Study Effection Of Tamoxifen On TGF-β1, Smad4 And ER Exprression Of Human Hepatocellular Carcinama Cells

Objective TGF-β1 suppressed the proliferation of cancer in the progress of cancer,Smad4 was the key factor in Smad signaling pathway, the study of domestic and abroad showed that it have important effectiveness in the progress of hepatocellular carcinoma. The abnormal of TGF-β1/Smad4 signaling pathway has important effectiveness in the progress of hepatocellular carcinoma. The metabolism and inactivation of Estradiol(E2) are majored in liver,it could induce activation of proto-oncogene of liver. Estrogen receptor (ER) is concerned with hepatocarcinogenesis,and it has relation with the development and progonosis of hepatocellular carcinoma. Antioestrogens tamoxifen has been widly used in the therapy of hepatocellular carcinoma in rencent years, the efection still has dispute,and the mechanisms of tamoxifen action in hepatocellular carcinoma, however, are not yet clearly understood.The effect of tamoxifen on TGF-β1,Smad4 and ER has been reported in other cancer,but the effect of tamoxifen on TGF-β1,Smad4 and ER of hepatocellular carcinoma has been little reported. The aim of this study was to research the effects of different concentration and act time of tamoxifen on proliferation,and TGF-β1,Smad4 and ER expression of human hepatocellular carcinoma cells,study the mechanism of TGF-β1,Smad4 and ER in the hepatocellular carcinoma progress and their relationship, the effction of tamoxifen on hepatocellular carcinoma whether or not and its mechanism,and give experimentation and theory support to use tamoxifen to cure hepatocellular carcinoma. Materials and methods HepG2 cells were maintained and passaged in RPMI 1640 containing 100 jig/ml streptomycin with 100μg/ml penicillin and 10% fetal calf serum (FCS) at 37℃ in 5% CO₂.(1)Observe the effect of different concentration and act time of tamoxifen on HepG2 cells: examine OD by MTT,then calculated suppression rate;(2)Observe the cellular morphology of HepG2 cells:cell growth were examined by inverted phase contrast microscope and HE;(3)Immunohistochemistry to observe the effect of different concentration tamoxifen acted 24h,48h and 72h,the exprission TGF-β1,Smad4 and ER of HepG2 cells.Results (1) Tamoxifen suppressed the proliferation of human hepatocellular carcinoma cells, accompanying the rise in drug's concentration or prolong drug's action time, suppression rate of human hepatocellular carcinoma cell rose gradually, that did time and concentration dependent (F_time =140.234,.P_time=0.000; F_{concentration}=44.031,P_{concentration}=0.002), time had more effection. There were positive significance among the group of 24 hour and 48 hour and 72 hour( all p < 0.05).The suppression rate of every different concentration group of tamoxifen was positive higher than control group( all p < 0.05). The suppression rate of group which tamoxifen concentration was 30μmol/L and action time was 72 hours was the highest.(2)It could find that the volume of cells become smaller,cell become round. and the number of cell become less. HE also found cell nucleus chromatosis disuniformity.(3) Tamoxifen encouraged the expression of TGF-β1, that did time and concentration dependent (F_time=34.144,P_time=0.003; F_{concentration} =18.510,P_{concentration} =0.010), time had more effection. TGF-β1 expression of 24 hours and 48 hours and 72 hours groups all higher than control group(all p < 0.05), and different tamoxifen concentration groups all encouraged the expression of TGF-β1 than control group(all p < 0.05). The expression of group which tamoxifen concentration was 30μmol/L and action time was 72 hours was the highest.(4)Tamoxifen encouraged the expression of Smad4, that does time and concentration dependent(F_time =44.644,P_time =0.012; F_{concentration} =16.062,P_{concentration}=0.002), time had moreeffection. Smad4 expression of the group of 24 hours and 48 hours and 72 hours all higher than control group(all p < 0.05), and different tamoxifen concentration groups all encouraged the expression of Smad4 than control group(all p < 0.05). The expression of group which tamoxifen concentration was 30μmol/L and action time was 72 hours was the highest.(5) Tamoxifen inhibited the expression of ER, that did time and concentration dependent(F_time =70.040, P_time =0.001; F_{concentration}=27.992,P_{concentration}=0.004), time had more effection. ER expression of 24 hours and 48 hours and 72 hours groups all lower than control group(all p < 0.05), and different tamoxifen concentration groups all suppressed the expression of ER than control group(all p < 0.05).The expression of group which tamoxifen concentration was 30μmol/L and action time was 72 hours was the lowest.(6)The effect of tamoxifen on TGF-β1 and ER in 24h,48h and 72h has negative correlation (r =-0.993,P=0.037;r =-0.994,P=0.030;r =-0.991,P=0.003),the expression of TGF-β1 and ER in 7.5μmol/L group,15μmol/L group and 30μmol/L group all changed with action time,and it has negative correlation (r =-0.999,P=0.012; r=-0.988,P=0.050; r=-1.000,P=0.004);(7) The effect of tamoxifen on Smad4 and ER in 24h,48h and 72h has negative correlation (r =-0.998,P=0.020; r =-1.000,P=0.008; r =-0.995,P=0.032), the expression of Smad4 and ER in 7.5μmol/L group, 15μmol/L group and 30μmol/L group all changed with action time,and it has negative correlation (r =-0.998,P=0.022; r =-1.00,P=0.008; r =-0.992,P=0.041);(8) The effect of tamoxifen on TGF-β1 and Smad4 in 24h, 48h and 72h has positive correlation (r =0.998,P=0.017;r =0.993,P=0.038;r =0.999,P=0.011), the expression of TGF-β1 and Smad4 in 7.5μmol/L group, 15μmol/L group and 30μmol/L group all changed with action time, and it has positive correlation (r =0.994,P=0.034; r =0.991,P=0.042; r =0.990,P=0.045). Conclusion (1) Tamoxifen suppressed the proliferation of human hepatocellular carcinoma cells, that did time and concentration dependent.(2) Tamoxifen encouraged the expression of TGF-β1, that did time and concentration dependent.(3) Tamoxifen encouraged the expression of Smad4, that does time and concentration dependent. (4)Tamoxifen inhibited the expression of ER, that did time and concentration dependent.(5)Tamoxifen suppressed the hepatocellular carcinoma cells by effect the expression of TGF-β1,Smad4 and ER in hepatocellular carcinoma cells.(6)The abnormal of TGF-β1/Smad4 signaling pathway and ER expression had synergistic effect in hepatocellular carcinoma.(7)Study the mechanism of TGF-β1,Smad4 and ER in the progress of hepatocellular carcinoma and mechanism of tamoxifen cure hepatocellular carcinoma, and give experimentation and theory support to use tamoxifen to cure hepatocellular carcinoma.