Manumycin对人肝癌HepG2细胞的生长抑制作用与Ras通路的关系

Correlation between Inhibitory Effect of Manumycin on HumanHepatoma Cancer Cell HepG2 and Ras Signal Transduction Pathway

背景与目的:目前肝癌药物治疗的临床疗效还不尽人意。在肝癌的发生发展过程中,Ras起着重要的作用。Ras必须在法尼基转移酶的作用下,经过法尼基化修饰才具有生物活性。我们观察了法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,并探讨其对Ras通路的影响。方法:应用氚标胸腺嘧啶脱氧核苷掺入实验观察法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,Westernblot技术检测Manumycin对HepG2细胞Ras通路相关蛋白,包括pan-Ras、N-Ras、membrane-pan-Ras、ERK1/2、p-ERK1/2、AKT、p-AKT及MKP-1表达的影响。结果:Manumycin(5、10、20、40、80μmol/L)处理24h对肝癌HepG2细胞株生长具有明显的抑制作用,并呈浓度依赖性,其IC50为(17.1±2.6)μmol/L。同时Westernblot分析显示,Manumycin对肝癌HepG2细胞pan-Ras蛋白和N-Ras蛋白及细胞膜pan-Ras蛋白表达均有抑制作用,且对细胞膜pan-Ras蛋白的抑制作用更明显。进一步研究发现Manumycin对Ras蛋白下游ERK1/2和AKT活性也有抑制作用,表现为ERK1/2和AKT磷酸化水平降低。Manumycin对AKT活性的影响与PI3K抑制剂Wortmannin作用相似,两者联用可加强对AKT的抑制。此外,Manumycin诱导MKP-1的表达具有浓度依赖性。

Background and Objective: Treatment with anti cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, Ras has been shown to play an important role. Ras requires a farnesyl moiety for activation .It has been found that farnesyltransferase inhibitor Manumycin inhibits farnesyl protein transferase,which catalyzes farnesylation. This study was designed to investigate the antitumor effect of Manumycin in human hepatoma cell line HepG2 and try to clarify its influence on Ras pathway. Methods:The growth inhibitory effect of farnesyltransferase inhibitor Manumycin on human hepatoma cell line HepG2 was observed by using thymidine incorporation assay. The relative protein expressions of pan Ras, N Ras, ERK1/2, AKT,and MKP 1 affected by Manumycin were determined by using Western blot analysis. Results:Manumycin(5, 10, 20, 40,and 80μmol/L) significantly inhibited cell growth of human hepatoma cell line HepG2 with IC50 value of (17.1±2.6)μmol/L. Manumycin could inhibit both pan Ras and N Ras in human hepatoma HepG2 cells, but its inhibitory effect on pan Ras of cell membrane was much stronger. Phospho MAPK and phospho AKT decreased significantly after treatment of HepG2 cells with Manumycin, while total MAPK and AKT were hardly affected. After treatment with 10 nmol/L wortmannin for 1 h,which had potent inhibitory effect on phosphorylation of AKT,Manumycin had stronger inhibitory effect on phosphorylation of AKT as compared to treatment without wortmannin, eventhough AKT protein levels were still unaffected. Furthermore, the expression of MKP 1 was elevated through manumycin treatment in a concentration dependent manner. Conclusion: Manumycin may significantly inhibit the growth of human hepatoma cell line HepG2, which was related to its inhibition on the combination of Ras and cell membrane and increasing the expression of MKP 1, accordingly inhibiting activation of ERK1/2 and AKT. These results suggest that Manumycin antagonizes the growth of HepG2 via the suppression of ras farnesylation blocking the function of oncogenic ras against and could be a potential new anti cancer agents human cancer, including hepatocellular carcinoma.