Investigation of the synergistic effect of combination therapy of MCL-1 inhibitor AZD5991 and genistein on MCF-7 breast cancer cells

Breast cancer is one of the most commonly diagnosed cancers worldwide and has the highest mortality rate among women. While early diagnosis has led to a reduction in mortality rates and increased survival rates for many patients, issues such as treatment costs, side effects, and resistance necessitate the exploration of alternative therapies. AZD5991 is an Mcl-1 protein inhibitor that induces apoptosis by targeting a gene involved in critical biological processes such as cell cycle regulation and apoptosis. Genistein, an isoflavone found in certain legumes, is also a potential chemotherapeutic agent known to regulate similar cellular processes in cancer cells. To date, there are no studies in the literature investigating the combined use of these two agents. In this context, our study comparatively investigates the cytotoxic, antiproliferative, and apoptotic effects of the combination therapy of the Mcl-1 inhibitor AZD5991 and the isoflavone genistein on the human breast cancer cell line MCF-7, including their effects on protein expression levels, compared to control groups. The data were analyzed using GraphPad Prism and ImageJ software. Cell viability was assessed using the SRB assay, and optimal efficacy was observed after 48 hours of incubation with 100 µM Genistein, 30 µM AZD5991, and a combination dose of 73 µM Genistein + 10 µM AZD5991. In single-agent treatments, cell viability was determined to be 63% for Genistein and 61% for AZD5991, whereas combination treatment reduced this rate to 43%. Cell migration was analyzed using the wound healing assay, and both Genistein and AZD5991 significantly inhibited migration (p<0.0001). Apoptosis was evaluated based on mitochondrial membrane potential using the JC-1 dye. The effects of Genistein, AZD5991, and their combination on the expression levels of target genes in MCF-7 cells were also assessed. ΔΔCt and fold change analyses of genes Bax, Bcl-2, Caspase-9, P21 and Caspase-8 revealed that Genistein treatment caused significant changes in the expression of Bax, Bcl-2, Caspase-9, P21 and Caspase-8. The expression levels of apoptosis-related proteins Mcl-1, Bax, P21, P53 and Parp-1 were examined using western blot analysis. In MCF-7 cells, both monotherapies and the combination therapy led to a decrease in Mcl-1 expression and an increase in Bax expression. The data obtained from our study are valuable in terms of creating preliminary data for further studies on the use of Genistein and AZD5991 in the treatment of breast cancer. Keywords: Breast Cancer; MCF-7; Apoptosis; AZD5991; Genistein; Mcl-1