In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down-regulation of Bcl-x L expression and apoptosis, whereas recurrence was systematically observed when Bcl-x L expression was maintained. Bcl-x L down-regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bclx L targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re-proliferate. Considering the possible cooperation between Bcl-x L and MCL-1, we investigated the possibility to avoid recurrence in vitro using a multi-targeted RNAi strategy directed against these two anti-apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi-targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl-x L and MCL-1 cooperate to constitute together a strong molecular ''bolt'', which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down-regulation of Bcl-x L and MCL-1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl-x L and MCL-1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.Ovarian cancer is the leading cause of death from gynecological malignancies worldwide and the fifth most common cause of cancer death in women. 1 Early diagnosis is difficult owing to the asymptomatic character of this disease in early stages, and more than 70% of these cancers are diagnosed in an advanced stage (FIGO stages III or IV). Patients with advanced ovarian cancer are treated initially with optimal debulking surgery and standard chemotherapy (platinumdrugs usually associated to taxanes). 2 Despite an initial 70-80% response rate, most patients will relapse within 1-2 years and develop resistance to chemotherapy 3 and the overall 5-year survival is less than 30%. The identification of new drugs or novel therapeutic strategies able to (re)sensitize ovarian carcinoma cells to existing chemotherapy thus appear as a major challenge.Cisplatin, a DNA-damaging agent that forms DNA adducts, is commonly used for the treatment of advanced ovarian cancers. It is widely accepted that these adducts lead to a cell cycle arrest followed by the induction of apoptotic cell death. 4 However, it should be considered that in resistant cells, a strong protection against apoptosis exist when the DNA damages reach their maximum level, to allow the reparation of these injuries and the r...

Chemoresistance of ovarian carcinoma has been associated previously to the absence of Bcl-x L expression downregulation in response to cisplatin. Among BH3-mimetic molecules constituting promising anticancer agents able to inhibit the activity of antiapoptotic Bcl-2 family proteins, we evaluated the effect of one of them, HA14-1, on various ovarian carcinoma cell lines. In response to HA14-1, the cisplatin-resistant IGROV1-R10 cell line underwent massive cell death, whereas other cell lines presented a partial response (IGROV1, SKOV3, and A2780) or did not respond to this molecule (OAW42 and OAW42-R). However, the expression of HA14-1 targets (Bcl-2 and Bcl-x L ) did not correlate to these different responses. In contrast, cell death was associated with the disappearance of Mcl-1 after exposure to HA14-1. We showed that, in the HA14-1 nonresponsive cell lines (SKOV3 and OAW42), small interfering RNA-mediated Mcl-1 downregulation allowed HA14-1-induced massive apoptosis in the absence of chemotherapy. Furthermore, cisplatininduced Mcl-1 downregulation was also able to sensitize highly chemoresistant SKOV3 cells to HA14-1. Taken together, these results show that Bcl-x L and Mcl-1 are able to cooperate to protect ovarian carcinoma cells against oncogenic stress or chemotherapy-induced apoptosis and suggest that the development of multitargeted strategies directed against these two antiapoptotic proteins may constitute a major challenge for the therapeutic care of chemoresistant ovarian carcinomas. BH3-mimetic compounds represent promising tools for this purpose either on their own (direct or indirect pan-inhibitors) or in combination with new drugs aiming to inactivate

Ovarian cancer is the leading cause of death from gynecological cancer. The anti-apoptotic protein Bcl-x(L) is frequently overexpressed in ovarian carcinoma which correlates with chemotherapy resistance. It has been demonstrated that Bcl-x(L) cooperates with another anti-apoptotic protein, Mcl-1, to protect ovarian cancer cells against apoptosis, and that their concomitant inhibition induces massive cell death. Here, we examined the interest of ABT-737, a potent BH3-mimetic molecule targeting Bcl-x(L), both alone and in combination with Mcl-1 modulators, in ovarian cancer cell lines. As a single agent, ABT-737 was ineffective at promoting cell death in the four cell lines we tested in vitro. However, the specific inhibition of Mcl-1 by siRNA dramatically increased the sensitivity of chemoresistant cells to ABT-737. Platinum compounds also sensitize to ABT-737 by dose-dependently decreasing Mcl-1 expression or by increasing the expression of pro-apoptotic BH3-only proteins Noxa and, to a lower extent, Bim. Furthermore, we demonstrated that Noxa accumulation was involved in apoptosis occurring in response to the combination of ABT-737 and platinum compounds, since cells were protected from apoptosis by its silencing. Moreover, the combination was also highly cytotoxic ex vivo in sliced SKOV3 tumor nodes. However we observed in these slices a strong basal expression of Noxa and apoptotic cell death in response to ABT-737 alone. Therefore, we have revealed that the modulation of the Mcl-1/Noxa axis by platinum compounds results in a strong sensitization of chemoresistant ovarian carcinoma cells to ABT-737, which could constitute a promising therapeutic in these cancers.

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In ovarian carcinoma, chemoresistance is the main responsible for the poor 5-year overall survival, remaining under 30%. Protection against apoptotic cell death is particularly involved in this chemoresistance, and among the observed alterations, overexpression of anti-apoptotic Bcl-2 proteins is of first importance. Among them, Bcl-xL et Mcl-1 appeared to cooperate to protect ovarian cancer cells against apoptosis, thus constituting together pertinent targets. In this context, the use of BH3-mimetic molecule ABT-737, that targets Bcl-xL, could constitute an alternative strategy to reverse the platinum chemoresistance in ovarian carcinoma. However, ABT-737 remaining unable to efficiently inhibit Mcl-1 activity, its clinical use in ovarian carcinoma thus requires defining of another tool able to inhibit Mcl-1. In this study, we investigated in vitro and ex vivo the capacity of platinum derivatives (cisplatin and carboplatin) to inhibit Mcl-1 expression or activity and therefore sensitize ovarian carcinoma cells to ABT-737. SKOV3 and IGROV1-R10 platinum-resistant ovarian cancer cell lines were exposed to these drugs, as single agents or associated, using various exposure protocols in vitro or ex vivo (SKOV3 and IGROV1-R10 xenografted tumor nodes developed in nude mice and subsequently used for slicing and ex vivo treatment). We thus studied apoptosis induction as well as Mcl-1 and pro-apoptotic BH3-only proteins expression. In vitro study: whereas neither cisplatin nor ABT-737 alone presented any toxicity, the association of ABT-737 and platinum was highly cytotoxic in both cell lines. We tested several protocols and showed that ABT-737 must be present during the 24h following carboplatin exposure to allow cell death. These observations are in agreement with the needed down-regulation or inactivation of Mcl-1 by platinum prior or concomitant with ABT-737 exposure to induce cell death. As expected, cisplatin as well as carboplatin were able either to inhibit Mcl-1 expression or to induce BH3-only expression (particularly Noxa and Puma). Moreover, we observed a synergetic effect of the association ABT-737/platinum on BH3-only Bim and Noxa expression, these events being correlated to the ability of platinum to sensitize to ABT-737. Ex vivo study: we confirmed these observations in SKOV3 and IGROV1-R10 ex vivo tumor slices models. Whereas ABT-737 and platinum derivatives remained poorly cytotoxic or completely ineffective as single agents, their association was highly cytotoxic. In conclusion, this strategy associating ABT-737 to platinum appears as an attractive way to reverse resistance to platinum derivatives that remain the most active drugs in ovarian cancer. Moreover, this study presents platinum derivatives as pertinent sensitizers to ABT-737, through its direct or indirect Mcl-1 inhibition, opening new perspectives for the clinical use of this promising BH3-mimetic molecule. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1777. doi:10.1158/1538-7445.AM2011-1777