Escin-sorafenib Synergy Up-regulates LC3-II and P62 to Induce Apoptosis in Hepatocellular Carcinoma Cells

Introduction: Hepatocellular carcinoma (HCC) is a common solid cancer and the leading cause of cancer deaths worldwide. Sorafenib is the first drug used to treat HCC but its effectiveness needs to be improved, and it is important to find ways to treat cancer that combine sorafenib with other drugs. Synergistic therapies lower effective drug doses and side effects while enhancing the anticancer effect.

Purpose: In the present study, the therapeutic potential of sorafenib in combination with escin and its underlying mechanism in targeting liver cancer has been established.

Study Design/methods: The IC of sorafenib and escin against HepG2, PLC/PRF5 and Huh7 cell lines were determined using MTT assay. The combination index, dose reduction index, isobologram and concentrations producing synergy were evaluated using the Chou-Talaly algorithm. The sub-effective concentration of sorafenib and escin was selected to analyze cytotoxic synergistic potential. Cellular ROS, mitochondrial membrane potential, annexin V and cell cycle were evaluated using a flow-cytometer, and autophagy biomarkers were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role. A DEN-induced liver cancer rat model was developed to check the synergy of sorafenib and escin.

Results: Different concentrations of escin reduced the IC of sorafenib in HepG2, PLC/PRF5 and Huh7 cell lines. Chou-Talaly algorithm determined cytotoxic synergistic concentrations of sorafenib and escin in these cell lines. Mechanistically, this combination over-expressed p62 and LC-II, reflecting autophagy block and induced late apoptosis, further reconfirmed by ATG5 knockdown. Sorafenib and escin combination  reduced HCC serum biomarker α-feto protein (α-FP) by 1.5 folds. This combination restricted liver weight, tumor number and size, also, conserved morphological features of liver cells. The combination selectively targeted the G /G phase of cancer cells.

Conclusion: Escin and sorafenib combination potentially up-regulates p62 to block autophagy to induce late apoptosis in liver cancer cells.