ANALYZING THE ROLE OF MDMX COMPARED TO MDM2 IN REGULATING CELL DEATH IN PROSTATE CANCER CELLS

Abstract

Prostate cancer is the most prominent cancer type and the second leading cause of cancer death among men in the USA. In this study, we evaluated the role of MDMX compared to MDM2, the two proto-oncogenes involved in the negative regulation of p53, in causing cell death and cell cycle arrest through p53 dependent and independent mechanisms. We observed that inhibition of MDMX decreased the cell viability of LNCaP prostate cancer cells in response to treatments with both NSC (NSC-207895) and SJ (SJ-172550), which are MDMX specific inhibitors. MDMX inhibition appears to induce necroptosis in LNCaP and PC3 prostate cancer cells by elevating the expression of receptor-interacting protein kinase 1 and receptor-interacting protein kinase 3 (RIP1 and pRIP3) and phosphor-mixed lineage kinase domain-like protein (pMLKL), which are key elements of the necroptotic pathway. These data were consistent with fluorescence images that were obtained using SYTOX® Green which is specific for detecting cells that undergo programmed or other forms of cell death. Moreover, the fluorescence images using DEVD staining, which is a fluorogenic substrate that can bind to caspases 3/7 during apoptosis, revealed that MDM2 inhibition by RG-7388 induced apoptosis through caspase activation. To further confirm the effects of MDMX on cell migration, NSC effectively blocked the cancer cell migration in LNCaP, not PC3 cells during the scratch assay. However, RG-7388 didn’t have a significant effect on cell migration but mainly caused cell death. Moreover, MDM2 inhibition by RG-7388 increases the expression of p21 and induces PARP cleavage, which highlights its role in cell cycle arrest and apoptosis. Analysis of the miRNA has revealed that inhibition of MDMX and MDM2 can significantly upregulate hsa-let-7d-5p, hsa-let- 7g-5p, hsa-miR-15a-5p, and hsa-miR-106b-5p levels, which have tumor suppressor characteristics. Furthermore, oncogenic miRNAs (OncomiRs) such as hsa-miR-10a-5p and hsa-miR-30b-5p were downregulated, which is in favor of the cell cycle arrest and cell death. The overall outcomes of our experiments provide additional knowledge and strategies for making an accurate assessment of the most suitable therapeutic strategies for achieving effective treatments for MDMX-positive prostate cancer.