桔梗皂苷D对人移行细胞癌5637细胞株的诱导凋亡作用及其分子机制
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摘要
目的探讨桔梗皂苷D对人移行细胞癌5637细胞诱导凋亡作用及分子机制。方法 HE染色和透射电子显微镜观察细胞的形态学变化;流式细胞学技术检测细胞凋亡率的变化;Western blot法检测凋亡通路相关蛋白的表达变化;PCR法检测p53、Bcl-2、Bax mRNA表达;免疫组织化学法检测凋亡相关蛋白表达的影响。结果通过HE染色和电子显微镜观察桔梗皂苷D组5637细胞,可见凋亡细胞,凋亡指数明显高于对照组(P<0.01);与对照组细胞相比,桔梗皂苷D组5637细胞Survivin、Livin表达量明显下降(P<0.05);Caspase-9、Caspase-8、Caspase-3、Cyt-c表达量与对照组比较,明显增加(P<0.05);p53、Bax mRNA水平明显升高,Bcl-2 mRNA表达下降(P<0.01);Bax、Caspase-9、Caspase-8以及Caspase-3表达较对照组增多,而Bcl-2表达则明显下降(P<0.01)。结论桔梗皂苷D能诱导人移行细胞癌5637细胞凋亡,其作用机制可能与上调Caspase-9、Caspase-8、Caspase-3、Cyt-c、p53和下调Bcl-2表达、激活线粒体途径和死亡受体途径有关。
Objective To explore the inducing effect of platycodin D on the apoptosis of human transitional cell carcinoma 5637 cell lines and related molecular mechanism. Methods The change of cell morphology was observed by HE staining and transmission electron microscope. The apoptosis was detected by flow cytometry. The change of apoptosis-related proteins expression was detected by Western blot. The mRNA expressions of p53,Bcl-2 and Bax related with apoptosis pathway were detected by PCR method. The expressions of apoptosis-related proteins in 5637 cells were detected by immunocytochemical method. Results From HE staining and transmission electron microscope,we found the apoptosis of 5637 cells treated with platycodin D. The number of apoptotic and dead cells was increased,with visible apoptotic bodies in platycodin D group. Compared with control group,the cell apoptosis index of platycodin D group was significantly higher(P<0.01); the expressions of Survivin and Livin were significantly decreased(P<0.05); the expressions of Caspase-9,Caspase-8,Caspase-3 and Cyt-c were increased(P<0.05); the expressions of p53 and Bax mRNA were greatly increased but Bcl-2 mRNA was decreased(P<0.01); the expressions of Bax,Caspase-9,Caspase-8 and Caspase-3 were increased,but Bcl-2 was decreased(P<0.01). Conclusion Platycodin D could induce the apoptosis of 5637 cells,which may be related to up-regulating Caspase-9,Caspase-8,Caspase-3,Cyt-c,p53 expression,down-regulating Bcl-2 expression and activating the mitochondrial pathway and death receptor pathway.
Objective To explore the inducing effect of platycodin D on the apoptosis of human transitional cell carcinoma 5637 cell lines and related molecular mechanism. Methods The change of cell morphology was observed by HE staining and transmission electron microscope. The apoptosis was detected by flow cytometry. The change of apoptosis-related proteins expression was detected by Western blot. The mRNA expressions of p53,Bcl-2 and Bax related with apoptosis pathway were detected by PCR method. The expressions of apoptosis-related proteins in 5637 cells were detected by immunocytochemical method. Results From HE staining and transmission electron microscope,we found the apoptosis of 5637 cells treated with platycodin D. The number of apoptotic and dead cells was increased,with visible apoptotic bodies in platycodin D group. Compared with control group,the cell apoptosis index of platycodin D group was significantly higher(P<0.01); the expressions of Survivin and Livin were significantly decreased(P<0.05); the expressions of Caspase-9,Caspase-8,Caspase-3 and Cyt-c were increased(P<0.05); the expressions of p53 and Bax mRNA were greatly increased but Bcl-2 mRNA was decreased(P<0.01); the expressions of Bax,Caspase-9,Caspase-8 and Caspase-3 were increased,but Bcl-2 was decreased(P<0.01). Conclusion Platycodin D could induce the apoptosis of 5637 cells,which may be related to up-regulating Caspase-9,Caspase-8,Caspase-3,Cyt-c,p53 expression,down-regulating Bcl-2 expression and activating the mitochondrial pathway and death receptor pathway.
引文
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[15]Zheng WY,Kang YY,Li LF,et al.Levels of effectiveness of gene therapies targeting survivin and its splice variants in human breast cancer cells[J].Drug Discov Ther,2011,5(6):293-8.
[16]Rodriguez-Berriguete G,Torrealba N,Ortega MA,et al.Prognostic value of inhibitors of apoptosis proteins(IAPs)and caspases in prostate cancer:caspase-3 forms and XIAP predict biochemical progression after radical prostatectomy[J].BMC Cancer,2015,15:809.
[17]Shiloach T,Berens C,Danke C,et al.t Livin displays flexibility by promoting alternative cell death mechanisms[J].PLo S One,2014,9(6):e101075.
[2]Yang HY,Wang JD,Lo TC,et al.Occupational exposure to herbs containing aristolochic acids increases the risk of urothelial carcinoma in Chinese herbalists[J].J Urol,2013,189(1):48-52.
[3]Zhao HL,Kim YS.Determinaion of the kinetic properties of platycodin D for the inhibition of pancreatic lipase using a1,2-diglyceride-based colorimetric assay[J].Arch Pharm Res,2004,27(9):968-72.
[4]Park DI,Lee JH,Moon SK,et al.Induction of apoptosis and inhibition of telomerase activity by aqueous extract from platycodin grandiflorum in human lung carcinoma cells[J].Pharmacol Res,2005,51(5):437-43.
[5]Luan X,Gao YG,Guan YY,et al.Platycodin D inhibits tumor growth by antiangiogenic activity via blocking VEGFR2-mediated signaling pathway[J].Toxicol Appl Pharmacol,2014,281(1):118-24.
[6]Li T,Chen X,Chen X,et al.Platycodin D potentiates proliferation inhibition and apoptosis induction upon AKT inhibition via feedback blockade in non-small cell lung cancer cells[J].Sci Rep,2016,6:37997.
[7]Tao W,Su Q,Wang H,et al.Platycodin D attenuates acute lung injury by suppressing apoptosis and inflammation in vivo and in vitro[J].Int Immunopharmacol,2015,27(1):138-47.
[8]Drosten M,Barbacid M.Ras and p53:An unsuspected liaison[J].Mol Cell Oncol,2015,3(2):e996001.
[9]Bisceglie F,Alinovi R,Pinelli S,et al.Autophagy and apoptosis:studies on the effects of bisthiosemicarbazone copper(ii)complexes on p53 and p53-null tumour cell lines[J].Metallomics,2016,8(12):1255-65.
[10]He Y,Chen W,Hu Y,et al.E.adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat[J].PLo S One,2015,10(9):e0138504.
[11]Sarkar A,Bhattacharjee S,Mandal DP.Induction of Apoptosis by Eugenol and Capsaicin in Human Gastric Cancer AGS Cells——Elucidating the Role of p53[J].Asian Pac J Cancer Prev,2015,16(15):6753-9.
[12]Dewanjee S,Dua T K,Khanra R,et al.Water Spinach,Ipomoea aquatic(Convolvulaceae),Ameliorates Lead Toxicity by Inhibiting Oxidative Stress and Apoptosis[J].PLo S One,2015,10(10):e0139831.
[13]Dua TK,Dewanjee S,Khanra R,et al.Cytoprotective and Antioxidant Effects of an Edible Herb,Enhydra fluctuans Lour.(Asteraceae),against Experimentally Induced Lead Acetate Intoxication[J].PLo S One,2016,11(2):e0148757.
[14]Chien CC,Wu MS,Shen SC,et al.Activation of JNK contributes to evodiamine-induced apoptosis and G2/M arrest in human colorectal carcinoma cells:a structure-activity study of evodiamine[J].PLo S One,2014,9(6):e99729.
[15]Zheng WY,Kang YY,Li LF,et al.Levels of effectiveness of gene therapies targeting survivin and its splice variants in human breast cancer cells[J].Drug Discov Ther,2011,5(6):293-8.
[16]Rodriguez-Berriguete G,Torrealba N,Ortega MA,et al.Prognostic value of inhibitors of apoptosis proteins(IAPs)and caspases in prostate cancer:caspase-3 forms and XIAP predict biochemical progression after radical prostatectomy[J].BMC Cancer,2015,15:809.
[17]Shiloach T,Berens C,Danke C,et al.t Livin displays flexibility by promoting alternative cell death mechanisms[J].PLo S One,2014,9(6):e101075.