Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation

Abstract

Ovarian carcinoma is the leading cause of death from gynecologic cancer in the developed world and is characterized by acquired chemoresistance leading to an overall 5-year survival rate of about 30 %. We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Despite BH3-mimetics represent promising drugs to target Bcl-xL, anti-Mcl-1 strategies are still in pre-clinical studies and required new investigations. Calcium is a universal second messenger and dysregulation of calcium signal is often observed during carcinogenesis. As change in cytosolic free calcium concentration [Ca(2+)]i is known to control the fate of the cell by regulating Bcl-2 family members, we wonder if calcium signal could impact on Mcl-1 expression and if its pharmacological inhibition could be useful to sensitize ovarian carcinoma cells to anti-Bcl-xL strategies. We therefore studied the effect of different calcium signals inhibitors in ovarian carcinoma cell lines SKOV3 and IGROV1-R10 and analysed their effects on proliferation and Mcl-1 expression. We also exposed these cells to these inhibitors in combination with anti-Bcl-xL strategies (siRNA or BH3-mimetic: ABT-737). We found that calcium signaling regulates Mcl-1 through translational events and a calmodulin-mediated pathway. BAPTA-AM and calmodulin inhibitor combination with ABT-737 leads to apoptosis, a process that is reversed by Mcl-1 enforced expression. As Mcl-1 represents a crucial hurdle to the success of chemotherapy, these results could open to new area of investigation using calcium modulators to directly or indirectly target Mcl-1 and thus efficiently sensitize ovarian carcinoma cells to anti-Bcl-xL strategies.

Fig. 1

Cytostatic effect of calcium chelator BAPTA-AM. IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with increasing concentrations of BAPTA-AM for 6 and 24 h. Response was appreciated by a morphological features b cell viability (assessed by the percentage of cell viability with respect to number of viable cells at 0 h) c assessment of sub-G1 peak (24 h). Data are representative of three independent experiments

Fig. 2

Dose-response and time course of BAPTA-AM-induced Mcl-1 decrease. a IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with increasing concentrations of BAPTA-AM for 6 h and expressions of Bcl-2 family members were appreciated by western blot and Mcl-1, Noxa and Puma expressions were quantified by Image J software. b IGROV1-R10 and SKOV3 cells were treated with 10 µM BAPTA-AM from 0 to 24 h. Expression of Mcl-1 was assessed by western blot. Data are representative of three independent experiments. Mcl-1 expression was quantified by Image J software. The relative intensity of each lane was calculated with respect to the sample at 0 h

Fig. 3

BAPTA-AM–induced Mcl-1 decrease does not result from transcriptional and post-translationnal events but is associated with mTORC1 pathway down-regulation. IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 10 µM BAPTA-AM for 6 h. a Mcl-1 mRNA level was determined by real time quantitative RT-PCR, b PARP and Caspase 3 cleavages were assessed by western blot. c IGROV1-R10 and SKOV3 cells were pre-treated 1 h with DMSO, 10 or 100 nM bortezomib. Then cells were treated or not (DMSO) with 10 µM BAPTA-AM for 6 h. Expression of Mcl-1 was followed by western blot. Data are representative of three independent experiments. d IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 5 and 10 µM BAPTA-AM for 6 h. AKT/mTOR as well as MAPK pathways were assessed for each condition and proteins expressions were quantified by Image J software. Data are representative of three independent experiments

Fig. 4

Calcium chelation combined with ABT-737 leads to apoptosis in ovarian carcinoma. a Real time analysis of cellular cytotoxicity of ABT-737/BAPTA-AM combination. Histogram was obtained using the xCELLigence System as described in “ Materials and methods” section. Cells were grown for 24 h and then treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM. Cell index was recorded every 2 h, with displayed standard error bars. IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 10 µM BAPTA-AM for 6 and 24 h. b Morphological features and c DNA contents were studied for each condition. d Cell viability was assessed by trypan blue exclusion at 24 h. e PARP and caspase 3 cleavages were studied by western-blot. Data are representative of three independent experiments

Fig. 5

Calmodulin antagonist W7 inhibits Mcl-1 expression and sensitizes ovarian carcinoma cells to ABT-737. a IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with increasing concentrations of W7 for 3 h and expressions of Mcl-1 and AKT/mTOR pathway were appreciated by western blot and proteins expressions were quantified by Image J software. Data are representative of three independent experiments. b Real time analysis of cellular cytotoxicity of ABT-737/W7 combination. Histogram was obtained using the xCELLigence System as described in “Materials and methods” section. Cells were grown for 24 h and then treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of 40 µM W7. Cell index was recorded every 2 h, with displayed standard error bars. c IGROV1-R10 and SKOV3 cells were treated or not (DMSO) with 10 µM ABT-737 in presence or not (DMSO) of increasing concentrations of W7 for 24 h. PARP cleavage was studied by western-blot. Data are representative of three independent experiments

Fig. 6

Mcl-1 enforced expression rescue ovarian carcinoma cells from apoptosis triggered by BAPTA-AM/ABT-737 or W7/ABT-737 combinations. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing Mcl-1 ORF (Mcl-1) for 40 h as described in “Materials and methods” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. a DNA contents were studied for each condition. b Cell viability was assessed by trypan blue exclusion. c Mcl-1 expression and Caspase 3 cleavage were studied by western-blot. IGROV1-R10 cells were transfected with empty plasmid pcDNA (Empty) or pcDNA containing EIF4E ORF (eIF4E) for 40 h as described in “Materials and methods” section. Then cell were treated with DMSO or cotreated either with 10 µM BAPTA-AM and 10 µM ABT-737 or 40 µM W7 and 10 µM ABT-737 for 24 h. d DNA contents were studied for each condition. e Cell viability was assessed by trypan blue exclusion. f Mcl-1, eIF4E expression and PARP and Caspase 3 cleavage were studied by western-blot