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

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.


Background
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][7][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.

Cell culture and treatment
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).

Real-time cellular activity assay (xCELLigence)
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.
Determination of cellular DNA content was performed by flow cytometry as described elsewhere [13].

Results
Effect of MR22388 on the proliferation and apoptosis of SKOV3 and IGROV1-R10 cisplatin-resistant cell lines 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.

MR22388 modulates MCL-1 expression and Bcl-x L phosphorylation
A 24 h exposure to MR22388 led to a half-decrease of Mcl-1 expression in both cell lines from 100 nM (Figure 2A and 2B), in absence of cell death. This Mcl-1 protein disappearance was not associated to transcriptionnal down-regulation ( Figure 2C). Otherwise, at the same time, MR22388 did not modified Bcl-x L expression but induced the appearance of a slower-migrating Bcl-x L band evocative of a phosphorylated form ( Figure 2D). We showed that this band was at least in part constituted by a Ser62-phosphorylated form of Bcl-x L ( Figure 2E).

MR22388 sensitizes to ABT-737
After a 24 h exposure to MR22388 (when Mcl-1 expression is decreased), cells were treated by ABT-737. Whereas 100 nM MR22388 alone allowed the recovery of proliferative population, as previously evocated, its association with ABT-737 suppressed this recovery in both cell lines ( Figure 3A and 3E), leading to the complete annihilation of the population. In contrast, ABT-737 had no (SKOV3) or little (IGROV1-R10) effect as a single agent.
This strong cytotoxic effect of the association is confirmed by morphological observations (Figure 3B and 3C). Moreover, the combination MR22388/ABT-737 led to a complete PARP and Caspase-3 cleavage, evocative of massive cell death ( Figure 3D and F), whereas MR22388 or ABT-737 alone did not. Similar results were obtained on both cell lines using siRNA directed against Bcl-x L instead of ABT-737 (data not shown).

Discussion
The tripentone MR22388 has been identified as a potential anti-tumour agent and demonstrated strong in vitro anti- proliferative and cytotoxic activities on various cell types [10,15,16]. We observed similar effects in two chemoresistant ovarian cancer cell lines. MR22388 exposure induced strong cell cycle perturbations and triggered apoptosis in a concentration dependent manner.
MR22388 has been shown to be able to exert various effects such as anti-tubulin effects [15] and inhibition of GSK3, CDK1 [11] and showed an affinity for FLT3-ITD [10].
However, whatever are the MR22388 direct targets, we focused our attention on possible indirect effects of MR22388 on Bcl-2 family members since we previously demonstrated that anti-apoptotic proteins Bcl-x L and Mcl-1 cooperate to protect ovarian carcinoma cells against apoptosis. Indeed, their concomitant inhibition leads to massive cell death, even in absence of chemotherapy [6,7]. Here we showed that Mcl-1 protein (but not mRNA) was drastically decreased in both cell lines after exposure to MR22388.
Mcl-1 is submitted to a rapid turnover, and the control of its expression could involve both transcriptional and  post-translational mechanisms. Its interactions with other Bcl-2 family members can favour its stabilization or its degradation, depending of the partner [17]. Mcl-1 degradation can involve the proteasome pathway following JNK and GSK3 associated Mcl-1 phosphorylations [18], but can also be a consequence of caspases activity [19]. This last possibility has been excluded in our model since Mcl-1 was decreased even in condition in which apoptosis is not detected (24 h).
We also detected the appearance of the ser62phosphorylated form of Bcl-x L . Phosphorylated forms have been previously described by others in response to various stresses [20,21]. Ser62-phosphorylated form has been associated with a loss of anti-apoptotic function [22] and to the appearance of a capacity of phospho-Bcl-x L to translocate in the nucleus to lead to a G2-M cell cycle arrest. Moreover, these events as well as Bcl-x L ser62-phosphorylation have been related to the activation of JNK, among other kinases [23].
Interestingly, it has also been shown that phosphorylation of Mcl-1 by JNK (itself activated by various apoptotic stresses) is required for subsequent docking of GSK3 on Mcl-1 allowing Mcl-1 phosphorylation, ubiquitinylation and degradation [24].
Altogether, our observations (G2-M blockade, Mcl-1 expression decrease and phosphorylation of Bcl-x L on ser62) argue in favour of an activation of cell-death related signalling pathways such as JNK pathway by MR22388.
In any case, the capacity of MR22388 to inhibit Mcl-1 could be positively exploited in the context of Bcl-x Ltargeting strategies. Here we showed that the association of MR22388 with ABT-737 was highly cytotoxic, in a manner correlated with the disappearance of Mcl-1.
Here we could hypothesize the existence of cooperation between tripentone that decrease Mcl-1 expression level and ABT-737 that inhibits Bcl-x L and consequently releases the BH3-only protein Bim from Bcl-x L. Bim subsequently becomes able to inactivate residual Mcl-1 protein, and to activate Bax/Bak apoptotic proteins. Indeed, a shuttling of Bim sequestration between Mcl-1 and Bcl-x L , depending of their respective expression level has been recently described [25], Bcl-x L being here presented as a determinant of the response to Mcl-1 inhibiting agents. In our model, MR22388 and ABT-737 should thus be considered as co-sensitizer agents.
In summary, although MR22388 activity is not yet fully understood, its interesting effect on the modulation of Bcl-2 family proteins could be positively exploited in the context of Bcl-x L -targeting strategies such as emerging BH3-mimetic molecules.