ИСТИНА

Contemporary oncotherapy focuses on the induction of apoptosis in tumor cells. However, cancer cells have mastered to develop resistance to apoptosis, which frequently accomplished through the elevated expression of Bcl-2 family proteins, particularly Mcl-1L. The increased level of Mcl-1L is shown in many hematological and solid neoplasms. A great effort has been made to target Mcl-1L using small molecules; this recently has culminated in the discovery of Mcl-1L-selective BH3-mimetics. Moreover, a conceptually new approach has emerged that allows not only to antagonize the antiapoptotic functions of Mcl-1L but also get Mcl-1 on the apoptotic track – the approach of alternative splicing (AS) switch toward Mcl-1S isoform. In the study, we evaluated the extent of sensitizing tumor cells to the apoptotic effect of a DNA damaging agent cisplatin upon Mcl-1L inhibition. The cancer cell lines (ovarian carcinoma Caov-4, cervical adenocarcinoma Hela) were selected as they are derived from the tumors that show clinically significant overexpression of Mcl-1L. The high-affinity BH3-mimetic А-1210477 was used to block the binding of BH3-motif of pro-apoptotic proteins to Mcl-1L. Beside the small-molecule mediated inhibition, the level of Mcl-1L was adjusted by siRNA knockdown technique. Analysis of apoptosis and monitoring of Mcl-1 inhibition were implemented using immunoblotting and flow cytometric Annexin V/PI measurement. The obtained data points to the significant sensitizing of the studied cell lines to cisplatin under siRNA knockdown. Namely, the percentage of viable tumor cells after siRNA and cisplatin (24h) treatment declined to 57% for Caov-4 and 35.7% for Hela. Nevertheless, chemical inhibition did not yield comparable effects: after А-1210477 and cisplatin treatment over the same time frame, alive cancer cells constituted 81.7% for Caov-4 and 64% for Hela. Such a difference in the efficacy of genetic and pharmacological inhibition can be attributed to the inability of А-1210477 to disrupt protein complexes with remarkably high‐binding affinity. The alternative way to address Mcl-1L-dependent tumors is to cause AS switch of pre-mRNA MCL-1 toward an exon-2 deficient form MCL-1S. Mcl-1S possesses only BH3-motif, thus acting as a proapoptotic BH3-only protein. In our work, it was confirmed that the core splicing protein SF3B1 inhibitor FR901464 increases the ratio of Mcl-1S/Mcl-1L and triggers significant cell death. However, the compound also changes the splicing of other proteins, such as AURKB. We also demonstrated that mitotic catastrophe upon microtubule-damaging agents (Nocodazole, Monastrol) and doxorubicin causes the AS switch toward Mcl-1S. Potentially, this effect can be used in oncotherapy for sensitizing tumors to apoptosis.