Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle
Abstract
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:
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.