Simultaneous Inhibition of MDM2 and XIAP by MX69 Induced Cell Cycle Arrest and Apoptosis in HUH7 and Hep3B Cell Lines.

Genomic instability not only drives tumor initiation and progression but also cooperates with apoptosis resistance to promote therapeutic evasion in hepatocellular carcinoma (HCC). Activation of MDM2, a negative regulator of p53, together with XIAP overexpression, represents a critical axis underlying this resistance. Simultaneous targeting of MDM2 and XIAP by MX69, a small molecule inhibitor, may therefore offer a potent interventional strategy to suppress cell proliferation and enhance pro-apoptotic signaling in HCC in vitro models. To evaluate the effects of MX69, cell viability was assessed via CVDK-8, colony formation, and real-time cell analysis. Oxidative stress levels and DNA damage were examined using fluorescence imaging and comet assays, respectively, while mitochondrial membrane potential was monitored through JC-1 staining. Furthermore, flow cytometry was employed to quantify apoptotic cell death and cell cycle distribution, while Western blot analysis was used to characterize the expression of apoptosis-related proteins. In vitro cytotoxicity assays revealed that MX69 reduced the viability of HUH7 and Hep3B cells in a dose-dependent manner, suppressed colony formation, and exerted anti-proliferative effects in real-time proliferation assays. Cell viability and IC50 values were evaluated using CVDK-8 and RTCA assays. Furthermore, MX69 induced oxidative stress and mitochondrial dysfunction, as evidenced by elevated ROS levels and loss of mitochondrial membrane potential. This was accompanied by significant DNA damage, detected by comet assay and γ-H2AX immunofluorescence, and G0-G1 cell cycle arrest. Moreover, MX69 triggered apoptotic cell death, demonstrating potent anticancer activity. Collectively, our findings identify MDM2/XIAP dual inhibition by MX69 as a promising therapeutic approach in HCC, with potential to overcome apoptosis resistance linked to genomic instability.