新型硫色满酮衍生物抑制胃癌细胞生长与诱导胃癌细胞凋亡机制研究

Effect of Novel Thiochromanone Derivatives on Growth and Apoptosis of Gastric Cancer Cells

[目的]研究(顺)-3(氯代亚甲基)-5,7-二氯-硫色满-4-酮[(Z)-3(chloromethyl-ene)-5,7(dichloro)-thiochroman-4-ketone,CMDCT]对体外低、中、高分化的人胃癌细胞株生长抑制作用与诱导胃癌细胞凋亡机制研究。[方法]MTT法测试CMDCT对3种胃癌细胞株的生长抑制;以中分化胃癌细胞(SGC-7901)为例,流式细胞术(FCM)和缺口末端核苷酸标记法(TUNEL)检测胃癌SGC-7901细胞的凋亡;ELISA法测定胃癌SGC-7901细胞内被激活的人半胱氨酸蛋白酶3(Caspase-3)的量;PCR检测Bcl-2、Bax、p53、Caspase-3基因表达;Western blot检测胃癌SGC-7901细胞中Bcl-2、Bax、p53、Caspase-3蛋白表达情况。[结果]CMDCT对3种胃癌细胞的生长具有显著抑制作用,随CMDCT浓度升高细胞生长的抑制率越高,当药物浓度为40μmol/L时,抑制率在(66.53%±2.47%)^(76.12%±1.35%)之间,和同浓度顺铂(DDP)组相比有较大差异;TUNEL检测胃癌SGC-7901细胞的凋亡指数:对照组为1.61%±0.23%;低浓度组为12.55%±1.44%;高浓度组61.15%±1.77%,加药组细胞凋亡指数明显高于对照组;流式细胞术结果显示,对照组凋亡率为4.78%±0.41%;加药组加入浓度10μmol/L、20μmol/L、40μmol/L药物后,凋亡率分别为(9.42%±1.27%),(21.07%±1.35%),(55.8%±10.03%),明显高于对照组细胞凋亡率;PCR和Western blot检测,与对照组对比,加药组的Bax、p53和Caspase-3基因和蛋白表达量均增高,均具有上调作用,Bcl-2基因和蛋白表达降低,呈下调趋势。[结论]CMDCT对体外人低、中、高分化的3种胃癌细胞株均具有生长抑制作用,且可以诱导细胞凋亡,通过使具有促凋亡作用的p53、Bax、Caspase-3高表达,及抗细胞凋亡作用的Bcl-2的低表达,有效地诱导了胃癌SGC-7901细胞凋亡。

[Purpose]To investigate the effect of(cis)-3(chloromethylene)-5,7-dichloro-thiochroman-4-ketone(CMDCT)on the growth and apoptosis of gastric cancer cells.[Methods]The low,medium and high differentiated human gastric cancer MKN-28,SGC-7901 and BGC-823 cells were treated with CMDCT.MTT assay was used to detect the growth gastric cancer cells;flow cytometry(FCM)and nick end nucleotide labeling(TUNEL)were used to detect the apoptosis of the SGC-7901 cells,and ELISA was used to determine the amount of Caspase-3 activated in SGC-7901 cells.The expressions of Bcl-2,Bax,p53 and Caspase-3 genes were detected by PCR,and the expressions of Bcl-2,Bax,p53 and Caspase-3 proteins in SGC-7901 cells were detected by Western blot.[Results]CMDCT inhibited the growth of MKN-28,SGC-7901 and BGC-823 cells in a concentration-effect manner.When the concentration of CMDCT was 40μmol/L,the inhibition rate was between(66.53%±2.47%)and(76.12%±1.35%),which was significantly higher than that of the same concentration of cisplatin(DDP).There were significant differences in apoptotic index of SGC-7901 cells among control group(1.61%±0.23%),low concentration group(12.55%±1.44%)and high concentration group(61.15%±1.77%).Flow cytometry showed that the apoptotic rates of the control group and 10μmol/L,20μmol/L,40μmol/L CMDCT treated groups were(4.78%±0.41%),(9.42%±1.27%),(21.07%±1.35%)and(55.8%±10.03%),respectively.Compared with the control group,the expressions of Bax,p53 and Caspase-3 genes and proteins in the CMDCT treated groups were increased,while expression of Bcl-2 gene and protein decreased as demonstrated by PCR and Western blot methods.[Conclusion]CMDCT can inhibit the growth of human gastric cancer cell lines with low,medium and high differentiation in vitro,and induce apoptosis.By increasing the expressions of p53,Bax,Caspase-3 and decreasing bcl-2,CMDCT can effectively induce the apoptosis of SGC-7901 cells.