Natural Phenolic Compounds Enhance the Lethality of the Multi-kinase Inhibitor Sorafenib in Human Colorectal and Hepatocellular Cancer Cells.

Abstract

Cancer is a leading cause of death worldwide. However, drug resistance and the persistent unenviable side-effects are the major obstacles to successful chemotherapeutic treatment. Sorafenib (Sora), a multi-targeted tyrosine kinase inhibitor (TKIs), with potent antiangiogenic and antitumor activities, has been approved by FDA for the treatment of several types of cancer. Effective strategies to reduce the undesirable side-effects and enhance the anticancer activity of TKIs are much needed. Flavonoids act as anticancer therapeutic agents due to their capacity to inactivate carcinogenesis, inhibit angiogenesis and halt cell proliferation or promote apoptosis. In this study, we endeavored to investigate the effect of combination treatment with Sora and various natural phenolic flavonoids, on human colorectal (CRC) and advanced hepatocellular carcinoma (HCC) cell growth, as well as the intracellular transduction pathways that regulate cell cycle and apoptosis. Four phenolic compounds ; curcumin, quercetin, kaempferol and resveratrol were selected on the basis of their % Cytotoxicity (0 ≤ 20) against CRL1554 human fibroblast cells, for further investigations using CRC cell lines (SW1116 and SW837) and HCC cell lines (Hep3b and HepG2). The synergistic lethality of Sora in combination with the selected phenolic compounds was tested against all four cancer cell lines, in three different treatment regimens; sequential, reverse sequential and simultaneous. The combination treatments proved to be cytotoxic to all cancer cell lines in a dose- and time-dependent manner. Flow cytometric analysis of the cell cycle revealed that CRC and HCC cells were arrested mainly at the S and G2/M phases following the combination treatment. Apoptotic DNA ladder assay revealed DNA fragmentation in a dose and type of treatment dependent manner. Annexin V assay, demonstrated that combined treatment with Sora and the selected phenolic compounds induced apoptotic cell death; a high percentage of cells displayed early and late apoptotic phenotypes. Furthermore, flow cytometric assessment of mitochondrial membrane potential in CRC and HCC cell lines demonstrated extensive mitochondrial membrane damage, due to active apoptosis, following combined treatment. Finally, western blot analysis showed that expression of cell cycle (p27, cylcin A2, cyclin B, cyclin D & pRB), pro- v (Bax, cleaved caspase 3 & cleaved caspase 9) and anti-apoptotic (Bcl-xL) proteins is articulated in a dose-dependent manner. The obtained results clearly show promising enhancement of sorafenib efficacy and suggest further in vivo and clinical studies of the combination treatment as a novel therapeutic strategy in hepatoma, colorectal cancer and other solid tumors.