Molecular Mechanism Study Of Simvastatin Inducing Apoptosis Of K562 Cells

Objective: To investigate the effects of simvastatin on apoptosis of K562 cells and to elucidate its molecular mechanisms.Methods: K562 cells had been treated with various concentrations of simvastatin for 72 h. 1. Morphological change of apoptotic cells was observed by Hoechst33258 fluorescent staining under fluorescent microscope. 2.Apoptosis rate of cells was determined with annexinⅤ-FITC/PI double staining by flow cytometry. 3.Apoptotic bands were detected by DNA Ladder experiment. 4.Intracellular calcium concentration ([Ca2+]i) was measured by Laser Scanning Confocal Microscope(LSCM). 5.Cytosolic, microsomal and mitochondrial proteins were isolated by QproteomeTM mitochondria isolation kit. 6.The expression levels of Bcl-2,Bax,Bak,IP3R,SERCA,GRP78,caspase-9,-7,-6,-3,GADD153,Calpain,MAGE -3 mRNA were determined by RT-PCR. 7.The expression levels of GRP78,Calpain,Caspase-3,-6,-7,-9,-12,GADD153 and cytochrome C proteins were evaluated by western blot.Results: 1. Simvastatin could induce apoptosis of K562 cells in concentration dependent manner. 2. K562 cells could be induced to undergo apoptosis after 10μmol/L, 20μmol/L and 30μmol/L simvastatin treatment for 72h,the apoptotic rate was(12.41±0.32)%,(19.08±0.26)% and ( 23.41±0.36 ) %, respectively. Compared with the control group(4.2±0.19), it was significantly highe(rP<0.01).3. After the treatment of simvastatin for 72h, a typical apoptotic morphological change and DNA ladder were observed. 4. After the treatment of simvastatin for 72h,Simvastatin induced the increase of [Ca2+]i in K562 cells,fluorescent intensities were (43±2.9),(54±2.7),(64±2.6),respectively. Compared with the control group(36±2.7), it was significantly higher(P<0.01). 5. RT-PCR results showed that Bax,Bak,IP3R,SERCA,GRP78,caspase-9,-7,-6,-3,GADD153,Calpain mRNA were up-regulated and Bcl-2,MAGE -3 mRNA were down-regulated. 6. Western blot results indicated that cleavage and activation of caspase-12, -9,-7,-6,-3, upregulation of GRP78 and GADD153 expression ,releasing of cytochrome c from mitochondria were induced in k562 cells . Caspase-12 that localizes in the endoplasmic reticulum was determined by western blot.Conclusions: 1. Simvastatin could induce apoptosis of K562 cells in concentration dependent manner. 2. Simvastatin could control Ca2+ leak from endoplasmic reticulum (ER) by modulating Bcl-2,Bax,Bak,IP3R,SERCA and induce apoptosis of k562 cells. 3. Simvastatin could induce apoptosis of k562 cells by upregulation of GADD153 mRNA expression and downregulation of MAGE -3 mRNA expression. 4. Simvastatin could induce K562 cells to undergo apoptosis; the underlying mechanism might be related to upregulation of caspase-9,-7,-6,-3 mRNA expression and cleavage and activation of caspase-12 protein, which subsequently transforms caspase-9,-7,-6,-3 into its active form. 5. Simvastatin induces apoptosis of k562 cells though endoplasmic reticulum pathway as cleavage and activation of caspase-12 protein, upregulation of GRP78 expression. 6. Our research profoundly explores the anti-leukemia mechanisms of simvastatin, which might be useful for clinical therapy and treatment of chronic myelocytic leukemia.