重楼复方对人胃癌细胞MKN-45增殖抑制作用及分子机制研究

Inhibitive Effect of Chonglou Recipe on Proliferation of Human Gastric Cancer Cell Line MKN-45 and Its Possible Molecular Mechanism

目的研究重楼复方对人胃癌细胞株MKN-45增殖抑制作用,及其对细胞周期、细胞凋亡的影响,并探讨可能的分子机制。方法不同浓度的重楼复方作用于MKN-45细胞48 h后,采用四甲基偶氮唑蓝(MTT)显色法检测细胞增殖抑制率,采用流式细胞仪检测细胞凋亡及细胞周期,RT-PCR法检测凋亡相关基因Sur-vivin、c-IAP1、c-IAP2、Bcl-2 mRNA的表达。结果重楼复方以剂量依赖方式抑制人胃癌细胞株MKN-45的细胞增殖,其半数抑制浓度(IC50值)为0.67 mg.mL-1。重楼复方0.2,0.4,0.8 mg.mL-1作用于MKN-45细胞48h后,其早期凋亡率分别为7.0%、13.8%、36.3%,而对照组为3.4%,其诱导凋亡作用呈现剂量依赖性。重楼复方0.8 mg.mL-1作用于MKN-45细胞24 h后,细胞积聚在G0/G1期(对照组63.30%,重楼复方73.16%),同时S期细胞比例减少(对照组32.48%,重楼复方22.52%)。重楼复方0.8 mg.mL-1作用MKN-45细胞48 h后,Survivin、c-IAP1、c-IAP2、Bcl-2的mRNA水平均下调。结论重楼复方具有抑制人胃癌细胞株MKN-45增殖及诱导细胞凋亡作用,并能阻滞细胞于G0/G1期,下调Survivin、c-IAP1、c-IAP2、Bcl-2 mRNA的表达可能为其诱导细胞凋亡的主要机制之一。

Objective To investigate the effect of Chonglou Recipe（CP） on proliferation,cell cycle and aopotosis of human gastric cancer cell line MKN-45,and to explore the possible molecular mechanism.Methods After treating human gastric cancer cell line MKN-45 with CP at various concentrations（0.025～1.6 mg·mL-1） for 48 h,the proliferation rate of cell line MKN-45 was detected with methyl thiazolyl tetrazolium（MTT） method.The apoptosis and cell cycle phases were examined by flow cytometery（FCM）.Reversed transcriptase polymerase chain reaction（RT-PCR） method was used to detect the mRNA expression of survivin,c-IAP1,c-IAP2 and Bcl-2.Results CP inhibited the proliferation of human gastric cancer cell line MKN-45 in a dose-dependent manner.The median effective concentration IC50 after incubation with CP for 48 hours was 0.67 mg·mL-1.After treatment with CP at the dosage of 0.2,0.4,and 0.8 mg·mL-1 for 48 hours,the apoptotic rate of cell line MKN-45 was 7.0 %,13.8 % and 36.3 %,respectively,showing a dose-dependent trend.The apoptotic rate was 3.4 % in the control group.After treatment with CP at the dosage of 0.8 mg·mL-1 for 24 h,the percentage of MKN-45 at G0/G1 phase was 73.16 %,and was 63.30 % in the control group,which was higher than the percentage of MKN-45 at S phase（22.52 % in CP group and 32.48 % in the control group）.After MKN-45 cells were exposed to CP at the indicated concentration of 0.8 mg·mL-1 for 48 h,the mRNA expression of survivin,c-IAP1,c-IAP2 and Bcl-2 were down-regulated.Conclusion CP can inhibit the proliferation and induce apoptosis in human gastric carcinoma cell line MKN-45.Disturbing the cell cycle and down-regulating the mRNA expression of survivin,c-IAP1,c-IAP2 and Bcl-2 may be one of its therapeutic mechanisms.