积雪草酸对人肝癌SMMC-7721细胞体内外增殖和凋亡的影响
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摘要
目的:研究积雪草酸(AA)体内外抗肝癌效应及其凋亡机制。方法:将人肝癌SMMC-7721细胞分为溶剂(0.1%DMSO溶剂)对照组和不同浓度(20μmol/L、30μmol/L、40μmol/L、50μmol/L、60μmol/L)AA组,作用于SMMC-7721细胞24 h后,采用CCK-8法检测细胞活性,Annexin V-APC/7-AAD双染色流式分析检测细胞凋亡率,JC-1染色检测细胞线粒体膜电位,同时采用Western blotting检测线粒体凋亡相关蛋白的表达。建立SMMC-7721细胞裸鼠皮下移植瘤模型,分为对照组、AA 50 mg/kg组和AA 100 mg/kg组,每组5只,观察AA灌胃给药21 d后移植瘤的生长情况。结果:AA浓度依赖性地抑制SMMC-7721细胞增殖(IC_(50)=38.31μmol/L)并促使细胞产生凋亡样改变。与溶剂对照组相比,AA 20μmol/L组、AA 40μmol/L组、AA 60μmol/L组均可提高SMMC-7721细胞凋亡率,并降低细胞线粒体膜电位(均P<0.01),具有一定的浓度依赖性;此外,AA 40μmol/L组明显上调Bax、Cyt-C和Cleaved-Caspase-3蛋白的表达,下调Bcl-2蛋白的表达(P<0.05或P<0.01),但对p53蛋白表达无明显影响(P>0.05)。AA 50 mg/kg组和AA 100 mg/kg组给药第14、第21天时的肿瘤体积明显小于对照组,给药第21天时的肿瘤质量小于对照组(均P<0.01)。结论:AA具有体内外抗肝癌作用,其机制可能与其诱导非p53依赖的线粒体途径的凋亡有关。
Objective:To investigate the effect and mechanisms of asiatic acid(AA) on the proliferation and apoptosis in human hepatoma SMMC-7721 cells in vitro and in vivo.Methods:Human hepatoma SMMC-7721 cells were divided into solvent control group and different concentrations(20～60 μmol/L) of AA groups.After 24 h of treatment,the cell viability,apoptosis rate,and the mitochondrial membrane potential changes were examined by CCK-8 method,Annexin V-APC/7-AAD double staining flow cytometric assay and JC-1 staining,respectively.The expression of the mitochondrial apoptosis related proteins were detected by western blotting.After the establishment of subcutaneous xenograft model bearing SMMC-7721 cells, the nude mice were divided into the control group,AA 50 mg/kg group and AA 100 mg/kg group(n=5 per group).The growth of xenograft was observed after 21 days of treatment.Results:AA inhibited the proliferation of SMMC-7721 cells in a dose-dependent manner with IC_(50)of 38.31 μmol/L and induced the apoptosis-like morphological change.Compared with the control group,AA dose-dependently elevated the apoptosis rate and lower the mitochondrial membrane potential(P<0.01).Additionally,at the concentration of 40 μmol/L,AA significantly up-regulated the protein expression levels of Bax,Cyt-C,and Cleaved-Caspase-3,while down-regulated the expression level of Bcl-2 as compared to the control group(P<0.05 or P<0.01),and it had no influence on the expression of p53(P>0.05).Intragastric administration of AA(50 mg/kg and 100 mg/kg) for 21 d significantly decreased the tumor volume and tumor weigh of nude mice(P<0.01).Conclusion:AA can inhibit the proliferation of human hepatoma SMMC-7721 cells in vitro and in vivo,and the mechanisms may be related to the mitochondrial apoptosis in a p53-indepandent manner induced by AA.
Objective:To investigate the effect and mechanisms of asiatic acid(AA) on the proliferation and apoptosis in human hepatoma SMMC-7721 cells in vitro and in vivo.Methods:Human hepatoma SMMC-7721 cells were divided into solvent control group and different concentrations(20～60 μmol/L) of AA groups.After 24 h of treatment,the cell viability,apoptosis rate,and the mitochondrial membrane potential changes were examined by CCK-8 method,Annexin V-APC/7-AAD double staining flow cytometric assay and JC-1 staining,respectively.The expression of the mitochondrial apoptosis related proteins were detected by western blotting.After the establishment of subcutaneous xenograft model bearing SMMC-7721 cells, the nude mice were divided into the control group,AA 50 mg/kg group and AA 100 mg/kg group(n=5 per group).The growth of xenograft was observed after 21 days of treatment.Results:AA inhibited the proliferation of SMMC-7721 cells in a dose-dependent manner with IC_(50)of 38.31 μmol/L and induced the apoptosis-like morphological change.Compared with the control group,AA dose-dependently elevated the apoptosis rate and lower the mitochondrial membrane potential(P<0.01).Additionally,at the concentration of 40 μmol/L,AA significantly up-regulated the protein expression levels of Bax,Cyt-C,and Cleaved-Caspase-3,while down-regulated the expression level of Bcl-2 as compared to the control group(P<0.05 or P<0.01),and it had no influence on the expression of p53(P>0.05).Intragastric administration of AA(50 mg/kg and 100 mg/kg) for 21 d significantly decreased the tumor volume and tumor weigh of nude mice(P<0.01).Conclusion:AA can inhibit the proliferation of human hepatoma SMMC-7721 cells in vitro and in vivo,and the mechanisms may be related to the mitochondrial apoptosis in a p53-indepandent manner induced by AA.
引文
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[2] LV J,SHARMA A,ZHANG T,et al.Pharmacological review on asiatic acid and its derivatives:A potential compound[J].SLAS Technol,2018,23(2):111-127.
[3] HSU Y L,KUO P L,LIN L T,et al.Asiatic acid,a triterpene,induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells[J].J Pharmacol Exp Ther,2005,313(1):333-344.
[4] TANG X L,YANG X Y,JUNG H J,et al.Asiatic acid induces colon cancer cell growth inhibition and apoptosis through mitochondrial death cascade[J].Biol Pharm Bull,2009,32(8):1399-1405.
[5] PARK B C,BOSIRE K O,LEE E S,et al.Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells[J].Cancer Lett,2005,218(1):81-90.
[6] 周军,高静,方春钱,等.积雪草酸对肝癌细胞增殖作用的影响[J].江苏大学学报(医学版),2009,19(6):475-479.
[7] LEE Y S,JIN D Q,KWON E J,et al.Asiatic acid,a triterpene,induces apoptosis through intracellular Ca2+ release and enhanced expression of p53 in HepG2 human hepatoma cells[J].Cancer Lett,2002,186(1):83-91.
[8] 郑德枢.细胞凋亡与细胞程序性死亡[J].动物学研究,2000(1):17-22.
[9] 郑婷婷,焦丽静,阙祖俊,等.中药诱导肿瘤凋亡相关机制研究进展[J].上海中医药杂志,2016,50(10):103-109.
[10] 冯浩,张箴,沙保勇,等.线粒体与肿瘤发生发展[J].生命科学,2014,26(8):799-803.
[11] LIU W,ZHAO H,WANG Y,et al.Calcium-calmodulin signaling elicits mitochondrial dysfunction and the release of cytochrome c during cadmium-induced apoptosis in primary osteoblasts[J].Toxicol Lett,2014,224(1):1-6.