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
enzyme-linked
immunosorbent assay (ELISA), and
DNA agarose gel electrophoresis. ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCHF-DA) 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 population (P < 0.05), reaching 39.0% +/- 2.8% of HepG2 cells after 48 h of treatment with BrMC
at 10 micromol/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 micromol/L and similar to
5-fluorouracil (33.0% +/- 2.1% for HepG2 cells and 29.3% +/- 2.3% for Bel-7402 cells)
at 10 micromol/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-3-specific inhibitor, prevented the activation of
caspase-3. Treatment with BrMC
at 10 micromol/L for 48 h resulted in the formation of
a DNA ladder. Treatment of cells with BrMC (10 micromol/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 micromol/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.