Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle.
OBJECTIVE
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 150 micromol/L or genistein 10 micromol/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.


CONCLUSIONS
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.
Yang
Ming
M
Institute of Pharmacy and Pharmacology, Nanhua University, Hengyang 421001, China.
Huang
Hong-Lin
HL
Zhu
Bing-Yang
BY
Tuo
Qin-Hui
QH
Liao
Duan-Fang
DF
D000818:Animals; D001013:Aorta, Thoracic; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification / Q000494:pharmacology*; D029624:Ferns / Q000737:chemistry; D044950:Flavanones / Q000302:isolation & purification / Q000494:pharmacology*; D016193:G1 Phase; D019833:Genistein / Q000494:pharmacology; D008297:Male; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D010946:Plants, Medicinal / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011728:Pyridones / Q000302:isolation & purification / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D019927:Cyclin E; D004365:Drugs, Chinese Herbal; D044950:Flavanones; D011728:Pyridones; D019938:Cyclin D1; C054955:onychine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D048052:Mitogen-Activated Protein Kinase 3