Abstract
OBJECTIVE: Hepatocellular carcinoma (HCC) represents one of the most fatal cancers worldwide, characterized by high mortality rates and poor prognosis. ABT-737, a small-molecule antagonist of the Bcl-2 family, has shown promise as an anticancer agent. However, the potential of ABT-737 to induce PANoptosis-a unique inflammatory programmed cell death pathway encompassing apoptosis, necroptosis, and pyroptosis-remains unexplored in HCC. This study aimed to investigate the potential of ABT-737 to induce PANoptosis in hepatocellular carcinoma cells and elucidate the underlying mechanisms governing its effects on proliferation, migration, and invasion. METHODS: Two human HCC cell lines (SK-HEP-1 and BEL-7402) were treated with ABT-737 at various concentrations (5, 10, and 20 µM) for 24 and 48 h. Cell morphology was examined under microscopy prior to each MTS assay to document cell death characteristics. Cell viability was assessed using MTS assay. BrdU incorporation assays were performed to specifically assess cell proliferation. Migration and invasion capabilities were evaluated through wound healing and transwell assays, respectively. To investigate PANoptosis pathway involvement, cells were co-treated with ABT-737 and specific inhibitors: Z-VAD-FMK (pan-caspase inhibitor, 10 µM), Necrostatin-1 (necroptosis inhibitor, 30 µM), or VX-765 (caspase-1 inhibitor, 10 µM). Flow cytometry analysis using Annexin V/PI staining was performed to directly assess cell death. Xenograft models were established in BALB/c nude mice using SK-HEP-1 and BEL-7402 cells to evaluate tumor formation with combination treatments. Tumor volumes were measured twice weekly, and tumor weights were recorded at the experimental endpoint. Ki-67 immunohistochemistry and H&E staining were performed on tumor sections to evaluate proliferation and necrosis. Western blot analysis was performed to examine the expression of PANoptosis-related proteins including phosphorylated MLKL (pMLKL) in both cultured cells and xenograft tumor tissues. RESULTS: ABT-737 demonstrated significant dose- and time-dependent inhibition of HCC cell proliferation. Microscopic examination revealed characteristic cell death morphology including cell shrinkage, membrane blebbing, and detachment in ABT-737-treated cells. Compared with the control group, ABT-737 treatment groups showed significantly reduced BrdU incorporation in a dose-dependent manner. Annexin V/PI flow cytometry demonstrated that compared with the control group, ABT-737 treatment groups exhibited significantly increased total apoptosis rates in a dose-dependent manner, confirming that the decrease in MTS absorbance reflects both reduced proliferation and increased cell death. Compared with the control group, ABT-737 treatment groups showed significantly reduced migration rates and invasive cell numbers. Co-treatment with PANoptosis pathway inhibitors partially restored cell viability compared with ABT-737 alone as measured by MTS assay. In xenograft models, compared with the control group, ABT-737 treatment group showed significantly reduced tumor volumes and tumor weights, which were partially reversed by pathway-specific inhibitors compared with ABT-737 alone. Compared with the control group, ABT-737-treated tumors showed significantly reduced Ki-67 positive rates, while H&E staining demonstrated markedly increased necrotic areas. Western blot analysis revealed that compared with the control group, ABT-737 treatment group showed upregulation of apoptosis markers (cleaved caspase-3, Bax), necroptosis markers (RIPK1, RIPK3, MLKL, and pMLKL), and pyroptosis markers (NLRP3, ASC, caspase-1), with concurrent downregulation of Bcl-2 in both cell lines and tumor tissues. CONCLUSION: ABT-737 exerts potent antitumor effects through the induction of PANoptosis in hepatocellular carcinoma, providing a promising therapeutic strategy for HCC treatment.