The Inhibitory Effect Of DodFMG In MCF-7 Cell Line In Vitro

Objective: To investigate the effects of the genistein derivative 5,4'-Di-n-octoxyl-7-gem-Difluoromethylenegenistein(DodFMG) on the inhibitor effect of human breast cancer cell line(MCF-7) in vitro,and explore the mechanism of DodFMG to induce human breast cancer cell apoptosis by upgrading PTEN, inhibitting pAkt, and activating Caspase-3.Methods: Human breast cancer cell line(MCF-7) cells were cultured in vitro. The proliferative inhibitory effect of DodFMG on MCF-7 cells was examined by MTT assay. Plate clone-formation method was used to determine the effect of DodFMG on the proliferation in MCF-7 cell line. DNA agarose gel electrophoresis was used to test apoptosis induced by DodFMG in MCF-7 cell line. Effects of DodFMG on the expressions of PTEN,PAKT and caspase-3 protein in MCF-7 cells were detected by western blotting, to explore molecular mechanism of the anti-breast cancer action of DodFMG..Results: MTT assay showed that DodFMG ihibited proliferation and growh of MCF-7 cells in a dose-dependent manner. the proliferation inhibitory rates were 27.76%,49.13%,65.71%and 55.38% respectively after treatment with DodFMG at various concentrations of 3.0,10.0,30.0μmol/L and Genistin at 3??蘭ol/L for 48h.The proliferation inhibition rate of DodFMG at 30.0μmol/L(65.71%) is higher than that of the lead compound Genistin (55.38%)at the same concentration. Plate clone-formation method showed that the clone inhibitory rates were 36.23%,54.13%,74.08%,59.63% and 87.61% after treatment with DodFMG (3.0,10.0,30.0μmol/L), gen30.0μmol/L, TAM5.0μmol/L for 48h respectively. The clone inhibitory rate of DodFMG(74.08%) at 30.0μmol/L is higher than that of the lead compound Gen (59.63%) at corresponding concentration, and lower than that of the positive control drug TAM(5.0μmol/L). DNA agarose gel electrophoresis showed that the DNA ladder bands could appear after treatment with DodFMG(10.0,30.0μmol/L) for 48h, this showed DodFMG could induce apoptosis significantly. Western-blotting analysis indicated that after exposures to DodFMG(3.0,10.0,30.0μmol/L) for 24h , the protein expressions of PTEN and Caspase-3 were up-regulated by 15.06%, 31.67%, 45.63% and 11.22%, 26.80%, 36.73%, and the protein expression of p-Akt was down-regulated by 8.34%, 27.50%, 39.77% respectively in comparison with the control group. After MCF-7 cells were treated with DodFMG at 30.0μmol/L for 6,12 and 24h, the protein expressions of PTEN and Caspase-3 were up-regulated by 10.41%, 25.64%, 50.45% and 9.10%, 16.62%, 37.00%, and the protein expression of p-Akt was down-regulated by 9.33%, 27.50, 61.19%, respectively in comparison with the control group. These showed the expressions of PTEN and Caspase-3 were inceased and the expression of p-Akt was deceased in a time and dose-dependent manner after treatment with various concentrations of DodFMG. In comparison with the lead compound Gen, DodFMG was more effective.Conclusions:(1) DodFMG posses the inhibitory effect of the proliferation and growth of MCF-7 cells cultured in vitro in a dose-dependent man??(2) DodFMG can induce apoptosis of human breast cancer MCF-7 cells significantly.(3) The inhibition of growth and induction of apoptosis of MCF-7 cells by DodFMG may be associated with upregulations of PTEN and Caspase-3 protein expression and downregulation of p-Akt protein expression.