花椒毒素通过死亡受体途径诱导胃癌SGC-7901细胞凋亡的研究
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摘要
目的:探讨花椒毒素对人胃癌SGC-7901细胞体外增殖抑制作用,并从死亡受体途径对其进行机制研究。方法:不同浓度花椒毒素作用于胃癌SGC-7901细胞48h后,MTT法检测花椒毒素对胃癌SGC-7901细胞的增殖抑制作用;Hoechst 33258染色及Annexin V-FITC/PI双染法观察花椒毒素诱导细胞凋亡的形态学变化和早中晚期凋亡的区别;流式细胞术检测死亡受体及其配体Fas/Fas L、DR5/TRAIL及Bid蛋白,Caspase-8检测试剂盒检测Caspase-8活性。结果:花椒毒素呈剂量依赖性抑制SGC-7901细胞的增殖,IC50为75.6μg/ml;花椒毒素作用后贴壁细胞减少,凋亡小体的数量增加,且呈剂量依赖性;给药48h后,DR5/TRAIL的表达升高,在一定浓度范围内Fas/Fas L蛋白的表达升高,Caspase-8的活性增强,Bid蛋白活化增多。结论:花椒毒素通过上调DR5/TRAIL的表达,在一定浓度范围内上调Fas/Fas L蛋白的表达,启动死亡受体途径,促进Caspase-8的活化,进而激活下游效应因子;活化的Caspase-8可激活Bid蛋白,进入线粒体发挥作用,可能是花椒毒素诱导SGC-7901细胞凋亡的途径之一。
Objective: To investigate the mechanism on( 8-MOP)-induced apoptosis in human gastric carcinoma SGC-7901 cells in vitro. Methods: using MTT to measure the inhibition of proliferation of gastric cancer SGC-7901 cells by 8-MOP; use Hoechst 33258 staining and Annexin V / PI staining to observe gastric cancer SGC-7901 cells apoptosis morphology and the difference between early,middle,and late apoptosis after the drug treated; using flow cytometry to test apoptosis related proteins of Fas / Fas L proteins,Bid proteins and DR5 / TRAIL proteins of gastric cancer SGC-7901 after treated by 8-MOP; The influence of 8-MOP to Caspase-8 proteins in the cell was determined by Caspase-8 Flouormetric assay kit. Results: After 48 h,8-MOP obviously inhibited the SGC-7901 cells proliferation,its inhibition was in relationship with concentration-response; the Hoechst staining study showed that the morphologic change happened to SGC-7901 cell; Flow cytometry results showed that in a certain concentration range,8-MOP can promote the Fas / Fas L protein expression. 8-MOP can promote the DR5 / TRAIL protein expression,and appear a dose-dependent relationship. After 48 h,the content of Bid protein in cells are increased,and appear a dose-dependent relationship. Conclusion: 8-MOP may induce SGC-7901 cells apoptosis in the certain concentration range through the Fas / Fas L protein mediated death receptor pathway,or by DR5 / TRAIL mediated death receptor pathway,by raising death receptor protein expression,activation Caspase-8,activating downstream effect factor,inducing cell apoptosis,or activate Caspase-8 cutting activate protein Bid,and then enter the mitochondrial pathway,induction of apoptosis.
Objective: To investigate the mechanism on( 8-MOP)-induced apoptosis in human gastric carcinoma SGC-7901 cells in vitro. Methods: using MTT to measure the inhibition of proliferation of gastric cancer SGC-7901 cells by 8-MOP; use Hoechst 33258 staining and Annexin V / PI staining to observe gastric cancer SGC-7901 cells apoptosis morphology and the difference between early,middle,and late apoptosis after the drug treated; using flow cytometry to test apoptosis related proteins of Fas / Fas L proteins,Bid proteins and DR5 / TRAIL proteins of gastric cancer SGC-7901 after treated by 8-MOP; The influence of 8-MOP to Caspase-8 proteins in the cell was determined by Caspase-8 Flouormetric assay kit. Results: After 48 h,8-MOP obviously inhibited the SGC-7901 cells proliferation,its inhibition was in relationship with concentration-response; the Hoechst staining study showed that the morphologic change happened to SGC-7901 cell; Flow cytometry results showed that in a certain concentration range,8-MOP can promote the Fas / Fas L protein expression. 8-MOP can promote the DR5 / TRAIL protein expression,and appear a dose-dependent relationship. After 48 h,the content of Bid protein in cells are increased,and appear a dose-dependent relationship. Conclusion: 8-MOP may induce SGC-7901 cells apoptosis in the certain concentration range through the Fas / Fas L protein mediated death receptor pathway,or by DR5 / TRAIL mediated death receptor pathway,by raising death receptor protein expression,activation Caspase-8,activating downstream effect factor,inducing cell apoptosis,or activate Caspase-8 cutting activate protein Bid,and then enter the mitochondrial pathway,induction of apoptosis.
引文
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2 Autore G,Marzocco S,Formisano C,et al.Cytotoxic activity and composition of petroleum ether extract from Magydaris tomentosa(Desf.)W.D.J.Koch(Apiaceae).Molecules,2015;20(1)∶1571~1578
3 杨倩,杨俭.8-甲氧补骨脂素药理作用研究进展.中国临床药理学与治疗学,2014;19(10)∶1177~1182
4 Mishra,BB,Tiwari VK.Natural products:An evolving role in future drug discovery.Eur J Chem,2011;46(10)∶4769~4807
5 Peng Yan,Liu Wei,Xiong Jing,et al.Down regulation of differentiated embryonic chondrocytes 1(DEC1)is involved in 8-methoxypsoralen-induced apoptosis in Hep G2 cells.Toxicology,2012;301(1~3)∶58~65
6 Sumiyoshi M,Sakanaka M,Taniguchi M,et al.Antitumor effects of various furocoumarins isolated from the roots,seeds and fruits of Angelica and Cnidium species under ultraviolet A irradiation.J Nat Med,2014;68(1)∶83 ~94
7 杨秀伟,徐波,冉福香,等.40种香豆素类化合物对人表皮癌细胞系A432细胞株和人乳腺癌细胞系BCAP细胞株增殖抑制活性的筛选.中国现代中药,2006;8(12)∶9~10
8 Zhang Xiujuan,Lv Zongyan,He Lijuan,et al.Effect of xanthotoxin on SGC-7901 cells proliferation and Autophagy.J Chem Pharm Res,2014;6(10)∶545~548
9 王宏婷,王存琴.27-O-(E)-香豆酰基-乌索酸通过调控JNK/SAPK通路诱导人乳腺癌细胞MDA-MB-231细胞凋亡.中国中药杂志,2015,40 (4)∶722~726
10 周青,连磊凡,吴丽珍,等.佛甲草对S180小鼠肿瘤组织IL-10、TNF-α、NF-κB表达的影响.中药药理与临床,2015;31(2)∶52~54
11 Chaudhry P,Srinivasan R,Patel FD,et al.Serum soluble Fas levels and prediction of response to platinum-based chemotherapy in epithelial ovarian cancer.Int J Cancer,2008;122(8)∶1716~1721
12 贺艳杰,李玉华,卢会芳,等.线粒体通路和死亡受体通路在中华眼镜蛇毒组分诱导KG1a细胞凋亡中的作用.中国药理学通报,2013;29 (3)∶356~360