Low molecular weight peptides derived from Iranian Scorpion (Odontobuthus bidentatus) Venom Induces Apoptosis in the Hepatocellular Carcinoma Cell Line (HepG2) in 3D Cell Culture

Abstract Background Hepatocellular carcinoma (HCC) is a deadly form of liver cancer with limited treatment options. Natural compounds, such as venom-derived peptides, have emerged as potential sources of anticancer agents. 3D cell models, which closely resemble the architectural characteristics of natural tumors, serve as an appropriate system for investigating the cytotoxic and apoptotic effects of scorpion venom on cancer cells. In this study, we investigated the apoptotic effects of low molecular weight peptides isolated from the venom of Odontobuthus bidentatus on HepG2 cells in a 3D cell culture model. Methods and Results The O. bidentatus venom was subjected to high-performance liquid chromatography (HPLC) for fractionation and purification of the low molecular weight peptides. Subsequently, the isolated peptides were evaluated for their impact on cell viability and apoptosis induction in HepG2 cells within a 3D cell culture system and were compared to crude venom. To create a 3D cell culture, HepG2 cells were enclosed within alginate hydrogel. Subsequently, the cytotoxic effects of scorpion venom were evaluated using MTT and neutral red uptake assays. Changes in the redox potential of HepG2 cells were assessed by measuring accumulated nitric oxide (NO) in the cell culture media, as well as levels of glutathione (GSH) and catalase activity. To determine the induction of apoptosis in cells treated with scorpion venom, various assays including alkaline comet assay, caspase-3 enzyme activity, and cytochrome c release were employed. Additionally, the expression of the pro-apoptotic gene BAX and the anti-apoptotic gene BCL -2 was evaluated using qRT-PCR. The results obtained from the MTT and neutral red uptake assays demonstrated that O. bidentatus crude venom and isolated fractions (5, 6, and 10) had cytotoxic effects on HepG2 cells in the 3D cell culture. The concentration of NO released into the culture media increased, while the levels of reduced glutathione and catalase decreased in a dose-dependent manner within the 3D culture. The findings from the caspase-3 enzyme activity, cytochrome c release assay, comet assay, and Bax / Bcl -2 gene expression ratio supported the conclusion that O. bidentatus scorpion venom induces apoptosis through the mitochondrial pathway. Conclusion This finding highlights the potential of scorpion venom-derived peptides as novel therapeutic agents for hepatocellular carcinoma.