积雪草酸对人肝癌SMMC-7721细胞增殖和自噬的影响
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摘要
目的探讨积雪草酸(asiatic acid,AA)对人肝癌SMMC-7721细胞增殖和自噬的影响。方法不同浓度AA作用于人肝癌SMMC-7721细胞24 h后,MTT法检测细胞活性并观察自噬抑制剂3-MA对40μmol/L AA的干预作用;MDC染色检测自噬泡的形成,Western blot检测自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、p62、mTOR、p-mTOR及p53的表达。结果 MTT检测结果显示,不同AA浓度均可抑制人肝癌SMMC-7721细胞增殖,并呈浓度依赖性(F=46.790,P=0.006),IC50=37.313μmol/L。与单独使用40μmol/L AA相比,自噬抑制剂3-MA可部分逆转40μmol/L AA对SMMC-7721细胞增殖的抑制作用[(46.400±9.099)%vs(22.000±3.391)%,P<0.001]。MDC染色实验表明40μmol/L AA干预可增加自噬泡形成。Western blot检测发现,与对照组比较,40μmol/L AA可明显降低LC3-Ⅰ的蛋白表达,而提高LC3-Ⅱ表达(1.744±0.108 vs 1.529±0.065,t=2.928,P=0.043;0.113±0.031 vs 0.380±0.036,t=-9.754,P<0.001),降低p62蛋白表达(0.522±0.024 vs 0.123±0.019,t=22.565,P<0.001)和p-mTOR蛋白表达(1.252±0.039 vs 0.353±0.028,t=30.775,P<0.001),但对mTOR和p53的蛋白表达无影响(1.713±0.111 vs 1.556±0.076,t=1.555,P=0.190;0.671±0.040 vs 0.726±0.055,t=-1.555,P=0.210)。结论 AA能抑制人肝癌SMMC-7721细胞增殖,可能与其通过非p53依赖方式负调控mTOR通路诱发自噬有关。
Objective To investigate the effects of asiatic acid(AA) on cell proliferation and autophagy of human hepatoma SMMC-7721 cells. Methods The human hepatoma SMMC-7721 cells were exposed to different concentrations of AA alone or combined with autophagy inhibitor 3-MA for 24 h,after that,the cell viability was examined by MTT method,the formation of autophagic vacuoles were detected by MDC staining,the expression of the autophagy-related proteins including LC3-Ⅰ,LC3-Ⅱ,p62,mTOR,p-mTOR,and p53 were detected by Western-blot. Results MTT assay results showed that different concentrations of AA could inhibit the proliferation of human hepatocarcinoma SMMC-7721 cells in a concentration-dependent manner(F=46.790,P=0.006)with IC50= 37.313 μmon/L.When combined with autophagy inhibitor 3-MA,the cell proliferation inhibition rate(%)of AA to SMMC-7721 cells was significantly reduced compared with the group treated with 40 μmol/L AA alone[(22.000±3.391)% vs(46.400±9.099)%,P<0.001)]. AA increased the amount of autophagic vacuoles detected by MDC staining. Compared with the control group,40 μmol/L AA significantly down-regulated the protein expression level of LC3-I while up-regulated the protein expression of LC3-Ⅱ(1.744±0.108 vs 1.529 ±0.065,t =2.928,P =0.043;0.113 ± 0.031 vs 0.380 ± 0.036,t =-9.754,P <0.001),but down-regulated the p62 protein expression(0.522±0.024 vs 0.123±0.019,t=22.565,P<0.001). Besides,AA significantly reduced the protein expression level of p-mTOR(1.252±0.039 vs 0.353 ±0.028,t =30.775,P <0.001),but had no influence on the protein expression of mTOR and p53(1.713±0.111 vs 1.556±0.076,t=1.555,P=0.190;0.671±0.040 vs 0.726±0.055,t=-1.555,P=0.210). Conclusions AA can inhibit the proliferation of human hepatoma SMMC-7721 cells,which may be related to the AA-inducted autophagy via the suppression of mTOR signaling pathway in a p53-indepandent manner.
Objective To investigate the effects of asiatic acid(AA) on cell proliferation and autophagy of human hepatoma SMMC-7721 cells. Methods The human hepatoma SMMC-7721 cells were exposed to different concentrations of AA alone or combined with autophagy inhibitor 3-MA for 24 h,after that,the cell viability was examined by MTT method,the formation of autophagic vacuoles were detected by MDC staining,the expression of the autophagy-related proteins including LC3-Ⅰ,LC3-Ⅱ,p62,mTOR,p-mTOR,and p53 were detected by Western-blot. Results MTT assay results showed that different concentrations of AA could inhibit the proliferation of human hepatocarcinoma SMMC-7721 cells in a concentration-dependent manner(F=46.790,P=0.006)with IC50= 37.313 μmon/L.When combined with autophagy inhibitor 3-MA,the cell proliferation inhibition rate(%)of AA to SMMC-7721 cells was significantly reduced compared with the group treated with 40 μmol/L AA alone[(22.000±3.391)% vs(46.400±9.099)%,P<0.001)]. AA increased the amount of autophagic vacuoles detected by MDC staining. Compared with the control group,40 μmol/L AA significantly down-regulated the protein expression level of LC3-I while up-regulated the protein expression of LC3-Ⅱ(1.744±0.108 vs 1.529 ±0.065,t =2.928,P =0.043;0.113 ± 0.031 vs 0.380 ± 0.036,t =-9.754,P <0.001),but down-regulated the p62 protein expression(0.522±0.024 vs 0.123±0.019,t=22.565,P<0.001). Besides,AA significantly reduced the protein expression level of p-mTOR(1.252±0.039 vs 0.353 ±0.028,t =30.775,P <0.001),but had no influence on the protein expression of mTOR and p53(1.713±0.111 vs 1.556±0.076,t=1.555,P=0.190;0.671±0.040 vs 0.726±0.055,t=-1.555,P=0.210). Conclusions AA can inhibit the proliferation of human hepatoma SMMC-7721 cells,which may be related to the AA-inducted autophagy via the suppression of mTOR signaling pathway in a p53-indepandent manner.
引文
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[3] 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.
[4] REN L,CAO Q X,ZHAI F R,et al.Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling[J].Pharm Biol,2016,54(11):2377-2382.
[5] HAO Y,HUANG J,MA Y,et al.Asiatic acid inhibits proliferation,migration and induces apoptosis by regulating Pdcd4 via the PI3K/Akt/mTOR/p70S6K signaling pathway in human colon carcinoma cells[J].Oncol Lett,2018,15(6):8223-8230.
[6] WU T,GENG J,GUO W,et al.Asiatic acid inhibits lung cancer cell growth in vitro and in vivo by destroying mitochondria[J].Acta Pharm Sin B,2017,7(1):65-72.
[7] LEE YS,JIN DQ,KWON EJ,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,2012,186(1):83-91.
[8] JANKU F,MCCONKEY D J,HONG D S,et al.Autophagy as a target for anticancer therapy[J].Nature Reviews Clinical Oncology,2011,8:528-539.
[9] YAZDANI H O,HUANG H,TSUNG A.Autophagy:dual response in the development of hepatocellular carcinoma[J].Cells,2019,8(2):E91.
[10]耿骥,郭文洁,刘佳,等.积雪草酸对结肠癌HCT116细胞增殖及自噬水平的影响[J].江苏大学学报(医学版),2015,25(2):133-136,141.
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[12]TIAN T,LI X,ZHANG J.mTOR Signaling in Cancer and m TOR Inhibitors in Solid Tumor Targeting Therapy[J].Int J Mol Sci,2019,20(3):E755.
[13]JUANJUAN TANG,JIEHUI DI,HUAN CAO,et al.p53-mediated autophagic regulation:A prospective strategy for cancer therapy[J].Cancer Letters,2015,363(2):101-107.
[14]聂雪坤,曾莉莉,牛培广,等.p53蛋白介导m TOR信号途径影响肿瘤细胞自噬的研究进展[J].中国肿瘤生物治疗杂志,2013,20(6):739-743.