Mechanism Of Simvastatin-Induced Apoptosis Of K562 Cells

OBJECTIVE1. To detect the influence of cell proliferation,cell cycle and apoptosis of K562 cells treated with simvastatin in vitro and in vivo.2. To investigate the molecular mechanism of apoptosis of K562 cells treated with simvastatin in vitro and in vivo.METHODS1.The effects of simvastatin on the cell proliferation,cell cycle,apoptosis of K562 cell were detected by MTT and flow cytometry (FCM)in vitro.2.The expression genes of Ras-MAPK,PI3K-AKT and NF-κBpathway were analyzed by RT-PCR to look for markedly differential genes.Meanwhile,the expression of differential genes were analyzed by immunohistochemistry LDP method and western blot in protein level.3.K562 cells were cultured in vitro for the construction of transplantation tumor model of K562 cell in nude mice. The changes of cell cycle of K562 cells induced by different concentration of simvastatin were detected by FCM and the early apoptosis of K562 cells was detected by TUNEL.4.The genes expression of Ras-MAPK,PI3K-AKT and NF-κBpathway were analyzed by RT-PCR in vivo to look for the genes with significantly differential expression. The expression of simvastatin-induced genes was further analyzed by immunohistochemistry LDP method and western blot.RESULTS1.MTT analysis showed that cell proliferation inhibition rate increased makedly with the prolongation of treatment time of 20μmol/L simvastatin(p<0.01).2. After K562 cells being treated with simvastatin for 24h,its apoptosis rate changed obviously(11.33±0.86%)and increased gradually with the prolongation of simvastatin-treatment time of(p<0.01) .3. Compared with control,G0/G1 arrests were detected after K562 cells being treated with simvastatin for 24h,48h and 72h respectively and the percentage of G0/G1 arrest of K562 cells treated with simvastatin for 48h increased significantly(52.5%). The percentage of G2/M(1.4%) and PI (0.4750) of K562 cells treated with simvastatin for 48h decreased significantly.4.N-Ras mRNA down-regulated with the prolongation of simvastatin -treated time of K562 cells especially after 72h. The differential expression of N-Ras mRNA of K562 cells treated with simvastatin for 24h,48h and 72h differed markedly(p<0.01). c-Raf-1 mRNA also showed down-regulated tendency with the prolongation of simvastatin-treated time especially after K562 cells being treated with simvastatin for 72h. The differential expression of c-Raf-1 mRNA of K562 cells treated with simvastatin for 24h,48h and 72h also differed markedly(p<0.01). ERK1 mRNA also showed down-regulated tendency with the prolongation of simvastatin-treated time especially for 72h. The differential expression of ERK1 mRNA of K562 cells treated with simvastatin for 24h,48h and 72h also differed significantly(p<0.01).Also, immunohistochemistry results showed that ERK1 protein down-regulated with the prolongation of simvastatin-treated time.5. PI3K mRNA began to down-regulate after K562 cells being treated with simvastatin for 24h and maintained down-regulated tendency and PI3K mRNA of K562 cells induced by simvastatin expressed differentially after being treated for 24h,48h and 72h respectively(p<0.01).AKT1mRNA also presented down-regulated tendency with the prolongation of simvastatin-treated time and AKT1 mRNA expressed differentially after K562 cells being treated with simvastatin for 24h,48h and 72h respectively(p<0.01).BCL-XLmRNA showed a certain up-regulated tendency with the prolongation of simvastatin-treated time and there was obvious difference between the BCL-XLmRNA of K562 cells treated with simvastatin for 48h and that of 72h(p<0.01).6. IKK-βmRNA began to down-regulate after K562 cells being treated with simvastatin for 24h and maintained down-regulated tendency with the prolongation of simvastatin-treated time. There was statistical difference of differential expression of IKK-βmRNA between K562 cells treated with simvastatin for 24h and 48h. There was statistical difference of differential expression of IKK-βmRNA between K562 cells treated with simvastatin for 24h and 72h.NFκ-B1 also presented down-regulated tendency with the prolongation of simvastatin-treated time and there was significantly differential expression among NFκ-B1 of K562 cells treated with simvastatin for 24h,48h and 72h respectively(p<0.01).IκB-αbegan to down-regulate after K562cells being treated with simvastatin for 48h(p<0.01) and maintained down-regulated tendency until 72h(p<0.01).7. Compared with control, 0.05mg/L simvastatin can induce markedly increasing percentage of G0/G1of K562cells (41.27±4.59%,p<0.05)and G0/G1 arrest of K562 cells. The percentage of G2/M.of T1group decreased significantly.8. The apoptotic cells among K562 cells of tumor tissues of control , T1 and T2 group were detected by TUNEL and the integrated optical density (IOD) of apoptotic cells were analyzed by Image pro-plus5.1 and there was statistical difference among the IOD of control , T1 and T2 group(p<0.01).9. N-Ras mRNA down-regulated obviously with the increase of simvastatin dose and there were statistical difference among the differential expression of N-Ras mRNA of control and T1 and T2 group(p<0.01).c-Raf-1 mRNA showed to be down-regulated with the multiplication of simvastatin dose and there was statistical difference of c-Raf-1 mRNA expression between control and T2 group(p<0.01).ERK1 mRNA also down-regulated with the increasing of simvastatin dose and the differential expression of ERK1 mRNA of K562 cells between control and treated groups have statistical difference(p<0.01). Immunochemistry results and western blot result showed p-ERK protein ,with accumulation of simvastatin dose ,expressed down-regulated and there was statistical difference between p- ERK protein of control and treated groups(p<0.01).10. There was statistical difference of differential expression of PI3K mRNA between control and treated groups(p<0.01).And there was statistical difference of differential expression of AKT1 mRNA between control and treated groups(p<0.01). Simvastatin also induced down-regulation of NFκB1and IKK-βof K562 cells. There was obviously differential expression of NFκB1 between control and treated groups(p<0.01). There was statistical difference of differential expression of IKK-βbetween control and treated groups (p<0.01). There was also statistical difference of differential expression of IκB-αmRNA between control and treated groups (p<0.01). There was statistical difference of differential expression of caspase-9 between control and treated groups (p<0.01).CONCLUSION1. Simvastatin can induce significant apoptosis of K562 cells in vitro and in vivo and induce marked proliferation inhibition and G0/G1 arrest of K562 cells in vitro.Simvastatin of low dose can induce obvious G0/G1 arrest of K562 cells in nude mice.2.Simvastatin can induce significant down-regulation of N-Ras of Ras-MAPK pathway and PI3K,BCL-XL of PI3K-AKT pathway and IKK-βof NFκB pathway in vitro. N-Ras, PI3K , BCL-XL and IKK-βprobably are the obvious targets of cell proliferation inhibition and apoptosis of K562 cells induced by simvastatin.2.The animal experiments demonstrate that simvastatin can induce marked down-regulation of N-Ras ,c-Raf-1 and ERK1 of Ras-MAPK signal pathway in gene level and/or protein level,which illustrate Ras-MAPK pathway proablely is important pathway that simvastatin induce the proliferation inhibition and apoptosis of K562 cells. Simvastatin can also induce the significant down-regulation of NFκB1and IKK-βand IκB-αof NFκB pathway in gene level,which explain that NFκB pathway proablely is also the important pathway that simvastatin induce the apoptosis of K562 cells.Simvastatin can not induce the down-regulation of PI3K and AKT1 of PI3K-AKT pathway in gene level,however can induce the up- regulation of caspase-9 of downstream of PI3K-AKT pathway to participate the apoptosis of K562 cells induced by simvastatin in vivo. 4. The differential expression of genes of Ras-MAPK, NFκ-B and PI3K-AKT pathway of K562 cells induced by simvastatin in vitro maintain similar tendency with that in vivo. However,the differential expression of PI3K and AKT1 in vitro differs greatly that in vivo,which illustrate that there probably is a different apoptosis-induced mechanism in K562 cells by simvastatin between in vitro and in vivo.