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

Ovarian cancer is the leading cause of death by gynecological malignancies [1]. Standard first line treatment (debulking surgery followed by platinum/taxane based chemotherapy) and various second line chemotherapies appear generally unable to cure the disease [2], the therapeutic failure being mainly due to a multifactorial intrinsic or acquired chemoresistance [3, 4]. Thus, there is an urgent need of new strategies for the treatment of refractory disease.

Our previous work showed that platinum resistance was associated with the capacity of ovarian cancer cells to maintain a high level of two anti-apoptotic proteins of the Bcl-2 family, Bcl-x_L and Mcl-1 [5]. These proteins cooperate to protect ovarian cancer cells against apoptotic cell death and their concomitant inhibition leads to massive cell death even in the absence of chemotherapy. We also demonstrated that Mcl-1 down-regulation or inactivation leads to a strong sensitization to Bcl-x_L-targeting strategies, such as treatment by the BH3-mimetic ABT-737 [6‐8].

The 8H-thieno[2,3-b]pyrrolizinones, also named tripentones, constitute a promising family of novel compounds for cancer therapeutics [9]. Among them, the 3-(3-hydroxy-4-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one (MR22388, abbreviated TP) has shown a significant cell growth inhibitory activity in the nanomolar range over a panel of human tumor cell lines, including ovarian carcinoma cells, and can be considered as the lead of this family [9, 10]. To date, the precise mechanisms of action of MR22388 remain unknown.

Here we investigated the cytotoxic activity of MR22388 on cisplatin-resistant ovarian carcinoma cells and its potential as a modulator of the expression or activity of Bcl-x_L and Mcl-1. We also tested its cytotoxic activity when used in combination with ABT-737.

MR22388 [11] was purchased from Syntheval (Caen, France). ABT-737 was purchased from Selleckchem (Euromedex, Souffelweyersheim, France).

SKOV3 cell line obtained from ECACC (Cerdic, Nice, France) and chemoresistant IGROV1-R10 subline obtained from IGROV1 parental cells [12] were cultured as described [13]. Cells were continuously treated with MR22388 or ABT-737. Cell viability was determined by Trypan blue exclusion assay using Cedex XS device (Roche, France).

Compound-mediated cytotoxicity was monitored using Real-Time Cell Analyzer (RTCA) multi-plate Instrument, xCELLigence System (Roche Applied Science, Mannheim, Germany) [13]. The cell index reflects changes in cell viability as described elsewhere [14]. Briefly, cells were left to grow for 24 h before treatment in 96-well E-Plate and impedance was continuously measured until the end of the treatment. Standard deviations of well replicates were analyzed with the RTCA Software.

RNA isolation and quantitative reverse transcripase-PCR (RT-PCR) were performed as described elsewhere [7].

Determination of cellular DNA content was performed by flow cytometry as described elsewhere [13].

Preparation of cell extracts and western blot analysis were performed as described elsewhere [13]. The following antibodies were used: PARP, Caspase-3 and Bcl-x_L (Cell Signaling Technology by Ozyme, Saint-Quentin-en-Yvelines, France), MCL-1 (Santa Cruz by CliniSciences, Nanterre, France), phospho(Ser62)-Bcl-x_L (AssaybioTech by Tebu-bio, Le Perray-en-Yvelines, France) and Actin (Sigma-Aldrich, Saint-Quentin Fallavier, France).

MR22388 induced a strong cytostatic effect from 100 nM on both cell lines, with a slight dose-effect between 100 and 1000 nM (Figures  1A and 1B). Moreover, MR22388 exposure induced an S phase elongation in IGROV1-R10 cells and a G2-M phase blockade in both cell lines (Figure  1C). Massive cell death occurred in a dose-dependent manner after a 72 h exposure to MR22388 (Figure  1C), as shown by cell detachment and sub-G1 peak appearance on DNA content histograms. PARP and Caspases 9 and 3 cleavages have been observed in a time and concentration dependent manner (data not shown). However, the exposure to 100nM MR22388 allowed a slow recovery of a proliferating cell population in both cell lines (Figure  1A). At this concentration, MR22388 also induced a transient cell detachment and rounding of SKOV3 cells that was not associated to massive cell death, responsible for a transient decrease cell index on impedancemetry curves (Figure  1B, left panel). After 24 h, the cells recovered a normal adhesion but were unable to proliferate, the cell index thus reaching a plateau after 48 h.