Inhibition of Mcl-1 expression by citrate enhances the effect of Bcl-xL inhibitors on human ovarian carcinoma cells

Abstract

The inhibition of two major anti-apoptotic proteins, Bcl-xL and Mcl-1, appears essential to destroy chemoresistant cancer cells. We have studied their concomitant inhibition, using ABT 737 or siRNA targeting XL1 and citrate, a molecule which reduces the expression level of Mcl-1.Two cisplatin-chemoresistant ovarian cell lines (SKOV3 and IGROV1-R10) were exposed to ABT 737 or siRNA targeting XL1 and citrate at various individual concentrations, or combined. Cell proliferation, cell cycle repartition and nuclear staining with DAPI were recorded. Western blot analyses were performed to detect various proteins implied in apoptotic cell death pathways.Mcl-1 expression was barely reduced when cells were exposed to citrate alone, whereas a mild reduction was observed after ABT 737 treatment. Concomitant inhibition of Bcl-xL and Mcl-1 using ABT 737 or siXL1 associated with citrate was far more effective in inhibiting cell proliferation and inducing cell death than treatment alone.Given that few, if any, specific inhibitors of Mcl-1 are currently available, anti-glycolytic agents such as citrate could be tested in association with synthetic inhibitors of Bcl-xL.

Figure 1

Effect of citrate on two ovarian cancer cell lines: SKOV3 (A) and IGROV1-R10 (B). Evolution of viable cells up to 72 H after continuous exposure to 5, 10 and 20 mM of citrate (CT). Exponentially growing SKOV3 cells (A) or IGROV1-R10 cells (B) were treated with different citrate concentrations in the medium with SVF. The number of viable cells was assessed by the trypan blue exclusion test at various times after treatment. Results are expressed as the mean values of two independent experiments. Analysis of variance was used to determine significance. **: p < 0.05; ***: p < 0.001.

Figure 2

Effect of various citrate (CT) concentrations (5, 10, 20 mM) in IGROV1-R10 cells after 24 and 72 H exposure. (A) The morphological features of cell layers were observed by photon microscopy. (B) DNA content was determined at 24 H or 72 H by flow cytometry after propidium iodide staining (for each condition, percentages of cells in the different phases of the cell cycle are indicated). Protein expression levels of PARP cleavage and caspase 3 cleavage and Mcl-1 and Bcl-x_L on SKVO3 cells (C) and on IGROV1-R10 cells (D) by western blot analysis in response to citrate at 6 and 24 H. Mcl-1 mRNA expression in SKOV3 cells (E) and IGROV1-R10 cells (F) treated with citrate for 6 H or 24 H was assessed using real-time quantitative reverse transcription PCR. GAPDH was used as a housekeeping reference gene for normalisation. Each relative mRNA expression level was calculated in comparison to the control cell expression level.

Figure 3

Effect of siXL1 in response to citrate in the SKOV3 cell line. Cells were transfected with 20 nM siXL1 24 H before exposure to 10 mM of citrate and cultured for an additional 48 H (i.e. 72 H post transfection). Evolution of viable cells up to 72 H (A) in SKOV3. Results are expressed as a percentage of viable cells compared to the 100% control cells. Results are expressed as the mean values of two independent experiments. Analysis of variance was used to determine significance. **: p < 0.05; ***: p < 0.001. (B) Cell cycle repartition was studied by flow cytometry after propidium iodide staining at 72 H after transfection in SKOV3 cell lines. (C) The nuclear morphology of SKOV3 cells was analysed by DAPI staining at 72 H. Bars 20 μm.

Figure 4

Effect of siXL1 on response to citrate in the IGROV1-R10 cell line. Cells were transfected with 20 nM siXL1 24 H before exposure to 10 mM of citrate and cultured for an additional 48 H (i.e. 72 H post transfection). (A) Evolution of viable cells up to 72 H in IGROV1-R10. Results are expressed as a percentage of viable cells compared to the 100% control cells. Results are expressed as the mean values of two independent experiments. Analysis of variance was used to determine significance. **: p < 0.05; ***: p < 0.001. (B) Cell cycle repartition was studied by flow cytometry after propidium iodide staining at 72 H after transfection in IGROV1-R10 cell lines. (C) Nuclear morphology of IGROV1-R10 cells was analysed by DAPI staining at 72 H. Bars 20 μm.

Figure 5

Protein expression in SKOV3 (A) and IGROV1-R10 (B). Levels of PARP cleavage and caspase 3 cleavage and Mcl-1 and Bcl-x_L were detected by western blot analysis in response to combined treatment with siXL1 and citrate 20 mM, compared with no treatment (control) or siGFP (siRNA control) or citrate alone at 72 H. Actin was used as a loading control.

Figure 6

Effect of citrate on response to ABT-737 in IGROV1-R10 cell lines. Cells were treated with 10 mM citrate 24 H before exposure to 10 μM of ABT-737 and cultured for an additional 24 H (i.e. 48 H post citrate exposure). (A) Morphological features of cell layers at 72 H after citrate exposure. (B) Nuclear morphology of IGROV1-R10 cells was analysed by DAPI staining at 72 H. Bars 20 μm. (C) Cell cycle repartition was studied by flow cytometry after propidium iodide staining at 72 H after transfection in IGROV1-R10 cell lines. (For each condition, percentages of cells in the different phases of the cell cycle are indicated). (D) Protein expression levels of PARP cleavage, Mcl-1 and Bcl-x_L on IGROV1-R10 cells. Actin was used as a loading control.