Sensitization of ovarian carcinoma cells to Bcl-xL-targeting strategies through indirect modulation of Mcl-1 activity by MR22388, a molecule of the tripentone family

Abstract

Background: Our work has been carried out in the context of the therapeutic failure in ovarian carcinoma, which remains the leading cause of death by gynecologic malignancy. In these tumours, recurrence and subsequent acquired chemoresistance constitute major hurdles to successful therapy. Here we studied the interest of a member of the tripentone chemical family, MR22388, for the treatment of chemoresistant ovarian cancer cells.

Findings: MR22388 activity has been assessed in vitro on cisplatin-resistant (SKOV3 and IGROV1-R10) ovarian cancer cell lines by conventional analysis, alone or combined to a BH3-mimetic molecule, ABT-737. MR22388 exerts its activity on cisplatin resistant cells, and we showed that it induces a decrease of the Mcl-1 anti-apoptotic protein expression. Considering our previous work demonstrating that the efficiency of Bcl-xL targeting strategies is conditioned to the concomitant inhibition of Mcl-1 we studied the interest of the association of this MR22388 with ABT-737, and showed that this combination was highly cytotoxic in chemoresistant cells.

Conclusions: This work thus opens new perspectives for the use of this promising molecule for the treatment of highly chemoresistant ovarian cancer cells and for sensitization of emerging Bcl-xL targeting strategies such as the use of BH3-mimetic molecules.

Figure 1

MR22388 exerts a cytostatic and cytotoxic effect on cisplatin-resistant cell lines. Exponentially growing SKOV3 and IGROV1-R10 cells were treated with MR22388 at 10, 100 and 1000 nM. [A] Percentages of cell viability, assessed by trypan blue exclusion test after 24, 48 and 72 h exposure to MR22388, as compared to the initial number of viable cells in the flask. [B] Real time analysis of cellular activity of MR22388 obtained using the xCELLigence system. Cells were grown for 24 h, then treated with MR22388 (10, 100 and 1000 nM). Cell index was recorded every 2 h. The results are the mean of three replicates. [C] DNA content histograms and cellular morphologies, assessed 72 h after the beginning of the MR22388 exposure.

Figure 2

MR22388 modulates MCL-1 expression and Bcl-x_L phosphorylation. Cells were treated with MR22388 (100 and 1000 nM) during 24 h. [A] PARP cleavage and Mcl-1 expression, analyzed by western blot. Actin was used as loading control. [B] Mcl-1 expression quantified by Image J software. The relative intensity of each lane was calculated as compared to the DMSO sample. [C] Mcl-1 mRNA was quantified by qRT-PCR. [D] Bcl-x_L expression and [E] Ser62-phospho-Bcl-x_L detected by western blot.

Figure 3

MR22388 sensitizes ovarian cancer cells to ABT-737. Real time analysis of cellular activity of the combination of MR22388 and ABT-737 was performed using the xCELLigence system. Cells were grown for 24 h, then treated with MR22388 (10, 100 and 1000 nM). 24 h after the beginning of the exposure, IGROV1-R10 (top panel) and SKOV3 (bottom panel) were treated by 10 μM ABT-737. [A,E] Cell index, recorded every 30 min. The results are the mean of three replicates. [B] 4 h after the ABT-737 exposure, IGROV1-R10 DNA content histograms and cellular morphology were assessed. [C] 72 h after ABT-737 exposure, IGROV1-R10 cellular morphology was assessed. PARP and Caspase 3 cleavages were determined by western blot in IGROV1-R10 [D] and SKOV3 cells [F].