羽扇豆醇对人肝癌细胞HepG2和SK-HEP-1侵袭、转移的影响及机制研究  

Effect of lupeol on invasion and metastasis of human hepatoma HepG2 and SK-HEP-1 cells and its mechanism

作　　者：刘博佳 宁青[2] 钟荣玲[2] 夏智[2] 姜子瑜 宋捷[1,2] 韦英杰[1,2] LIU Bo-jia;NING Qing;ZHONG Rong-ling;XIA Zhi;JIANG Zi-yu;SONG Jie;WEI Ying-jie(Third School of Clinical Medical of Nanjing University of Chinese Medicine,Nanjing 210028,Chirui;Key laboratory of Traditional Chinese Medicine Drug Release System,State Administration of Traditional Chinese Medicine,Jiangsu Provincial Academy of Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学第三临床医学院,江苏南京210028 [2]江苏省中医药研究院国家中医药管理局中药释药系统重点研究室,江苏南京210028

出　　处：《中国中药杂志》2020年第24期6028-6035,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(81873055);全国中医药创新骨干人才培训项目(19ZYCXGG-2);北京希思科临床肿瘤学研究基金会项目(Y-L2019-05);江苏省卫生计生委项目(CXTDB2017003)。

摘　　要：上皮-间质转化(EMT)广泛存在于胚胎发育中,与细胞的迁移、侵袭密切相关。肿瘤中EMT水平升高表现为上皮型钙黏附素(E-cadherin)被神经型钙黏附素(N-cadherin)取代,间质标志物α-平滑肌肌动蛋白(α-SMA)、波形蛋白(vimentin)表达上调等。据报道,羽扇豆醇可降低基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)和N-cadherin蛋白表达抑制人肉骨瘤细胞转移,调控MMP-9 mRNA表达抑制Huh-7细胞侵袭。但羽扇豆醇在肝癌方面的研究较少,因此该文探讨羽扇豆醇对肝癌细胞体内外侵袭转移的作用及其可能机制。采用MTT法和Annexin V/PI双染法检测羽扇豆醇对HepG2细胞和SK-HEP-1细胞活力、凋亡的影响。Transwell法检测羽扇豆醇对细胞侵袭能力的影响。Western blot法检测迁移相关蛋白E-cadherin,N-cadherin,α-SMA,vimentin和MMP-9的表达水平。建立裸鼠皮下移植瘤模型以及H22肝癌细胞肺部转移模型,HE染色结合免疫组化法观察羽扇豆醇体内抗肿瘤生长和抑制肺转移的疗效。结果显示,羽扇豆醇(50,100μmol·L^(-1))显著抑制细胞活性,诱导细胞凋亡;羽扇豆醇(5,10,20μmol·L^(-1))可剂量依赖性地抑制HepG2细胞和SK-HEP-1细胞侵袭,同时诱导上皮-间质转化(EMT)标志蛋白E-cadherin表达,降低N-cadherin,α-SMA,vimentin和MMP-9蛋白表达。体内实验显示,羽扇豆醇抑制异种移植HepG2细胞荷瘤小鼠肿瘤的生长,上调肿瘤组织中E-cadherin蛋白表达,降低N-cadherin蛋白表达;同时减少肝癌H22细胞在小鼠肺部转移灶的形成。以上结果表明,羽扇豆醇抑制肝癌细胞侵袭转移的机制可能与其调控EMT过程相关。Epithelial-mesenchymal transformation(EMT)exists in embryonic development and is closely related to cell migration and invasion.The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin,and the expression of interstitial markers such asα-SMA and vimentin was up-regulated.It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9)and N-cadherin to inhibit the metastasis of osteoma cells.However lupeol has been less studied in liver cancer.Therefore,this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism.MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells.Moreover,the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay.The expressions of E-cadherin,N-cadherin,α-SMA,vimentin and MMP-9 were measured by Western blot.The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay.The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis.Western blot showed that the expression of E-cadherin,a landmark protein for EMT,was induced by lupeol,and the expressions of N-cadherin,α-SMA,vimentin and MMP-9 were decreased.In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft.In addition,immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice,reduce the expression of N-cadherin,and inhibit the metastasis of liver cancer H22 cells in the lungs of mice.The above results indicated that the mechanism of

关 键 词：肝癌细胞 羽扇豆醇 侵袭 转移 上皮-间质转化(EMT) HEPG2细胞 SK-HEP-1细胞 

分 类 号：R285[医药卫生—中药学]