The Effects Of Tamoxifen On Smad3, Smad4 And Smad7 Expressions Of Human Hepatocellular Carcinoma HepG2 Cells

Objective: To research the effects of different concentration and acting time of tamoxifen (TAM) on proliferation of human hepatocellularcarcinoma cells, and Smad3,Smad4 and Smad7 expression. To study the mechanisms and relationship among Smad3, Smad4, Smad7 and hepatocellular carcinoma.Materials and methods:Human HepG2 cells were cultured in RPM1 1640 containing 100μg/ml streptomycin, 100μg/ml penicillin and 10% fetal calf serum (FCS) at 37℃in 5% CO2. The three groups were divided according to TAM concentrations:(1) Low concentration group:TAM's concentration is at 7.5μmol/L; (2) Middle concentration group. TAM's concentration is at 15μmol/L; (3) High concentration group,TAM's concentration is at 30μmol/L.Each well were added 0.1 ml TAM of different concentrations; (4) the control group:Each well was added 0.1 ml PBS. Each group is set 6 parallel wells. After tamoxifen treating, HepG2 cells proliferation changes were calculated with MTT; the cellular morphological changes were observed by inverted phase contrast microscope and HE staining; smad3,smad4 and smad7 expression were examined with immunohistochemistry.Results:(1) Tamoxifen suppressed the proliferation of human HepG2 cells, with increasing concentration and prolonged treating time, suppression rate of human HepG2 cells increased, time-does dependent (Ftime=929.656, P=0.000; Fdose=487.992,P=0.002), longer time were more effective. The suppression rates treated by tamoxifen was significent higher than the control group(all P<0.05). The suppression rate of group which tamoxifen concentration was at 30μmol/L and 72 hours reached the highest. (2)The volume of HepG2 cells became smaller and round,and the number of HepG2 cells become less.The cell nucleus were found chromatosis disuniformity by HE. (3) Tamoxifen increased the expression rates of Smad3, does-time concentration dependent (FTime=141.979,P=0.000; Fdose=126.82, P=0.000). Smad3 expression rates at 24 h,48 h and 72 h, were higher than the control group(all P<0.05),and the expression rates of Smad3 with different concentrations of tamoxifen were higher than the control group(all P<0.05). The expression ratess, at 30μmol/L and treated for 72 hours, reached the highest. (4)Tamoxifen increased the expression of Smad4, does-time dependent (Ftime=397.199, P=0.000; Fdose=261.016, P=0:000).Smad4 expression rates at 24 h,48 h and 72 h were higher than the control group(all P<0.05), and at different concentrations of tamoxifen the expression rates of Smad4 were higher than the control group(all P<0.05). The expression rate reached the highest,at 30μmol/L and 72 h; (5) Tamoxifen reduceed the expression rates of Smad7, does-time dependent (Ftime=332.91, P=0.000; Fdose=275.961,.P=0.000), Smad7 expression rates at 24 h,48 h and 72 h were lower than the control group(all P<0.05), and at different concentration of tamoxifen Smad7 expression rates were lower than the control group(all P<0.05).The expression rates of Smad 7 reached the lowest at 30μmol/L and 72 h.Concluions:(1) Tamoxifen suppressed the proliferation of human hepatocellular carcinoma cells, at time-dose dependent mode. (2) Tamoxifen increased the expression rates of Smad3 and Smad4, does-time dependent. Tamoxifen rduceed the expression of Smad7, does-time dependent. (3)Tamoxifen suppressed the hepatocellular carcinoma cells by up-regulating the expression of Smad3 and Smad4,down-regulating the expression of Smad7. The data suggeats that tamoxifen may be effective to treat hepatocellular carcinoma.