Inhibition Of Proliferation And Induction Of Apoptosis In Human Liver Cancer SMMC-7721 Cell Line By 5-ally-7-gen-difluormethylenechrysin

Objective : The aim of this work was to investigate effects of 5-ally-7-gen-difluormethylenechrysin(ADFMChR) on the proliferation inhibition and apoptosis induction in human liver cancer(SMMC-7721) cell line and its mechanism. Methods:Human liver cancer(SMMC-7721) cells were cultured in vitro.The influence of various concentrationss of ADFMChR on the proliferation of SMMC-7721 cells and heptocyte (L-02 ) cells were evaluated by clone formation assay .Cell counting method was used to test the influence of growth curve in SMMC-7721 cells treated with different concentrations of ADFMChR.The apoptosis of SMMC-7721 cells was determined by AO/EB fluorescence stain and analyzed with flow cytometry by PI staining . Moreover, Western blot analyzed the expression of PPARγ, NF-κB, Bcl-2, Bax and Caspase-3 proteins.Results:Clone formation test have shown that ADFMChR inhibited significantly growth of SMMC-7721 cells and L-02 cells in a dose- and a time–dependent manner. The IC₅₀ value of ADFMChR was 3.56μM and 23.88μM respectively,meanwhile the IC₅₀ value of ChR was 9.28μM. Cell counting showed that average doubling time retarded from 23.81h in normal culture SMMC-7721 cells to 25.99h in 0.3uM ADFMChR experimented SMMC-7721 cells,even to 61.22 h in 30.0uM ADFMChR experimented SMMC-7721 cells.AO/EB fluorescence stain data provided evidence that the apoptosis of SMMC-7721 cells induced by ADFMChR in a dose- and a time–dependent manner.PI staining flow cytometry revealed that increased G1 percentage of cell cycle was accompanied with the raised apoptotic percentage in SMMC-7721 cells after treatment with ADFMChR. Adding 1.0μM,3.0μM,10.0μM ADFMChR for 48h increased G1 percentage of cell cycle from 45.9% in normal culture SMMC-7721 cells to 56.3%,60.9%,64.2%,respectively.Adding 3.0μM ADFMChR(0,12,24,48,72h) increased G1 percentage of cell cycle from 45.9% in normal culture SMMC-7721 cells to 50.8%,53.2%,60.9%,62.9%(P <0.05 VS control).Western blotting analysis indicated that ADFMChR of various concentrations could down-regulate expression of Bcl-2, NF-кB proteins , meanwhile up- regulate expression of PPARγ,Bax and Caspase-3 protein in SMMC-7721 cells (P<0.05﹚.Conclusions: Our results showed that ADFMChR strongly inhibited cell growth and proliferation human liver cancer (SMMC-7721) cells. ADFMChR down-regulate expression of NF-κB,up regulate expression of Caspase-3,decreasing the value of Bcl-2 /Bax protein via activating of PPARγ.