The Effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo  

钙调素拮抗剂EBB抗小鼠肝癌的作用及其机制的研究(英文)

作　　者：刘杰文[1] 齐淑玲[1] 朱惠芳[1] 李卓[1] 王彤[1] 张金红[2] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所,天津300020 [2]南开大学分子生物研究所,天津300071

出　　处：《Chinese Medical Journal》2002年第5期759-762,157,共4页中华医学杂志（英文版）

基　　金：ThisProjectwassupportedbyTianjinnaturalfoundationofsciences(9414 0 90 17);theMinisterofPublicHealthofChina (94 2 0 2 4)

摘　　要：OBJECTIVE: To evaluate the anti-hepatoma effect of Calmodulin antagonist 0 - 4-ethoxyl-butyl-Berbamine (EBB), one of the berbamine derivatives. METHODS: Monotetrazolium (MTT) method was used to analyze the effect of EBB on the proliferation and growth inhibition effect. Of a hepatoma cell line in vitro. A mouse hepatoma model was induced by injection of hepatoma cells (H22) in the abdominal cavity. The effect of EBB on survival at different concentrations as well as in combination with 5-FU were investigated in vivo. Flow cytometry analysis, dot blot hybridization, western blot, immunochemistry, enzyme-linked lectin assay (ELISA), trifluoperazine (TFP) and electron microscopic observation were used to study the effect of EBB on cell cycle process, P53 mRNA and protein levels, calmodulin content and ultrastractural changes of hepatoma cells. RESULTS: EBB exerts a very strong inhibitory effect on human hepatoma cell line 7402 and mouse hepatoma cell line H22 in vitro. The IC(50) value of EBB for the two cell lines are 3.312 microg/ml and 1.167 microg/ml, respectively. The sensitivity of H22 cells to 5-FU can be markedly enhanced: The IC(50) dosage of 5-Fu can be decreased from 0.75 microg/ml down to 0.15 microg/ml, when jointly administered with nontoxic dosages of EBB (IC(10)). In vivo, EBB can prolong the lifespan of mice with ascites H22 to more than three months. 64% of mice survived, while all animals in the control group died by the 18th day. When EBB (5 mg x kg(-1) x d(-1)) is jointly used with 5-FU (25 mg x ml(-1) x d(-1)), 73% of mice with ascites H22 survived, much higher than 27% in the 5-FU treated group. EBB can enhance the anti-hepatoma ability of 5-Fu treatment. EBB mechanism against hepatoma: P53 expression in the EBB treated group is substantially higher than that in the control group. EBB increased the translation of P53. As a calmodulin antagonist, EBB decreases amount of the CaM in hepatoma cells and blocked the hepatoma cell proliferation cycle at the G(2)M phase. Before the G(0)/G(1) phase, a 目的　本项目研究了国产新型CaM拮抗剂EBB的抗肝癌作用。方法　体外实验表明EBB对人肝癌细胞系 74 0 2和鼠肝癌细胞系H2 2均有明显抑制作用 ,并有剂量依赖关系 ,其IC5 0分别为 3 312 μg/ml和 1 16 7μg/ml,无毒剂量 (IC10 )的EBB与 5 Fu(IC5 0 )联合应用可明显提高H2 2细胞对5 Fu的敏感性 ,5 Fu的IC5 0由 0 75 μg/ml降至 0 15 μg/ml,敏感性提高 5倍。体内实验证明 ,EBB可使腹水型肝癌小鼠动物长期健康存活 (3个月以上 ) ,长期存活率高达 6 4% ,对照组均在 18天左右死亡。EBB的无毒剂量 (5mg·kg 1·d 1)与 5 Fu (2 5mg·kg 1·d 1)联合应用 ,EBB组的长期存活率 (73% )明显高于 5 Fu组 (2 7% ) ,证明EBB对5 Fu的抗肝癌有增敏作用 ,统计学上有显著性。结果　EBB作用机制的研究显示 ,EBB组P5 3蛋白表达水平明显高于对照组 ,提示EBB对P5 3在翻译水平上有较强的上调作用 ;EBB作为拮抗剂使肝细胞内CaM含量下降 ;使增殖的细胞阻断于G2 M期 ,在G0 /G1期间 ,有二倍体峰以及超微结构可见凋亡细胞。结论　CaM拮抗剂EBB具有强抗肝癌作用 ,对 5 Fu有增敏作用 ,对肝癌细胞凋亡有诱导作用 ,它可上调P5 3蛋白表达及下调CaM含量 ,提示它是一种新型的、有前途的化疗药物 ,有进一步研究和开发价值。

关 键 词：BENZYLISOQUINOLINES Alkaloids Animals Antimetabolites  Antineoplastic CALMODULIN Carcinoma  Hepatocellular Cell Division Cell Survival Chromatography  Thin Layer Dose-Response Relationship  Drug Drug Synergism Fluorouracil Inhibitory Concentration 50 Liver Neoplasms  Experimental Mice Neoplasm Transplantation RNA  Messenger Research Support  Non-U.S. Gov't Tumor Cells  Cultured Tumor Suppressor Protein p53 

分 类 号：R735.7[医药卫生—肿瘤] R965[医药卫生—临床医学]