Cao Ying, Cao Jianbo, Yu Binbin, Wang Shusheng, Liu Lili, Tao Li, Sun Wanping
Department of Pharmacy, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, Jiangsu 215600, P.R. China.
Department of Surgery, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, Jiangsu 215600, P.R. China.
Exp Ther Med. 2018 Feb;15(2):1894-1901. doi: 10.3892/etm.2017.5637. Epub 2017 Dec 15.
Hepatocellular carcinoma (HCC) is a primary malignancy in the liver, which is a global health problem. The present study aimed to observe the apoptotic effects of berbamine on SMMC-7721 cell lines and to investigate the effects of berbamine on induction of the intrinsic apoptotic pathway. The human HCC SMMC-7721 cells were cultured and cell morphology observed using a phase contrast microscope. SMMC-7721 cell apoptosis was examined by employing a flow cytometry assay. The nuclei of SMMC-7721 cells were stained with DAPI and observed by utilizing a laser fluorescence microscope. Cytochrome (Cyto ) levels were evaluated by using immunofluorescence staining. The reverse transcription-semi-quantitative polymerase chain reaction (RT-sqPCR) and western blot analysis were used to examine the mRNA and protein levels of B-cell lymphoma 2, (Bcl-2), Bax, Bcl-2-associated X, apoptosis regulator, p53 and survivin, respectively. Berbamine inhibited SMMC-7721 cell growth at 20 and 0 µmol/l, compared with control group (0 µmol/l berbamine). DAPI results demonstrated that berbamine affected the nucleus morphology of SMMC-7721 cells. Berbamine at a concentration of 20 µmol/l (P<0.05) and 40 µmol/l (P<0.01) significantly enhanced apoptosis rate compared with control group. Berbamine triggered Cyto release from SMMC-7721 cell nuclei to the cytoplasm. Berbamine (10, 20, 40 µmol/l) significantly enhanced Bax and p53 levels and decreased Bcl-2 and survivin levels compared with control group, according to RT-sqPCR and western blot assay findings. In conclusion, berbamine induced SMMC-7721 cell apoptosis, through upregulating p53 expression and downregulating survivin expression, which further triggered mitochondria signaling pathway-mediated apoptosis.
肝细胞癌（HCC）是肝脏的一种原发性恶性肿瘤，是一个全球性的健康问题。本研究旨在观察小檗胺对SMMC - 7721细胞系的凋亡作用，并探讨小檗胺对诱导内源性凋亡途径的影响。培养人肝癌SMMC - 7721细胞，并用相差显微镜观察细胞形态。采用流式细胞术检测SMMC - 7721细胞凋亡情况。用DAPI对SMMC - 7721细胞的细胞核进行染色，并用激光荧光显微镜观察。通过免疫荧光染色评估细胞色素c（Cyto c）水平。分别采用逆转录 - 半定量聚合酶链反应（RT - sqPCR）和蛋白质免疫印迹分析检测B细胞淋巴瘤2（Bcl - 2）、Bax、Bcl - 2相关X蛋白、凋亡调节因子、p53和生存素的mRNA和蛋白水平。与对照组（0 μmol/l小檗胺）相比，20 μmol/l和40 μmol/l的小檗胺抑制了SMMC - 7721细胞的生长。DAPI结果表明，小檗胺影响了SMMC - 7721细胞的细胞核形态。与对照组相比，浓度为20 μmol/l（P<0.05）和40 μmol/l（P<0.01）的小檗胺显著提高了凋亡率。小檗胺促使Cyto c从SMMC - 一、7721细胞核释放到细胞质中。根据RT - sqPCR和蛋白质免疫印迹分析结果，与对照组相比，小檗胺（10、20、40 μmol/l）显著提高了Bax和p53水平，降低了Bcl - 2和生存素水平。总之，小檗胺通过上调p53表达和下调生存素表达诱导SMMC - 7721细胞凋亡，进而触发线粒体信号通路介导的凋亡。
