Investigation of Apoptotic Mechanisms in Combination of IAP Inhibitor and Cisplatin in Epithelial Ovarian Cancer Cells

Project no. 30810 · Status: Kapanmış · 2023

Summary

Ovarian cancers are gynecologic cancers with high mortality and epithelial ovarian cancers are the most common subtype. In most metastatic patients chemotherapy resistance, especially to platinum-based drugs, leads to recurrent tumor relapses. Response to platinum-based chemotherapy depends on several parameters such as tumor biology and histology, molecular alterations and tumor microenvironment. Platinum agents cause DNA damage and subsequently induce apoptosis in cancer cells. Resistance to apoptosis is one of the most important targets of cancer therapy. Apoptosis is regulated by IAP proteins through inhibition and modulation of caspases and transcription factor (NF-κB). Cisplatin continues to be used clinically in the treatment of many epithelial ovarian cancers in various malignancies. Due to their anti-apoptotic activity, association with poor prognosis and correlation with treatment resistance in multiple malignancies, IAP proteins have become an important target for the development of anticancer therapeutics. SMAC (second mitochondrial activator of caspase) mimetics are being studied in clinical trials as monotherapy and in combinations of different chemotherapeutic drugs in various cancers. When cIAP1 levels are low, it activates cell death pathways more effectively, especially when combined with an increase in TNF-α, the potential for response to treatment is reported. TNF-α as an important component of the immune system, is a cytokine that regulates inflammation processes and cell death and high gene expression may increase susceptibility to increased inflammatory signaling and apoptosis. TNF-α may cause selective cell death in target cells, especially when used in combination with drugs that increase pro-apoptotic signaling in the treatment process. Based on this information, we investigated the effects of Birinapant, Cisplatin and their combinations on apoptotic mechanisms in epithelial ovarian cancer cells.For this aim, MTT assay was applied to in vitro epithelial ovarian cancer cell lines (SKOV3, MDAH2774) and healthy human endometrial stromal (HESC) cells to determine the IC50 application dose. Flow cytometry analysis was performed to control the amount of apoptosis in control and experimental groups. According to the IC50 results, immunofluorescent staining was performed in antibodies (cIAP1, MLKL, Caspase 3, Caspase 8, NFKB) determined for the relevant proteins in SKOV3 cells, the cell line with synergy in combination analysis and Imagej analyzes were performed by obtaining fluorescence intensities. In gene expression analyses, RT-PCR method was applied for gene expressions in related genes (MLKL, cIAP1, NFKB, Caspase 3, Caspase 8, TNF-α, MMP9, MMP2, CDH1, Vecadherin, Vimentin).In MTT and combination assays, IC50 doses were found to be synergistic for SKOV3 cells. Antagonist effects were found in MDAH2774 cell line. In immunofluorescence staining related to the Apoptosis and Necroptosis signaling pathway, it was found that the protein luminescence intensity Caspase 3, Caspase 8 were significantly higher and cIAP1 was significantly lower in the experimental groups compared to the control group. RT-PCR results in SKOV3 cell lines showed significant upregulation of Caspase 3, Caspase 8, CDH1 and MMP9 genes in the Cisplatin group compared to the control group. TNF-α and Vecadherin showed downregulation in the experimental groups. Significant upregulation was found in Caspase 3 and MMP9 genes in the birinapant group. NFKB, TNF-α, Vecadherin and MMP2 downregulation occurred in the experimental groups. Significant upregulation was found in TNF-α and MMP2 genes in the combination group. MMP9, cIAP1, Caspase 3, Caspase 8 and Vecadherin were downregulated in the experimental groups. The role of TNF-α in regulating apoptotic processes was found to be prominent in the combination group. It is thought that the increase in this cytokine may increase cancer cell death, especially with the activation of TNF-α-mediated apoptotic signals. Further bioinformatic analyses with genes showing differences in gene expression levels may provide important contributions to molecular targets and mechanisms for epithelial ovarian cancer treatment.Keywords: Apoptosis; Birinapant; Cisplatin; IAPs; Ovarian cancer.

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