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AIM:To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dosedependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of AD- FMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC50 was 9.27 μmol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were signif icantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 μmol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 μmol/L GW9662, a blocker of PPARγ. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFM-ChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells. CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.
AIM: To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose dependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 μmol / L and 191.55 μmol / L respectively, the potency of AD-FMChR to HepG2 cells, was found to be similar to 5-fluoro The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55 / 8.45), higher than 5-FU (SI was 7.05 (65.37 / 9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol / L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were signif icantly higher when treated with 30.0 μmol / L ADFMChR than when treated DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol / L ADFMChR (30.0 μmol / L ChR (16.0%)) (P <0.05) and similar to those obtained with 30.0 μmol / for 48 h and 72 h resulted in 24 h of treatment with 3.0, 10.0, 30.0 μmol / L ADFM-ChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol / L GW 9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol / L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells. CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF- κB, and increasing Bax expression.