Investigation of Ras/Raf/MEK/ERK signaling-mediated apoptotic effects of combination of cucurbitacin D and i with sorafenib in hepatocellular carcinoma cell line

Aim: Sorafenib, a multi-tyrosine kinase inhibitor, is a systemic drug used as first-line therapy in the treatment of HCC. In order to increase overall survival, it is necessary to develop combined drug therapies that will increase the potency of sorafenib and prevent drug resistance. Cucurbitacins are natural compounds that show anticancer effects by suppressing over-activated signaling pathways in many cancer types. In this study, the anticarcinogenic effects of sorafenib-CuD and CuI combination on HepG2 cell line were investigated in order to increase the efficacy of sorafenib and decrease the treatment dose. Material and Method: Cell inhibition, migration, apoptosis, cell cycle distribution, mitochondrial membrane potential (ΔΨm) were investigated by applying CuD and CuI alone or in combination with sorafenib to HepG2 cells. In addition, in order to reveal how cucurbitacins affect the signal pathways known to affect sorafenib; proteins and genes involved in VEGF, EGFR, MMP-2, caspase cascade, PI3K/AKT/mTOR, Raf/MEK/ERK signaling pathways were assessed by western blot and qRT-PCR. Results: It has been shown that CuD and CuI have an antiproliferative effect at low concentrations and show a synergistic effect when combined with sorafenib. Combined administrations induced apoptosis by increasing caspase-9, Bax activity and inhibiting Bcl‑xL activation, blocking the cell cycle in G2/M phase and causing loss of ΔΨm. The combinations also suppressed MMP-2 and VEGF, reduced cell migration. The combined cucurbitacin-sorafenib applications inhibited the expression of proteins and genes involved in EGFR and PI3K/AKT/mTOR, Raf/MEK/ERK signaling pathways. Conclusion: Due to showing the synergetic effect of cucurbitacins with sorafenib and their targeting of similar signaling pathways reveal that their combination may increase the efficacy of sorafenib by suppressing angiogenic, metastatic and proliferative activity in HCC.