PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways

Background: Aberrant Ras/Raf/MAPK and PHK/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study reports how sorafenib (a multi-kinase inhibitor) and PI-103 (a dual PI3K/mTOR inhibitor) alone and in combination inhibit the proliferation of the HCC cell line, Huh7. Materials and Methods: Huh7 proliferation was assayed by 3H-thymidine incorporation and by MTT assay. Western blot was used to detect phosphorylation of the key enzymes in the Ras/Raf and PI3K pathways. Results: Sorafenib and PI-103, as single agents inhibited Huh7 proliferation and epidermal growth factor (EGF)-stimulated Huh7 proliferation in a dose-dependent fashion; the combination of sorafenib and PI-103 produced synergistic effects. EGF increased phosphorylation of MEK and ERK, key Ras/Raf downstream signaling proteins; this activation was inhibited by sorafenib. However, sorafenib as a single agent increased AKT(Ser473) and mTOR phosphorylation. EGFstimulated activation of PBK/AKT/mTOR pathway components was inhibited by PI-103. PI-103 is a potent inhibitor of AKT(Ser473) phosphorylation; in contrast, rapamycin stimulated AKT(Ser473) phosphorylation. It was found that PI-103, as a single agent, stimulated MEK and ERK phosphorylation. However, the combination of sorafenib and PI-103 caused inhibition of all the tested kinases in the Ras/Raf and PI3K pathways. Conclusion: The combination of sorafenib and PI-103 can significantly inhibit EGF-stimulated Huh.7 proliferation by blocking both Ras/Raf/MAPK and PUK/AKT/mTOR pathways.