Acta Pharmacologica Sinica 2005 February; 26 (2): 205-211; doi: 10.1111/j.1745-7254.2005.00526.x

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 D₁ 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 G₁-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P<0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3% vs 16.4%, P<0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D₁ 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 G₁ phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D₁ and cyclin E, and sequentially inhibiting Rb phosphorylation.

Keywords: onychin; vascular smooth muscle; cell cycle; retinoblastoma; cyclin D₁; cyclin E; tyrosine kinase; mitogen-activated protein kinase