Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle1

Aim: To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS). Methods: Rat VSMCs were incubated with onychin 1–50 μmol/L or genistein 10 μmol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D 1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies. Results: The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G 1 -phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P vs 16.4%, P 1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS. Conclusion: Onychin inhibits the proliferation of VSMCs through G 1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D 1 and cyclin E, and sequentially inhibiting Rb phosphorylation.