Zhao Hongwei, Zhang Yubao, Sun Jianmin, Zhan Chao, Zhao Liang
Department of Hepatopancreatobiliary Surgery, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China.
Oncol Res. 2016;23(5):237-48. doi: 10.3727/096504016X14562725373671.
Raltitrexed (RTX) is an antimetabolite drug used as a chemotherapeutic agent for treating colorectal cancer, malignant mesothelioma, and gastric cancer. The antitumor capacity of RTX is attributed to its inhibitory activity on thymidylate synthase (TS), a key enzyme in the synthesis of DNA precursors. The current study is aimed at investigating the potential antitumor effects of RTX in liver cancer. Using the HepG2 cell line as an in vitro model of liver cancer, we evaluated the effects of RTX on cell proliferation employing both a WST-8 assay and a clone formation efficiency assay. In addition, we monitored the ultrastructure changes of HepG2 cells in response to RTX with transmission electric microscopy. To investigate the mechanism underlying the regulation of cell proliferation by RTX, we analyzed cell cycle using cell flow cytometry. Moreover, real-time PCR and Western blot analyses were conducted to examine expression levels of cell cycle regulatory proteins cyclin A and cyclin-dependent kinase 2 (CDK2), as well as their mediators tumor suppressor genes p53 and p16. Our results demonstrate that RTX inhibits HepG2 proliferation by arresting the cell cycle at G0/G1. This cell cycle arrest function was mediated via downregulation of cyclin A and CDK2. The observed elevated expression of p53 and p16 by RTX may contribute to the reduction of cyclin A/CDK2. Our study indicates that RTX could serve as a potential chemotherapeutic agent in the treatment of hepatocellular carcinoma.
雷替曲塞（RTX）是一种抗代谢药物，用作治疗结直肠癌、恶性间皮瘤和胃癌的化疗药物。RTX的抗肿瘤能力归因于其对胸苷酸合成酶（TS）的抑制活性，TS是DNA前体合成中的关键酶。本研究旨在探讨RTX在肝癌中的潜在抗肿瘤作用。我们使用HepG2细胞系作为肝癌的体外模型，采用WST-8法和克隆形成效率法评估RTX对细胞增殖的影响。此外，我们用透射电子显微镜监测了HepG2细胞对RTX反应的超微结构变化。为了研究RTX调节细胞增殖的机制，我们用细胞流式细胞术分析细胞周期。此外，进行了实时PCR和蛋白质印迹分析，以检测细胞周期调节蛋白细胞周期蛋白A和细胞周期蛋白依赖性激酶2（CDK2）及其介导因子肿瘤抑制基因p53和p16的表达水平。我们的结果表明，RTX通过将细胞周期阻滞在G0/G1期来抑制HepG2细胞增殖。这种细胞周期阻滞功能是通过下调细胞周期蛋白A和CDK2介导的。观察到RTX使p53和p16表达升高，这可能有助于降低细胞周期蛋白A/CDK2的水平。我们的研究表明，RTX可能作为一种潜在的化疗药物用于治疗肝细胞癌。
