Sanguinarine inhibits Rac1b-rendered cell survival enhancement by promoting apoptosis and blocking proliferation
Aim:Small GTPase Rac1 is a member of the Ras superfamily, which plays important roles in regulation of cytoskeleton reorganization, cell growth, proliferation, migration, etc. The aim of this study was to determine how a constitutively active Rac1b regulated cell proliferation and to investigate the effects of the Rac1b inhibitor sanguinarine.Methods:Three HEK293T cell lines stably overexpressing GFP, Rac1-GFP or Rac1b-GFP were constructed by lentiviral infection. The cells were treated with sanguinarine (1 μmol/L) or its analogue berberine (1 μmol/L) for 4 d. Cell proliferation was evaluated by counting cell numbers and with a BrdU incorporation assay. The levels of cleaved PARP-89 (an apoptosis marker) and cyclin-D1 (a proliferative index) were measured using Western blotting.Results:In 10% serum-containing media, overexpressing either Rac1 or Rac1b did not significantly change the cell proliferation. In the serum-starved media, however, the survival rate of Rac1b cells was significantly increased, whereas that of Rac1 cells was moderately increased. The level of cleaved PARP-89 was significantly increased in serum-starved Rac1 cells, but markedly reduced in serum-starved Rac1b cells. The level of cyclin-D1 was significantly increased in both serum-starved Rac1 and Rac1b cells. Treatment with sanguinarine, but not berberine, inhibited the proliferation of Rac1b cells, which was accompanied by significantly increased the level of PARP-89, and decreased both the level of cyclin-D1 and the percentage of BrdU positive cells.Conclusion:Rac1b enhances the cell proliferation under a growth-limiting condition via both anti-apoptotic and pro-proliferative mechanisms. Sanguinarine, as the specific inhibitor of Rac1b, is a potential therapeutic agent for malignant tumors with up-regulated Rac1b.