IgG] 2a) MAb against Salmonella typhi supplied by Dr. L. Ashman (Institute for Medical and Veterinary Science, Adelaide, Australia), the mouse IgG1 MAb purchased from Pharmingen (Bioclone), and rabbit IgG from Sigma Chemical, Co. Apoptosis Quantitation of apoptotic cells by measurement of sub-g 1 DNA content using the propidium iodide method was carried out as described elsewhere [19,28]. Mitochondrial Membrane Potential Melanoma cells were seeded at cells/well in 24-well plates and allowed to reach exponential growth for 24 hours before treatment. Changes in the mitochondrial membrane potential (ΔΨ m )werestudied by staining the cells with the cationic dye, JC-1, according to the manufacturer s instructions (Molecular Probes, Eugene, OR) as described previously [30]. Western Blot Analysis Western blot analysis was carried out as described previously [19,27]. Labeled bands were detected by Immun-Star HRP Chemiluminescent Kit, and images were captured and the intensity of the bands was quantitated with the Bio-Rad VersaDoc image system (Bio-Rad, Regents Park,NSW,Australia). Immunoprecipitation Methods used were as described previously with minor modification [31]. Briefly, 100 μl of lysates were precleared by incubation with 20 μl of a mixture of protein A and protein G sepharose packed beads (Santa Cruz Biotechnology) in a rotator at 4 C for 2 hours and then with 20 μl of fresh packed beads overnight. Twenty micrograms of anti Mcl-1 Ab or control immunoglobulin was then added to the lysates and rotated at 4 C for 2 hours. The beads were then pelleted by centrifugation. The resulting supernatants (nonprecipitated fractions) were collected. The beads

3 Neoplasia Vol. 11, No. 9, 2009 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. 947 Figure 1. Induction of ER stress renders melanoma cells more sensitive to obatoclax-induced apoptosis. (A and B) Obatoclax at nanomolar concentrations is relatively ineffective in induction of apoptosis in melanoma cells. (A) Mel-RM and MM200 cells were treated with obatoclax at indicated concentrations for 24 or 48 hours. (B) Melanoma cells and melanocytes were treated with obatoclax at 1 μm for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (C) Mel-RM and MM200 cells were treated with obatoclax at 1 μm (upper panel) or 100 nm (low panel), TM (3 μm), or the combination TM and obatoclax at 1 μm (upper panel) or 100 nm (low panel) for 24 or 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (D) Mel-RM and MM200 cells were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 24 hours. Whole cell lysates were subjected to Western blot analysis. The cleaved form of PARP was detected by an Ab that specifically recognized the cleaved PARP. Data shown are representative of three individual experiments. (E) Melanoma cells and melanocytes were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (F) Melanoma cells were treated with obatoclax (1 μm), TG (1 μm), or the combination of both for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments.

4 948 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. Neoplasia Vol. 11, No. 9, 2009 Figure 1. (continued). were washed five times with ice-cold lysate buffer before elution of the proteins from the beads in lysate buffer at room temperature for 1 hour. The resulting immunoprecipitates were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. Plasmid Vector and Transfection Stable Mel-RM and MM200 transfectants of Bcl-2 and Mcl-1 were established using the pef-puro vector carrying human Bcl-2 kindly provided by Dr. David Vaux ( Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) and p3xflag-cmv-10 carrying human Mcl-1 kindly provided by Dr. Xiaodong Wang (Howard Hughes Medical Institute, Dallas, TX), respectively, as reported before [19]. Briefly, Melanoma cells were seeded at cells per well in 24-well plate 24 hours before transfection. Cells were transfected with 0.8 μg of plasmid as well as the empty vector (Sigma-Aldrich) in Opti-MEM medium (Invitrogen, Carlsbad, CA) with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer s protocol. Six hours after transfection, the cells were switched into antibiotic-free medium containing 5% fetal calf serum for a further 24 hours. Cells were then passaged at 1:10 into fresh medium for a further 24 hours followed by G418 (Sigma-Aldrich) selection. Small RNA Interference The small RNA interference (sirna) constructs used were obtained as the sigenome SMARTpool reagents (Dharmacon, Lafayette, CO), the sigenome SMARTpool Bak (M ), the sigenome SMARTpool Bax (M ), the sigenome SMARTpool Mcl-1 (M ), the sigenome SMARTpool PUMA (M ), the sigenome SMARTpool Noxa (M ), and the sigenome SMARTpool Bim (M ). The nontargeting sirna control, SiConTRol- Non-targeting SiRNA pool (D ), was also obtained from Dharmacon. Transfection of sirna pools was carried out as described previously [19,27]. Results Obatoclax at Nanomolar Concentrations Is Relatively Ineffective in Induction of Apoptosis of Melanoma Cells Obatoclax as a single agent has been shown to be a potent apoptosis inducer in some types of cancer cells, such as myeloma cells, with a mean half-maximal inhibitory concentration value of 246 nm [26]. To study the apoptosis-inducing potential of obatoclax in melanoma

5 Neoplasia Vol. 11, No. 9, 2009 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. 949 cells, we treated Mel-RM and MM200 cells with the compound at varying concentrations up to 1 μm for 24 and 48 hours. As shown in Figure 1A, obatoclax did not induce apoptosis of the cells at 24 hours after treatment. By 48 hours, it caused apoptosis in approximately 32% and 26% of Mel-RM and MM200 cells, respectively, when used at 500 to 1000 nm. A summary of studies in a panel of melanoma cell lines and fresh melanoma isolates and a melanocyte line treated with obatoclax at 1 μm for 48 hours are shown in Figure 1B. Although obatoclax induced varying degrees of apoptosis, none of the melanoma cell lines or fresh isolates displayed a half-maximal inhibitory concentration value lower than 1 μm. These results suggest that obatoclax at nanomolar concentrations is relatively ineffective in the induction of apoptosis of melanoma cells. Of note, obatoclax did not induce significant apoptotic cell death in melanocytes (<10% apoptotic cells). Induction of ER Stress Renders Melanoma Cells More Sensitive to Obatoclax-Induced Apoptosis Previous studies have shown that most melanoma cell lines are not sensitive to ER stress induced apoptosis, and this is associated with the up-regulation of the antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-2 [18 20]. To test whether obatoclax can reverse the resistance of melanoma cells to apoptosis under ER stress [23,25], we Figure 2. Increased induction of apoptosis by cotreatment with obatoclax and TM is primarily due to inhibition of Mcl-1. (A) Mel-RM and MM200 cells were treated with ABT737 (2 μm), TM (3 μm), or the combination of both for 24 or 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (B) Mel-RM and MM200 cells were treated with TM (3 μm), ABT737 (2 μm), obatoclax (1 μm), the combination of TM and ABT737, or the combination of TM and obatoclax for 24 hours before measurement of ΔΨ m by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨ m. Data shown are representative of three individual experiments. (C) Mel-RM and MM200 cells were treated with TM (3 μm), ABT737 (2 μm), obatoclax (1 μm), the combination of TM and ABT737, or the combination of TM and obatoclax for 24 hours. Isolated cytosolic fractions were subjected to Western blot analysis. Data shown are representative of three individual experiments. (D) Mel-RM cells overexpressing Bcl-2 (left panel) were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours (right panel). Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (E) Mel-RM cells overexpressing Mcl-1 (left panel) were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours (right panel). Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments.

6 950 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. Neoplasia Vol. 11, No. 9, 2009 Figure 2. (continued). treated Mel-RM and MM200 cells with obatoclax at 1 μm or 100 nm in the presence of the classic ER stress inducer TM. As reported elsewhere, TM alone induced only moderate levels of apoptosis (<10% apoptotic cells) even at 48 hours after treatment [19,27]. Cotreatment with TM and obatoclax at 1 μm or 100 nm resulted in marked increases in apoptotic cell death in both cell lines at 24 and 48 hours in comparison with treatment with obatoclax alone (Figure 1C). The percentage of apoptotic cells at 48 hours after cotreatment reached 95% and 80%, respectively, in Mel-RM and MM200 when obatoclax was used at 1 μm. Enhancement of apoptosis by cotreatment with TM and obatoclax in melanoma cells was also evidenced by increased activation of caspase-3 and cleavage of its substrate PARP (Figure 1D). Figure 1E shows that cotreatment with TM and obatoclax for 48 hours resulted in enhanced apoptosis in a panel of melanoma cell lines and fresh melanoma isolates. Although TM alone induced apoptosis in approximately 28% of melanocytes [27], there was no further increase in the levels of apoptosis after cotreatment with obatoclax and TM (Figure 1E). Similar to TM, coadministration of another classic ER stress inducer TG and obatoclax also enhanced the induction of apoptosis in melanoma cells (Figure 1F). Together, these results indicate that obatoclax is more effective in the induction of apoptosis in melanoma cells undergoing acute ER stress. Increased Induction of Apoptosis by Obatoclax in Melanoma Cells Undergoing ER Stress Is Primarily Due to the Inhibition of Mcl-1 To differentiate the roles of inhibition of Mcl-1 and inhibition of Bcl-2 in the enhancement of obatoclax-induced apoptosis in melanoma cells cotreated with TM, we exposed Mel-RM and MM200 cells to TM in combination with another BH3 mimetic ABT737 that inhibits Bcl-2 but not Mcl-1 [22,23]. Figure 2A shows that even when used at 2 μm, ABT737 alone induced only moderate levels of apoptosis (<10%) in both cell lines at 48 hours after treatment. The combination of ABT737 and TM resulted in increased apoptotic cell death at 24 and 48 hours. However, the overall levels of apoptosis were markedly lower than those induced by cotreatment with obatoclax and TM (P <.01, 2-tailed Student s t test; Figures 1C and 2A). We compared mitochondrial apoptotic events induced by the combination of obatoclax and TM with those by cotreatment with ABT737 and TM. As shown in Figure 2 (B and C), relative to treatment with obatoclax alone, the combination of obatoclax and TM induced marked increases in the reduction in ΔΨ m and in the mitochondrial release of cytochrome c as shown by elevated cytosolic levels of cytochrome c in Mel-RM and MM200 cells. ABT737 alone induced a minimal reduction in ΔΨ m and mitochondrial release of cytochrome c,but the combination of ABT737 and TM resulted in enhanced induction of these mitochondrial apoptotic events. However, the overall levels of reduction in ΔΨ m and cytosolic expression of cytochrome c induced by cotreatment with ABT737 and TM were noticeably lower than those induced by the combination of obatoclax and TM. To further confirm the roles of inhibition of Mcl-1 and inhibition of Bcl-2 in obatoclax-induced apoptosis in melanoma cells submitted to ER stress, we cotreated Mel-RM cells that had been stably transfected with complementary DNA encoding Bcl-2 or Mcl-1 with obatoclax and TM (Figure 2, D and E) [19]. Overexpression of either Mcl-1 or Bcl-2 inhibited apoptosis induced by the combination of the compounds, but the degrees of inhibition by the overexpression of Mcl-1

7 Neoplasia Vol. 11, No. 9, 2009 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. 951 effects on apoptosis induced by obatoclax or obatoclax in combination with TM (Figure 4D). We examined the role of the multidomain Bcl-2 family protein, Bak, in apoptosis induced by cotreatment with obatoclax and TM in Mel-RM and MM200 cells in which Bak was knocked down by sirna (Figure 5A). Inhibition of Bak significantly inhibited apoptosis induced by either obatoclax alone or by cotreatment with obatoclax and TM (P <.01,2-tailedStudent s t test; Figure 5B). As shown in Figure 5 (C and D), inhibition of the other multidomain Bcl-2 family protein Bax by transfecting a sirna pool into Mel-RM and MM200 cells also blocked the induction of apoptosis by obatoclax or obatoclax in combination with TM, but the degrees of inhibition were less pronounced than those resulting from the inhibition of Bak. Figure 3. Obatoclax alters the expression profile of Bcl-2 family proteins in melanoma cells under ER stress. Mel-RM and MM200 cells were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 36 hours. Whole cell lysates were subjected to Western blot analysis. Data shown are representative of three individual experiments. were significantly greater than those by the overexpression of Bcl-2 (P <.01, 2-tailed Student s t test; Figure 2, D and E). Induction of ER Stress Alters the Expression Profile of Bcl-2 Family Proteins in Response to Obatoclax Induction of ER stress is known to result in increased expression of the BH3-only proteins PUMA and Noxa and the antiapoptotic proteins Bcl-2 and Mcl-1 [19]. As shown in Figure 3, exposure of melanoma cells to obatoclax caused even greater degrees of up-regulation of PUMA, Noxa, Bcl-2, and Mcl-1. In addition, the BH3-only protein Bim was also increased in melanoma cells treated with obatoclax. Notably, cotreatment with both agents did not cause any further changes in the levels of PUMA, Bcl-2, and Bim. However, the levels of expression of Noxa were markedly higher in cells cotreated with obatoclax and TM than those in cells treated with either compound alone (Figure 3). In contrast, the levels of Mcl-1 were even lower than those induced by obatoclax alone (Figure 3). Inhibition of Noxa or Bak Partially Blocks Obatoclax-Induced Apoptosis in Melanoma Cells under ER Stress To study the roles of BH3-only proteins in enhancement of induction of apoptosis by cotreatment with obatoclax and TM, we transfected sirna pools for Noxa, PUMA, and Bim into Mel-RM and MM200 cells, respectively. Figure 4 (A C) shows that sirna reduced their expression by at least 70% but had no effect on the expression of their relative Bcl-2 or Mcl-1. Knockdown of Noxa by sirna partially blocked apoptosis induced by obatoclax alone (Figure 4D). Similarly, inhibition of Noxa partially blocked apoptosis induced by cotreatment with obatoclax and TM (P <.01, 2-tailed Student s t test; Figure 4D). In contrast, knockdown of PUMA or Bim by sirna had only minimal Mcl-1/Bak Interaction Is Disrupted by Obatoclax More Efficiently in Melanoma Cells Undergoing ER Stress In the absence of apoptotic stimulation, Bak is known to be sequestered by Mcl-1 and Bcl-X L on the outer mitochondrial membrane [32]. Release of Bak from these antiapoptotic proteins by replacement with BH3-only proteins plays a key role in permeabilizing the outer mitochondrial membrane [32]. We studied the interactions between Mcl-1 and Bak and Noxa in Mel-RM and MM200 cells before and after treatment with obatoclax in the absence or presence of TM. As shown in Figure 6, Western blot analysis of precipitates resulting from immunoprecipitation with an Ab against Mcl-1 revealed that treatment with obatoclax led to reduced association between Bak and Mcl-1 and, intriguingly, increased interactions between Noxa and Mcl-1. Notably, there was little Bak coprecipitated with Mcl-1 in cells cotreated with obatoclax and TM (Figure 6). In contrast, the levels of Noxa in Mcl-1 precipitates from cells cotreated with obatoclax and TM seemed even higher than those from cells treated with obatoclax alone (Figure 6). Discussion Previous studies have shown that melanoma cells have largely adapted to ER stress and are largely resistant to ER stress induced apoptosis [18 20]. One of the major adaptive mechanisms seems to be upregulation of the antiapoptotic Bcl-2 family protein Mcl-1, which plays a critical role in preserving outer mitochondrial membrane integrity in melanoma cells submitted to ER stress [19]. In the present study, we show that the novel BH3 mimetic, obatoclax, potently disrupts the adaptive mechanism and overcomes resistance of melanoma cells to ER stress induced apoptosis. Moreover, we demonstrate that the BH3-only protein Noxa is preferentially increased by obatoclax, which in turn, along with Bak, plays a dominant role in induction of apoptosis by obatoclax in melanoma cells undergoing ER stress. Both Bcl-2 and Mcl-1 can be upregulated by the ER stress response in melanoma cells [19], but enhancement of apoptosis induced by obatoclax in melanoma cells undergoing acute ER stress induced by cotreatment with TM is primarily due to the inhibition of Mcl-1. This was shown by the limited ability of another BH3 mimetic ABT737, which is known to inhibit Bcl-2 but not Mcl-1 [22,23], to enhance mitochondrial apoptotic events and induction of apoptosis in melanoma cells under ER stress. In addition, overexpression of Mcl-1 almost completely blocked apoptosis, whereas overexpression of Bcl-2 had markedly fewer effects on apoptosis induced by the combination of obatoclax and TM. We have previously shown that overexpression of Bcl-2 could not rescue melanoma cells with deficient Mcl-1 expression from ER stress induced apoptosis [19]. Together, these findings are supported

8 952 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. Neoplasia Vol. 11, No. 9, 2009 Figure 4. Inhibition of Noxa partially inhibited enhancement of induction of apoptosis by cotreatment with obatoclax and TM. (A C) Mel-RM and MM200 cells were transfected with the control, Noxa (A), PUMA (B), or Bim (C) sirna. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of Noxa, PUMA, or Bim expression. Western blot analysis of Mcl-1 or Bcl-2 was included as controls. The relative expression levels of Noxa, PUMA, and Bim were inhibited by 86%, 90%, and 82%, respectively, in Mel-RM cells, and by 82%, 87% and 80%, respectively, in MM200 cells. Data shown are representative of three individual experiments. (D) Mel-RM and MM200 cells were transfected with the control, Noxa (A), PUMA (B), or Bim (C) sirna. Twenty-four hours later, cells were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. by reports showing that obatoclax can overcome Mcl-1 mediated resistance to apoptosis [21,23 25], whereas ABT737 is relatively ineffective in increasing sensitivity to apoptosis when Mcl-1 is involved in the resistance [22,23]. Intriguingly, obatoclax alone induced marked up-regulation of Bcl-2 and Mcl-1 in melanoma cells. This seemed to be melanoma cell specific, in that the compound did not cause any increase in Bcl-2 and Mcl-1 in other types of cancer cells [21,23,25]. It is conceivable that up-regulation of Bcl-2 and Mcl-1 may increase the amount of obatoclax needed to neutralize these proteins, thus rendering melanoma cells relatively resistant to obatoclax-induced apoptosis. Notably, obatoclax also upregulated the BH3-only proteins Noxa, PUMA, and Bim in melanoma cells. Although Bim has been reported to be increased by obatoclax [23], the present study seemed to be the first to show that Noxa and PUMA can be similarly upregulated by the compound in melanoma cells. The unexpected effect of obatoclax on the expression of multiple Bcl-2 family proteins including Bcl-2 and Mcl-1 indicates that this compound possesses more complex biologic effects on melanoma cells than just binding to and inhibiting antiapoptotic Bcl-2 family proteins [23,24]. Of note, the levels of Mcl-1 in melanoma cells cotreated with TM and obatoclax were lower than those induced by obatoclax alone, but the BH3-only protein Noxa, which was upregulated by TM or obatoclax alone, was further increased by cotreatment with the agents. Binding of Noxa to Mcl-1 has been shown to target the latter for proteasomemediated degradation [32,33], so the reduced Mcl-1 expression may be due to increased Noxa that precipitated Mcl-1 degradation. Another possibility is that Mcl-1 was degraded by caspase-3 activity that was enhanced in melanoma cells cotreated with obatoclax and TM [34,35]. Noxa seemed to be the main BH3-only protein involved in induction of apoptosis by obatoclax in melanoma cells undergoing ER stress, as shown by its strong up-regulation in response to cotreatment with obatoclax and TM and by inhibition of apoptosis when Noxa was knocked down by sirna. This is consistent with the finding that inhibition of

9 Neoplasia Vol. 11, No. 9, 2009 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. 953 Figure 5. Inhibition of Bak partially blocks enhancement in induction of apoptosis by obatoclax and TM. (A) Mel-RM and MM200 cells were transfected with the control or Bak sirna. Whole cell lysates were subjected to Western blot analysis. The relative expression levels of Bak were inhibited by 84% and 86% in Mel-RM and MM200 cells, respectively. Data shown are representative of three individual experiments. (B) Mel-RM and MM200 cells with Bak knocked down by sirna were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. (C) Mel-RM and MM200 cells were transfected with the control or Bax sirna. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. The relative expression levels of Bak were inhibited by 84% and 78% in Mel-RM and MM200 cells, respectively. Data shown are representative of three individual experiments. (D) Mel-RM and MM200 cells were transfected with the control or Bax sirna. Twenty-four hours later, cells were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. Data shown are the mean ± SE of three individual experiments. Figure 6. Obatoclax disrupts the Bak/Mcl-1 interaction more efficiently in melanoma cells undergoing ER stress. Mel-RM and MM200 cells were treated with obatoclax (1 μm), TM (3 μm), or the combination of both for 24 hours. Whole cell lysates were subjected to immunoprecipitation with an Ab against Mcl-1. The resulting precipitates and nonprecipitated fractions were then subjected to Western blot analysis of Mcl-1, Noxa, and Bak. The arrowhead points to bands generated by the immunoglobulin light chain. Western blot data are representative of three individual experiments.

10 954 Obatoclax Kills Melanoma Cells Under ER Stress Jiang et al. Neoplasia Vol. 11, No. 9, 2009 Mcl-1 played an essential role in enhanced induction of apoptosis because Noxa can only be antagonized by Mcl-1 but not Bcl-2 [36]. Mcl-1 has been suggested to have a unique role in protection against apoptosis, in that elimination of Mcl-1 was shown to be required for induction of apoptosis by various stimuli [37]. It seems that the reduced Mcl-1 expression would facilitate obatoclax-mediated inactivation of its function as an antiapoptotic effector. Obatoclax is known to overcome Mcl-1 mediated resistance to apoptosis induced by the proteasome inhibitor bortezomib in mouse melanoma and human lymphoma cells [23,24]. Interestingly, bortezomib has been shown to induce ER stress in a number of types of cells including melanoma cells [38,39]. However, as a proteasome inhibitor, bortezomib may elicit a variety of changes in gene expression in tumor cells that may not be necessarily related to induction of ER stress [40,41]. In melanoma, it has been shown that bortezomib induced apoptosis by up-regulation of Noxa through a c-myc mediated transcriptional mechanism [41]. The effect of obatoclax on melanoma cells treated with bortezomib may, therefore, not necessarily reflect its effect on melanoma cells undergoing ER stress, although in both cases, obatoclax as a BH3 mimetic would be expected to enhance induction of apoptosis. As far as we are aware, the changes in the expression profile of Bcl-2 family proteins in response to obatoclax in melanoma cells undergoing ER stress have not been previously observed in melanoma or other cancer cells treated with bortezomib [22,24]. sirna knockdown of Bak showed that it was also important for the induction of apoptosis by obatoclax in melanoma cells undergoing ER stress. It is believed that Bak is sequestered by Mcl-1 and Bcl-X L on the outer mitochondrial membrane in intact cells [32] and that competitive binding of these antiapoptotic proteins by BH3-only proteins frees Bak, allowing it to oligomerize and trigger outer mitochondrial permeability [32]. In this study, there was much less Bak coprecipitated with Mcl-1 in melanoma cells cotreated with obatoclax and TM than those treated with obatoclax alone, indicating that the interaction between Bak and Mcl-1 was disrupted more efficiently by obatoclax in melanoma cells undergoing ER stress. This, along with the reduced Mcl-1 expression, may cooperate with obatoclax to release Bak from Mcl-1. Because Bak-mediated apoptosis also requires neutralization of Bcl-X L [32], it is conceivable that other BH3-only proteins such as PUMA and Bim that can engage Bcl-X L also play roles in the induction of apoptosis by the combination of obatoclax and TM. However, in this study, inhibition of PUMA or Bim by sirna seemed to have only minimal effects on apoptosis induced by cotreatment with the compounds. The reduced levels of Mcl-1 in melanoma cells cotreated with obatoclax and TM argue against a major role of PUMA and Bim in antagonizing Mcl-1, in that engagement of Bim or PUMA is known to stabilize Mcl-1 against degradation [33,42]. Nevertheless, it is noteworthy that cotreatment with ABT737 and TM also resulted in a slight increase in apoptosis. This indicates that inhibition of antiapoptotic Bcl-2 family proteins other than Mcl-1 may be involved in enhancement of apoptosis induced by obatoclax in melanoma cells undergoing ER stress. Similarly, inhibition of Bax by sirna knockdown also reduced the levels of apoptosis, albeit to a lesser extent. Adaptation to ER stress is known to contribute to resistance of melanoma cells to various chemotherapeutic drugs [29,43]. The present study, along with our previous studies showing that up-regulation of Mcl-1 is an important adaptive mechanism by which melanoma cells survive ER stress [19], demonstrates that the BH3 mimetic obatoclax is a potent agent that targets this adaptive mechanism and overcomes resistance of melanoma cells to ER stress induced apoptosis. Importantly, these findings also applied to fresh melanoma isolates that may reflect more closely the susceptibility of melanoma cells in vivo. These results suggest that treatment with obatoclax in combination with agents that can induce ER stress such as cisplatin and sorafenib may be particularly effective in the treatment of melanoma [29,44]. Cotreatment with obatoclax and TM did not induce more killing of melanocytes than TM alone, suggesting the combination of obatoclax and ER stress inducers may have limited cytotoxicity toward normal tissues. 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