AdipoRon Promotes Mitochondrial Ca<sup><b>2+</b></sup> Overload and Apoptosis in Hepatocellular Carcinoma Cells by Activating the PLC-IP3-IP3R Signaling Pathway

Objective: Hepatocellular carcinoma (HCC) ranks among the most prevalent malignant tumors globally. Metabolically associated fatty liver disease is a significant risk factor for HCC. Adiponectin, a key regulatory protein in glucolipid metabolism, presents potential as an anti-tumor target in HCC cells. The study focused on evaluating the anti-HCC properties of AdipoRon, an agonist of the adiponectin receptor. Method: Cell viability and proliferation were assessed using the cell counting kit-8 and colony formation assays, respectively. AdipoRon’s effect on HCC cell damage was evaluated via flow cytometry, apoptosis, and (lactate dehydrogenase) LDH assays. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (MMP), ATP levels, and Complex I activity. Additionally, mitochondrial reactive oxygen species (ROS) and calcium (Ca2+) levels were analyzed using MitoSOX Red and Rhod-2 AM probes, respectively. Results: Our findings indicated that AdipoRon suppressed the proliferation of HCC cells and triggered apoptosis, with both effects being dose-dependent. Furthermore, AdipoRon caused a decrease in mitochondrial membrane potential, ATP levels, and Complex I activity, alongside the generation of mitochondrial ROS. Notably, AdipoRon disrupted intracellular Ca2+ homeostasis by causing mitochondrial Ca2+ overload due to release from the endoplasmic reticulum (ER). Additionally, AdipoRon promoted Ca2+ release from the ER by activating the PLC-IP3-IP3R pathway. The resulting mitochondrial Ca2+ overload enhances the anti-HCC effect when combined with chemotherapeutic drugs. Conclusions: Therefore, our study demonstrates that AdipoRon promotes mitochondrial Ca2+ overload and apoptosis in HCC cells by activating the PLC-IP3-IP3R signaling pathway. AdipoRon has the potential to become an effective anti-HCC drug.