The effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo.
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 diploid peak and apoptic cells in the treated groups were observed.


CONCLUSIONS
The CaM antagonist, EBB, has a strong anti-hepatoma effect and enhances the effect of 5-FU, induces hepatoma cell to apoptosis, promotes the P53 protein expression and decreases the amount of CaM in the cytoplasm. All these results demonstrate that EBB is a new and potentially useful drug against hepatoma and should be researched further.
Liu
Jiewen
J
Department of Pathology, Institute of Hematology, CAMS & PUMC, Tianjin 300020, China. LiuJW@Publictpt.tj.c
Qi
Shuling
S
Zhu
Huifang
H
Zhang
Jinhong
J
Li
Zhuo
Z
Wang
Tong
T
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D044182:Benzylisoquinolines; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005472:Fluorouracil / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009368:Neoplasm Transplantation; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D000470:Alkaloids; D000964:Antimetabolites, Antineoplastic; D044182:Benzylisoquinolines; D002147:Calmodulin; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C050387:O-(4-ethoxybutyl)berbamine; D005472:Fluorouracil