Inhibition of membrane anchorage of the Ras oncoprotein reduces hepatocyte proliferation in rats after partial hepatectomy

Background : The Ras oncoprotein seems to be over-expressed and/or mutated in hepato-cellular carcinoma (HCC). Studies in cell lines suggest that the ability of Ras to promote cell differentiation and proliferation is prevented by inhibiting its membrane anchorage for example after administration of the ras antagonist S-Farnesylthiosalycilic acid (FTS). In addition, Ras mediated activation of the MAPkinase pathway seems to play a role in promoting cell proliferation.
Aims : To evaluate in vivo the impact of the inhibition of Ras membrane anchorage, using treatment with FTS, on hepatocyte proliferation in rats after partial hepatectomy (PH).
Methods : Male Wistar rats were administered FTS intraperitoneally (50mg/kg, 1,8,16h after PH) and sacrificed 12 or 24 hours after PH. BrdU incorporation was visualized by immunohistochemisty and quantified by flow cytometry. Protein expression in different cell fractions was analysed by Western blotting. Real time PCR was used for mRNA expression.
Results : FTS treatment induced a significant reduction in BrdU incorporation and PCNA protein expression after PH in hepatocytes. Unlike control rats, the Ras protein was found in substantial amounts in the cytosol of FTS-treated animals suggesting the inhibition of Ras membrane anchorage that ultimately results in inactivation of Ras. In addition, no Raf membrane recruitment and phosporylation was observed in the membrane fractions of FTS-treated rats and consequently phosphorylation of Erk1/2 located down-stream in the ras-raf signalling pathway was found to be reduced, thereby, confirming the functional inhibition of Ras. Amplification of mRNA by real time PCR showed a similar increase in Ras mRNA after PH in control and FTS-treated animals suggesting that FTS does not affect Ras mRNA expression but only the mature protein.
Conclusions : Taken together, these observations show that the functional inhibition of Ras, by FTS, results in a significant reduction in liver cell proliferation even after a strong and highly synchronized proliferation stimulus such as PH. The inhibitory effect is at least in part mediated by inhibition of ras-dependent Raf-MAPkinase-ERK signalling. Therefore, it seems worthwhile to evaluate the impact of Ras inhibition on carcinogenesis and treatment of HCC.