Experimental Study On Induction Of Apoptosis In Human Lever Carcinoma HepG2 Cells Through Activation Of PPARγ By 6, 8-ditrifluoromethy-7-acetoxychrysin

Objective: The aim of this study is to investigate induction of the growth inhibition and apoptosis in human lever carcinoma cell line (HepG2) by the deviration of chrysin, 6, 8-ditrifluoromethy-7-acetoxychrysin (dFMAChR), and estimate the anti-tumor effect of dFMAChR in vivo using Human lever carcinoma xenograft in nude mouse model and provide a molecular mechanism of this effect so as to find a new chemical entity for tumor chemotherapy.Methods: Human lever carcinoma cell line (HepG2) cells and human embryo lever diploid cell line (L-02) cells were cultured in vitro. MTT assay was used to determine the effect of dFMAChR on the proliferation of HepG2 cells and L-02 cells and to evaluate the selectivity of action against tumor cells. Clone-formation assay in plate and soft agar were applied to test inhibition of clone formation of HepG2 by dFMAChR. Apoptosis of HepG2 cells was determined by DNA fragmentation assay and Flow cytometry (FCM) using PI staining. Hoechst 33258 staining was used to observe the morphologic changes of apoptosis induced by dFMAChR in HepG2 cells. Human lever carcinoma xenograft in nude mouse model was established to investigate the anti-tumor effect of dFMAChR in vivo. HE staining was utilized to observe the morphologic changes of the xenografts. The apoptosis of the human lever carcinoma xenograft cells in nude mouse was detected by TUNEL staining. The protein expression of PPARγ, NF-κB, Bcl-2, Bax and Caspase-3 were examined by Western blot to explore the molecular mechanism of anti-lever cancer action of dFMAChR. Results: dFMAChR inhibited proliferation and growth of HepG2 cells in a dose-dependent manner, its efficacy was stronger than that of Chrysin. While there was no significant effect in L-02 cells. The selective index was 42.96. Clone-formation assay in plate and soft agar showed that dFMAChR inhibited the clone formation of HepG2 in a concentration-dependent manner.Typical morphologic changes of apoptosis was observed after treatment with dFMAChR by fluorescence microscope using hoechst 33258 staining. Flow cytometry after PI staining indicated that apoptosis in HepG2 cells was significantly induced by dFMAChR. After treatment with dFMAChR at 10.0μM for 48h and 72h, the isolated DNA displayed a clear the ladder-shaped band in agarose gel electrophoresis. While con-treatment with dFMAChR 10.0μM and GW9662 10.0μM , this phenomenon did not appear.Therapeutic trial of human lever carcinoma xenograft in nude mouse model indicated that dFMAChR significantly inhibited the growth of human lever carcinoma xenografts in Balb/c-nu mice. The tumor weight inhibitory rate of dFMAChR at 20, 40, 80 mg/kg was 40.17%,47.41% ,66.81% respectively. HE staining and TUNEL staining confirmed the apoptosis of the human lever carcinoma xenograft cells in nude mouse after treatment with dFMAChR.Western blotting indicated that the expression of PPARγincreased 10.3%,29.9%,46.4% in comparison with the control group, whereas the protein level of NF-κB,Bcl-2 was down-regulated 5.1%,9.0%,14.6% and 1.9%,5.6%,9.4% respectively after exposure to dFMAChR at 3.0μM, 10.0μM, 30.0μM for 24h,. In the meanwhile ,the express of Bax and caspase-3 was up-regulated 5.6%,9.2%,14.1% and 9.3% respectively. When con-treated with dFMAChR and GW9662, a blocking agent of PPARγ, the protein level of PPARγand caspase-3 did not display an up-regulated tendency . The expression of NF-κB was not decreased sifnificantly as well. Conclusion:1. dFMAChR possess a significant inhibitory effect on the cell growth of human lever carcinoma cell line hepG2 in vitro, and it has less toxic activity on human embryo lever diploid cell line L-02. The toxic activity of the dFMAChR is highly selective to tumor cell in vitro.2. dFMAChR can significantly inhibit the growth of human lung carcinoma xenograft in nude mice in a dose-dependent manner.3. The growth inhibitory effect of dFMAChR in HepG2 cell line is associated with the induction of apoptosis.4. Induction of apoptosis by dFMAChR in HepG2 is associated with activication of PPARγ, down-regulation of NF-κB and Bcl-2, and activication of caspase-3.