Background: Natural venoms have biological activities including anti-inflammatory, antimicrobial, and anticancer effects.  Hepatocellular carcinoma (HCC) is still a worldwide problem and difficult to treat by chemotherapeutic agents especially sorafenib (SOR), as it evokes many harsh side effects and is disable to differentiate between normal and cancer cells. Objective: The present study aimed to test the hypothesis that combining crude venoms of the snake or the bee or the scorpion could synergistically enhance the antiproliferative effects of SOR in hepatocellular carcinoma cell line (HepG2). Experimental design: Separate crude venoms have been applied to HepG2 cells and normal human retinal cells (RBE1) for estimation of IC₅₀. The most effective venom has been combined with sorafenib in five nonconstant ratios and the combination index (CI) was estimated to expose their synergistic or antagonistic action. The best combination was used for downstream analysis. Results: The crude snake venom exhibited the most cytotoxic effect and the least IC₅₀. It has been combined with sorafenib, and the combination index (CI) was calculated. IC₂₅ SV + IC₁₀ SOR was the best combination with CI=0.209 indicating high synergistic cytotoxic activity against HepG2. The underlining molecular mechanisms of action, in terms of the expression level of apoptotic genes (p53, Bax, Caspase 3, and Bcl2), flow cytometric analysis of cell cycle, oxidative stress markers as well as the activity of some limiting enzymes in the glycolytic pathway (ALDOB, PK and LDH) have been investigated.
Conclusion: Our results suggest a novel synergistic, and anti-proliferative effect of snake venom with sorafenib on HepG2 cells.