Zhou Li, Yang Fan, Li Guobing, Huang Jingbin, Liu Yali, Zhang Qian, Tang Qin, Hu Changpeng, Zhang Rong
Department of Pharmacy, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.
Department of Orthopaedic, General Hospital of Tibetan Military Command Lhasa, Lhasa, China.
Front Pharmacol. 2018 May 18;9:517. doi: 10.3389/fphar.2018.00517. eCollection 2018.
Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Hence, new anti-liver cancer treatment strategies need to be urgently developed. Coptisine is a natural alkaloid extracted from rhizoma coptidis which exhibits anticancer activity in various preclinical models, including liver cancer. However, the molecular mechanisms underlying the anti-liver cancer effects of coptisine remains unclear. We used flow cytometry to assess the binding of coptisine to 67LR expressed on the surface of SMMC7721, HepG2, LO2 and H9 cells. Then SMMC7721, HepG2 and BEL7402 cells, belonging to the HCC cell lines, were treated with coptisine. The cell viability was detected using a cell counting kit-8 assay. Apoptosis was evaluated using flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assay. Apoptotic-related proteins and tumor death receptor 67-kDa laminin receptor (67LR) were detected using Western blot analysis. The cyclic guanosine 3',5'-monophosphate (cGMP) concentration was determined using enzyme-linked immunosorbent assay. sh67LR lentivirus, anti67LR antibody, and cGMP inhibitor NS2028 were used to determine how a 67LR/cGMP signaling pathway regulated coptisine-induced apoptosis. Tumor growth inhibited by coptisine was confirmed in a SMMC7721 cell xenograft mouse model. Coptisine selectively exhibited cell viability in human hepatoma cells but not in normal human hepatocyte cell line LO2 cells. Coptisine promoted SMMC7721 and HepG2 cell apoptosis by increasing 67LR activity. Both 67LR antibody and sh67LR treatment blocked coptisine-induced apoptosis and inhibition of cell viability. Coptisine upregulated the expression of cGMP. Moreover, cGMP inhibitor NS2028 significantly decreased coptisine-induced apoptosis and inhibition of cell viability. experiments confirmed that coptisine could significantly suppress the tumor growth and induce apoptosis in SMMC7721 xenografts through a 67LR/cGMP pathway. Coptisine-mediated 67LR activation may be a new therapeutic strategy for treating hepatic malignancy.
肝细胞癌（HCC）是最常见的原发性肝癌。因此，迫切需要开发新的抗肝癌治疗策略。黄连碱是从黄连根茎中提取的一种天然生物碱，在包括肝癌在内的各种临床前模型中均表现出抗癌活性。然而，黄连碱抗肝癌作用的分子机制仍不清楚。我们使用流式细胞术评估黄连碱与SMMC7721、HepG2、LO2和H9细胞表面表达的67LR的结合。然后，用黄连碱处理属于肝癌细胞系的SMMC7721、HepG2和BEL7402细胞。使用细胞计数试剂盒-8检测细胞活力。使用流式细胞术和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）检测法评估细胞凋亡。使用蛋白质免疫印迹分析检测凋亡相关蛋白和肿瘤死亡受体67-kDa层粘连蛋白受体（67LR）。使用酶联免疫吸附测定法测定环磷酸鸟苷（cGMP）浓度。使用sh67LR慢病毒、抗67LR抗体和cGMP抑制剂NS2028来确定67LR/cGMP信号通路如何调节黄连碱诱导的细胞凋亡。在SMMC7721细胞异种移植小鼠模型中证实了黄连碱对肿瘤生长的抑制作用。黄连碱在人肝癌细胞中选择性地表现出细胞活力，但在正常人肝细胞系LO2细胞中则不然。黄连碱通过增加67LR活性促进SMMC7721和HepG2细胞凋亡。67LR抗体和sh67LR处理均阻断了黄连碱诱导的细胞凋亡和细胞活力抑制。黄连碱上调了cGMP的表达。此外，cGMP抑制剂NS2028显著降低了黄连碱诱导的细胞凋亡和细胞活力抑制。实验证实，黄连碱可通过6
