| Objective: To investigate the effects of 20μmol/L simvastatin on the cell proliferation, apoptosis and the control factors, and elucidate the molecular mechanisms of apoptosis induced by simvastatin.Methods: 1. MTT assay was employed to evaluate the proliferative inhibition rate and the growth curve was draw. 2. The morphology change was observed under light microscope. 3. Flow cytometry assay was used to detect the apoptotic rate, the mitochondrial membrane potential (MMP), the ROS and Ca2+ levels. 4. Colorimetric method was used to measure the caspases-3, 8, 9 activity, the NO and GSH levels. 5. To presume the apoptosis pathway, the drug-treated groups were added caspase-9, NO inhibitor respectively, then the K562 cells were counted and its apoptosis ratio was detected at different times. 6. The apoptotic correlated gene of D28K, iNOS, Bcl-2, Bax, caspase-3, 8, 9 were analyzed the mRNA expression levels by RT-PCR (reverse transcription-PCR). 7. The protein of cyto C was detected by immunohistochemical and western blot method.Results: 1. 48 hours after been treated with 20μmol/L simvastatin, the morphology change of K562 cells such as karyopyconosis, nuclear fragmentation and apoptotic body emerged. 2. The growth of K562 cell was inhibited by 20μmol/L simvastatin in a time dependent manner. 3. AnnexinV-FITC/PI was used to detected the apoptic ratio of K562 cells, the apoptotic ratio of the treated groups were were(2.55±0.22)%,(6.1±0.35)%,(14.15±0.42)%,(30.70±0.65)% respectively compared with the control groups in time dependent manner. It was significantly higher (P<0.05). 4. The percentage of G0/G1 phase cells increased while the percentage of S phase cells decreased after being treated with simvastatin for 24h, arrestting cell cycle in G0/G1 phase in a time dependent manner. 5. The caspase-3, 8, 9 activities of K562 cells that had been treated with simvastatin for 48h and 72h elevated remarkably Compared with the control group. The caspase-3,8, 9 activities of 20μmol/L simvastatin-treated groups were markedly higher (P<0.05). 6. The nitrogen monoxidum leves were markedly higher (P<0.05) Compared with the control group after 20μmol/L simvastatin 24h in a time dependent manner. 7. The GSH leves in K562 cells decreased markedly being treated with 20μmol/L simvastatin for 24～72h Compared with the control group. 8. The Ca2+ was detected by Fluo-3AM and were markedly increased in treated groups compared with the control groups at different time points (P<0.05), the peak time point was 12h. 9. The changes of the cell percentage with MMP were (0.7±0.24)%, (39.6±4.80)%, (24.4±2.45)%, (6.7±1.62)% at different times (12h, 24h, 48h, 72h) respectively. It reached the highest peak in drug-treated groups compared with the control groups at 24h. 10. ROS was detected by 2', 7'-dichloro fluorescein diacetate (DCFH-DA) and were markedly increased in treated groups compared with the control groups, the peak time point was 24h. 11. There is no marked difference of cell number and apoptosis ratio between the groups treated with the caspase-9 inhibitor and the drug-treated group. 12. The mRNA of Apaf-1, D28K, iNOS and Bax expression levels in drug-treated group were up-regulated, while the mRNA of Bcl-2 was down regulated. 13. The quantity of protein of cyto C were increased markedly in treated groups detected by immunohistochemical and western blot method (P<0.05).Conclusions: 1. 20μmol/L Simvastatin can inhibit proliferation of K562 cells and induce their apoptosis in a time. 2. It can be presumed that the pathway of apoptosis induced by 20μmol/L Simvastatin is through the mitochondrial pathway. 3. There might be other apoptosis pathway involved except the mitochondrial pathway. 4. The free of Ca2+, nitrogen monoxidum, disequilibrium of redoxreaction and apoptosis gene are take partly in the mitochondrial pathway of apoptosis. 5. many factors are involved inducing the K562 cells apoptosis by the simvaststin. |
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