Investigating EGFR-VEGF-mediated apoptotic effect of cucurbitacin D and I combination with sorafenib via Ras/Raf/MEK/ERK and PI3K/Akt signaling pathways

Sorafenib, which is a type of systemic multi-kinase inhibitor drug, is used for first-line therapy in treating hepatocellular carcinoma (HCC). In this study, the anticarcinogenic effects of sorafenib–cucurbitacin D and sorafenib–cucurbitacin I combination on HepG2 cell line were investigated. Cell inhibition, migration, apoptosis, cell cycle distribution, mitochondrial membrane potential (ΔΨm), colony formation, and wound healing were investigated by applying cucurbitacin D and I 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. It has been shown that cucurbitacin D and I 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. 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. Graphical abstract