Anti-caspase-3 (sc-7272), anti-caspase-4 (sc-56056), anti-cleaved caspase-4 (sc-22173-R), and anti-caspase-9 (sc-56073) antibodies (Abs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase-3 (ab2302), anti-cleaved caspase-9 (ab2324), anti-PDI (ab2792), anti-VDAC1 (ab14734), anti-CHOP (ab11419), anti-IP3R (ab5804) Abs were purchased from Abcam Ltd. (Hong Kong, China). Anti-β-actin (60008-1-Ig), anti-Bax (50599-2-Ig), anti-Bcl-2 (12789-1-AP), anti-cytochrome c (Cyto C) (10993-1-AP), anti-Grp78/BIP (11587-1-AP), anti-Grp75 (14887-1-AP), anti-mitofusin 2 (Mfn2) (12186-1-AP), anti-β-tubulin (10068-1-AP), anti-calreticulin (10292-1-AP), peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (SA00001-1), peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (SA00001-2) and anti-IgG control (30000-0-AP) Abs were purchased from Proteintech Group, Inc. (Chicago, IL, USA). The anti-calpain-1 catalytic subunit (#31038-1) Ab was purchased from SAB (College Park, MD, USA). Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in normal saline (NS) for in vitro use and animal studies. ABT-737 was provided by Abbott Laboratories (Abbott Park, IL, USA) and dissolved in dimethyl sulfoxide (DMSO) for in vitro use and animal studies. 2-aminoethyl diphenylborinate (2-APB) (ab120124) was purchased from Abcam Ltd.

The cisplatin-resistant clone SKOV3/DDP was obtained from the Peking Union Medical College (Beijing, China). COC1/DDP and A2780/DDP cells were purchased from the American Type Culture Collection (Manassas, VA, USA). SKOV3/DDP, COC1/DDP and A2780/DDP cells were maintained in IMDM (Gibco; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), supplemented with 10% (v/v) fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin and 100 U/ml penicillin (each from Genview, Galveston, TX, USA). The cells were incubated at 37°C in an atmosphere containing 5% CO₂.

Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Beyotime Institute of Biotechnology, Haimen, China). The cisplatin-resistant ovarian cancer cells, during the exponential growth phase, were seeded into 96-well culture plates in 100 μl of IMDM at a density of 1.0×10⁴ cells/well. After a 24-h incubation, the indicated drugs were added for another 24 h in four parallel wells. The MTT assays were performed as follows: 20 μl of MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well, and the cells were incubated at 37°C for 4 h. At the end of the incubation, 150 μl of DMSO (Beijing Chemical Industry Co., Ltd., Beijing, China) was added to each well. The cells were agitated for 10 min prior to measuring the absorbance at 570 nm using a model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The growth inhibition rate was calculated as follows: Inhibition (%) = [1 − (absorbance of experimental group/absorbance of control group)] × 100. The mean value of four replicate wells was calculated for each treatment group.

The Muse™ Annexin V Dead Cell kit (Ref. MCH 100105, Merck Millipore, Darmstadt, Germany) was used to monitor cell death. Exponentially growing cisplatin-resistant ovarian cancer cells were seeded into 6-well culture plates at a density of 2×10⁵ cells/well. After exposure to different experimental conditions, the cells were trypsinized and resuspended in IMDM with 10% FBS at a concentration of 1×10⁶ cells/ml. Cells were incubated with Annexin V and Dead Cell reagent in a dark room at room temperature for 20 min. Finally, the samples were measured by flow cytometry (Muse Cell Analyzer, Merck Millipore).

Changes in the mitochondrial membrane potential (ΔΨm) during the early stages of apoptosis were assayed using the Muse MitoPotential assay (Ref. MCH 100110, Merck Millipore) in SKOV3/DDP cells treated with cisplatin alone or in combination with ABT737. Briefly, cells were harvested, and the cell pellet was suspended in assay buffer (1×10⁵ cells/100 μl). MitoPotential dye working solution was added, and the cell suspension was incubated at 37°C for 20 min. After the addition of Muse MitoPotential 7-AAD dye (propidium iodide) and incubation for 5 min, changes in ΔΨm and in cellular plasma membrane permeabilization were assessed on the basis of the fluorescence intensities of both the dyes, which were analyzed by flow cytometry (Muse Cell Analyzer, Merck Millipore).

Whole cell protein extracts from SKOV3/DDP cells were prepared with cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM NaF, 1 μg/ml leupeptin, and 1 mM PMSF) for western blotting. The protein extracts were quantified using a Bio-Rad kit (Pierce). Then, the protein extracts were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (GS0914, Millipore). After being blocked in 5% non-fat milk in buffer [10 mM Tris-HCl (pH 7.6), 100 mM NaCl and 0.1% Tween-20], the membranes were probed with primary antibodies at 37°C for 2 h. The membranes were then incubated with HRP-conjugated secondary antibodies at room temperature for 1.5 h. Immunodetection was performed using enhanced chemiluminescence reagents (Thermo Scientific, Rockford, IL, USA), and images were captured using a Syngene Bio Imaging System (Synoptics, Cambridge, UK). Specific proteins were quantified by densitometry using Quantity One software (Bio-Rad Laboratories), normalized to actin, and presented as the mean ± SD of three independent experiments.

The cytoplasmic Ca²⁺-sensitive fluorescent dye Fluo-4/AM (Molecular Probes) and the mitochondrial Ca²⁺-sensitive fluorescent dye Rhod-2/AM (AAT Bioquest, Sunnyvale, CA, USA) were used to measure the Ca²⁺ concentration according to the manufacturer’s instructions. Before exposure to different experimental conditions, the cells were incubated with Fluo-4/AM or Rhod-2/AM for 30 min at 37°C. Cell samples were then analyzed by confocal laser microscopy. All experiments were performed in triplicate.

Cells were seeded onto coverslips in 24-well plates at a density of 5×10⁴ cells/well and allowed to recover overnight. After treatment with the indicated drugs, cells were washed three times with cold 0.1 M PBS and fixed in 4% (w/v) paraformaldehyde/PBS for 20–30 min, stained with the nuclear stain Hoechst 33342/H₂O (2 μg/ml, Sigma) for 30 min, washed with 0.01 M PBS, and examined using Olympus FV1000 confocal laser microscopy to reveal chromatin condensation. The co-localization of IP3R and VDAC1 and the expression of PDI were examined by the indirect immunofluorescence method. Cells were cultured on coverslips overnight, then treated with the indicated drugs, and rinsed with 0.1 M PBS three times. After incubation, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min, washed three times with 0.01 M PBS, and then blocked for 30 min in 5% (w/v) non-immune animal serum (goat) (Beyotime Biotechnology, Shanghai, China) PBS, and incubated with primary antibody (IP3R, VDAC1, PDI) overnight at 4°C. The next day, the slides were incubated with the Alexa Fluor-488/546-conjugated secondary antibody (1:400 dilution; Invitrogen) for 1 h, then stained with Hoechst 33342 (2 μg/ml) for 2 min, and washed three times with PBS. After mounting, the cells were examined by Olympus FV1000 confocal laser microscopy.

The purification of the cytoplasmic fraction, ER fraction, pure mitochondrial fraction, crude mitochondrial fraction and the MAM fraction were performed as previously described (17). The above fractions were lysed in RIPA buffer [1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris (pH 8.0), 0.14 M NaCl] for immunoblotting with antibodies against IP3R, VDAC1, Bcl-2, calreticulin, Cyto C, Mfn2, Grp75 and tubulin. For co-immunoprecipitation analysis, the MAM lysates were pre-cleared with protein G agarose beads (Proteintech Group, Inc.) for 1 h at 4°C. Then, equal amounts of sample lysates were incubated with either 2 μg of IgG or anti-IP3R antibody for 20 h at 4°C, and this was followed by precipitation with protein G agarose beads. Immunoprecipitated proteins from MAM lysates and total MAM lysates were subjected to immunoblot analysis with the anti-IP3R antibody and anti-VDAC1 antibody. Total MAM lysates were used as the input control. The results are representative of three independent experiments.

Electron microscopy and morphometric analysis were performed as described previously (26). Cells were fixed for 30 min with ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, embedded in Epon, and processed for transmission electron microscopy by standard procedures. Representative areas were chosen for ultra-thin sectioning and examined on a transmission electron microscope at a magnification of ×20,000.

Small interfering RNA (siRNA) sequences targeting human Bcl-2 and a non-target sequence were constructed by Genechem (Shanghai, China). The Bcl-2 siRNA sequence was CCG-CAT-TTA-ATT-CAT-GGT-ATT and that of the non-target siRNA (Scramble) was TTC-TCC-GAA-CGT-GTC-ACG-T. Transfections with the siRNAs were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, cisplatin-resistant SKOV3/DDP cells were placed into 6-well plates and transfected the next day with 4 μg of Bcl-2 siRNA or siScramble, using 10 μl of Lipofectamine 2000 (at 1 μg/μl). Cells were harvested 2 days after transfection; whole cell lysates were isolated for western blots. For MTT assays, transfected cells were treated with cisplatin for 24 h, and then subjected to the MTT assay to determine cell viability.

BALB/c nude mice (4–6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., China. Animals were maintained in specific pathogen-free conditions and a controlled light and humidity environment. Animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals. SKOV3/DDP cells (5×10⁶) were subcutaneously injected into the right flank of each mouse. Tumor volume (mm³) was measured every 4 days using a Vernier caliper and calculated as 0.4 × (short length)² × long length. Treatment was initiated when tumors reached a volume of 45–55 mm³ (day 16). The mice were randomly divided into 4 groups (5 animals/group) and received NS (IP, daily), 4 mg/kg cisplatin (IP, every other day), 50 mg/kg ABT737 (IP, every other day), or a combination treatment for 20 days. The mice were sacrificed, and the experiment was terminated at the end of 36 days. Tumors were isolated, weighed, and imaged.

Immunohistochemical staining for cleaved caspase-3 was performed on 5-μm thick sections embedded in paraffin after formalin fixation. The sections were de-paraffinized in xylene and rehydrated in graded ethanol solutions (reducing concentration from 95 to 70%). The sections were incubated in H₂O₂ solution (3% H₂O₂ in PBS buffer) for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in retrieval buffer (pH 9.0, 20 mM Tris, 0.05 mM EDTA, 0.05% Tween-20 buffer) in a 99°C bath for 20 min. The sections were subsequently incubated at 4°C overnight with anti-cleaved caspase-3. After rinsing with PBS buffer, the secondary antibody (MaxVision™ HRP-Polymer Anti-Rabbit IHC kit, Maixin Bio, China) was applied for 15 min at RT. The DAB (Maixin Bio, China) solution was used as the chromogen. Finally, the sections were counterstained with hematoxylin (Sigma) to identify the nuclei. The images were observed and analyzed using a microscope (Leica DM 4000B) with Image-Pro Plus 6.0 software.

Apoptosis analysis was performed using an In Situ Cell Death Detection kit (Roche, Indianapolis, IN, USA) to identify DNA breaks according to the manufacturer’s instructions. Sections were incubated with TUNEL reaction mix containing 10 U of terminal deoxyribosyltransferase, 10 mM dUTP biotin, and 2.5 mM cobalt chloride in 1X terminal transferase reaction buffer for 1 h at 37°C in a humidified atmosphere. The DAB (Maixin Bio, China) solution was used as the chromogen. Finally, the sections were counterstained with hematoxylin (Sigma) to identify the nuclei. The images were observed and analyzed using a microscope (Leica DM 4000B) with Image-Pro Plus 6.0 software.

All values are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test with the software GraphPad Prism version 6.00 for Mac (GraphPad Software, La Jolla, CA, USA). A value of P<0.05 was considered statistically significant.

We treated cisplatin-resistant ovarian cancer cells (SKOV3/DDP, COC1/DDP and A2780/DDP cells) with ABT737 in combination with cisplatin to investigate whether ABT737 would enhance the antitumor effects of cisplatin. On the basis of our previous studies (unpublished data), the non-toxic dose of ABT737 in SKOV3/DDP, COC1/DDP and A2780/DDP cells for 24 h is 5 μM. Thus, we treated SKOV3/DDP, COC1/DDP and A2780/DDP cells with 15 μg/ml cisplatin and/or 5 μM ABT737 for 24 h. MTT assays indicated that ABT737 increased cisplatin-induced growth inhibition (Fig. 1A–C).

On the basis of these results, we treated cells with cisplatin and/or ABT737 for 24 h and then examined apoptotic chromatin condensation with Hoechst 33342 staining. These results showed that ABT737 increased cisplatin-induced apoptotic chromatin condensation (Fig. 1D–F). Additionally, flow cytometry analysis revealed that the apoptosis rate was clearly higher in SKOV3/DDP, COC1/DDP and A2780/DDP cells exposed to cisplatin and ABT737 for 24 h compared with cells treated with cisplatin alone (Fig. 1G–I). These results indicate that ABT737 restores sensitivity to cisplatin in cisplatin-resistant ovarian cancer cells.

After the in vitro experiments, in vivo experiments were performed to determine whether ABT737 contributes to suppressing tumor growth in combination with cisplatin. SKOV3/DDP cells were subcutaneously inoculated into BALB/c nude mice to establish a subcutaneous transplant tumor model. The tumor growth curve over time was recorded (Fig. 2A). As predicted, ABT737 plus cisplatin caused marked tumor growth inhibition compared with treatment with cisplatin-only (Fig. 2B). The final tumor weights were 1.01, 0.39, 0.97 and 0.19 g for BALB/c nude mice injected i.p. with NS, cisplatin, ABT737 or a combination treatment, respectively (Fig. 2C). Next, we used immunohistochemical methods to detect the expression of the apoptosis-related protein cleaved caspase-3 in mice exposed to cisplatin and/or ABT737 for 20 days. As shown in Fig. 2D, ABT737 combined with cisplatin treatment led to the upregulation of cleaved caspase-3 compared with treatment with cisplatin only. Furthermore, TUNEL assays were used to determine whether ABT737 had a synergistic effect with cisplatin in inducing cell apoptosis in mouse models of ovarian cancer. These results showed that the number of apoptotic cells in the cisplatin plus ABT737 group was significantly greater than that of the cisplatin group (Fig. 2E and F). Together, these data suggest that ABT737 and cisplatin have a synergistic effect in the treatment of human ovarian cancer xenograft mouse models.

First, we investigated whether ABT737 would enhance cisplatin-induced mitochondrial apoptosis. To determine the effect of cisplatin and/or ABT737 on mitochondrial function, we measured ΔΨm by using flow cytometry after 6 h. As shown in Fig. 3A, the combination treatment further reduced the cisplatin-induced decrease of ΔΨm. Moreover, western blot analysis showed that the combination treatment increased the ratio of Bax/Bcl-2 and enhanced the expression of mitochondrial apoptosis-related proteins (calpain-1, Cyto C, cleaved caspase-9 and cleaved caspase-3), as compared with the cisplatin treatment (Fig. 3B and C).

A growing body of research suggests that cisplatin triggers ER stress and induces ER stress-mediated apoptotic events (2). Hence, we further investigated whether ABT737 enhanced cisplatin-induced ER stress-mediated apoptosis in SKOV3/DDP cells. Using confocal microscopy, we found that ABT737 increased the expression of PDI induced by cisplatin (Fig. 3D). We assessed the expression of ER stress-related proteins (PDI, Grp78, CHOP and cleaved caspase-4) by western blot analysis. These results showed that the combination treatment enhanced the expression of PDI, Grp78, CHOP and cleaved caspase-4, as compared with the cisplatin treatment (Fig. 3E and F). Thus, we demonstrated that the intrinsic mitochondrial apoptotic pathway and ER-mediated apoptosis are actively involved in the ABT737-mediated increase in the cytotoxic effects of cisplatin on SKOV3/DDP cells.

Ca²⁺ is not only a key regulator of cell survival but also triggers cell apoptosis in response to various physiological and pathological conditions. A growing body of literature shows that cytoplasmic and mitochondrial Ca²⁺ overload can contribute to ER-mediated apoptosis and mitochondrial apoptosis (22,27). As described in Introduction, the ER is the most important Ca²⁺ storage compartment in eukaryotic cells. Therefore, we determined whether mitochondrial and cytoplasmic Ca²⁺ overload is a result of Ca²⁺ release from the ER. We used 2-APB, an IP3R antagonist that inhibits ER Ca²⁺ efflux (27). The fluorescent dyes Fluo-4/AM and Rhod-2/AM were used to detect cytoplasmic and mitochondrial Ca²⁺ levels in SKOV3/DDP cells, respectively. As shown in Fig. 4, the cytoplasmic and mitochondrial Ca²⁺ elevation induced by cisplatin or/and ABT737 in SKOV3/DDP cells was markedly reduced in the presence of 2-APB. These results strongly suggest that ABT737 increases cisplatin-induced Ca²⁺ transfer from the ER to the cytosol and mitochondria.

We showed that ABT737 enhanced cisplatin-increased mitochondrial Ca²⁺ levels in the previous experiments (Fig. 4). Therefore, we next explored whether the increased mitochondrial Ca²⁺ levels were mediated by increasing the ER-mitochondria contact sites. IP3R and VDAC1 localize to the mitochondria and ER, respectively (13–15,28). We used confocal microscopy to analyze the co-localization of IP3R and VDAC1. The yellow areas represent co-localization between IP3R and VDAC1, and these results demonstrated that ABT737 enhanced cisplatin-induced ER-mitochondria contact sites (Fig. 5A).

Next, we detected the changes in the ER-mitochondria contact sites by western blotting and immunoprecipitation. Before studying the changes in ER-mitochondria interactions, we assessed the quality of each fraction by immunoblotting for subcellular organelle marker proteins: IP3R and calreticulin for the ER and MAM; VDAC1 for the mitochondria and MAM; Bcl-2 which enriches mitochondria and ER and is also present in MAM; Cyto C for the mitochondria; tubulin for the cytoplasm (17,24,28). As expected, the data showed that we obtained highly pure fractions, and there was no cross-contamination (Fig. 5B). By analyzing MAM composition, we found that ABT737 enhanced the cisplatin-induced interactions of VDAC1 with Grp75 and Mfn2 (Fig. 5C and E), thus suggesting that ER-mitochondria contact sites induced by cisplatin were enhanced in the combination treatment. Then, we treated cells with cisplatin and/or ABT737 for 24 h. The MAM fraction was immunoprecipitated using the anti-IP3R antibody followed by western blotting using the anti-VDAC1 antibody. These results showed that ABT737 enhanced the cisplatin-induced IP3R-VDAC1 interactions (Fig. 5D and F).

Finally, we used electron microscopy to examine the ultrastructural changes in MAM in the cisplatin and/or ABT737 group for 24 h. White arrowheads indicate the ER-mitochondria interactions. The contact points between the two organelles were significantly increased in the combination-treatment group compared with the control group (Fig. 5G and H). In summary, we concluded that ABT737 enhances cisplatin-induced mitochondrial Ca²⁺ levels by increasing ER-mitochondria contact sites.

To better clarify the role of Bcl-2 in cisplatin resistance in SKOV3/DDP cells, we depleted the endogenous Bcl-2 using a specific siRNA in SKOV3/DDP cells and observed that the Bcl-2 protein expression was clearly downregulated (Fig. 6A). MTT assays showed that Bcl-2 siRNA combined with cisplatin for 24 h markedly decreased cell viability compared with that in the cisplatin plus siScramble group (Fig. 6B). Additionally, flow cytometry analysis revealed that the apoptosis rate was substantially higher in SKOV3/DDP cells exposed to cisplatin combined with Bcl-2 siRNA for 24 h compared with cells treated with cisplatin plus siScramble (Fig. 6C and D). Consistently with the results in Fig. 4, Bcl-2 siRNA also enhanced the cisplatin-induced cytoplasmic and mitochondrial Ca²⁺ elevations (Fig. 6E). Finally, we used electron microscopy to examine the ultrastructural changes of MAM after treatment with cisplatin and/or Bcl-2 siRNA. White arrowheads indicate the ER-mitochondria interactions. The contact points between the two organelles were significantly increased after the cisplatin plus Bcl-2 siRNA treatment for 24 h compared with the cisplatin plus siScramble treatment (Fig. 6F and G). Together, these findings suggest that Bcl-2 siRNA improves the sensitivity of ovarian cancer cells to cisplatin by increasing cytoplasmic and mitochondrial Ca²⁺ levels.