摘要
AIM:To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin(BrMC) involve reactive oxygen species(ROS) generation and c-Jun N-terminal kinase(JNK) activation in human hepatocellular carcinoma cells(HCC).METHODS:HepG2,Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry(FCM) after propidium iodide(PI) staining,caspase-3 activity using enzymelinked immunosorbent assay(ELISA),and DNA agarose gel electrophoresis.ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCHFDA) probe labeling.The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.RESULTS:FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell pop-ulation(P < 0.05),reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L.The potency of BrMC to HepG2 and Bel-7402(32.1% ± 2.6%) cells was found to be more effective than the lead compound,chrysin(16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil(33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L.BrMC had little effect on human embryo liver L-02 cells,with the percentage of sub-G1 cell population 5.4% ± 1.8%.Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3,in a concentration-dependent manner.z-DEVD-fmk,a caspase-3specific inhibitor,prevented the activation of caspase-3.Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder.Treatment of cells with BrMC(10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h.BrMC did not affect ROS generation in L-02 cells.BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine(10 mmol/L).In addition,in HepG2 cells treated with BrMC(2.5,5.0,10.0 μmol/L) for 12 h,JNK activation was observed.Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h.The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment,but GW9662 had no effect.SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells.N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.CONCLUSION:BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.
AIM:To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin(BrMC) involve reactive oxygen species(ROS) generation and c-Jun N-terminal kinase(JNK) activation in human hepatocellular carcinoma cells(HCC).METHODS:HepG2,Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry(FCM) after propidium iodide(PI) staining,caspase-3 activity using enzymelinked immunosorbent assay(ELISA),and DNA agarose gel electrophoresis.ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCHFDA) probe labeling.The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.RESULTS:FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell pop-ulation(P < 0.05),reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L.The potency of BrMC to HepG2 and Bel-7402(32.1% ± 2.6%) cells was found to be more effective than the lead compound,chrysin(16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil(33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L.BrMC had little effect on human embryo liver L-02 cells,with the percentage of sub-G1 cell population 5.4% ± 1.8%.Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3,in a concentration-dependent manner.z-DEVD-fmk,a caspase-3specific inhibitor,prevented the activation of caspase-3.Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder.Treatment of cells with BrMC(10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h.BrMC did not affect ROS generation in L-02 cells.BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine(10 mmol/L).In addition,in HepG2 cells treated with BrMC(2.5,5.0,10.0 μmol/L) for 12 h,JNK activation was observed.Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h.The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment,but GW9662 had no effect.SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells.N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.CONCLUSION:BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.
基金
Supported by Hunan Provincial Natural Science Foundation of China,No.03JJY5009
