8-Bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK.
OBJECTIVE
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 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.


CONCLUSIONS
BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.
Yang
Xiao-Hong
XH
Laboratory of Medicine Engineering, Medical College, Hunan Normal University, Changsha 410013, Hunan Province, China.
Zheng
Xing
X
Cao
Jian-Guo
JG
Xiang
Hong-Lin
HL
Liu
Fei
F
Lv
Yuan
Y
D000111:Acetylcysteine / Q000494:pharmacology; D000813:Anilides / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D004789:Enzyme Activation; D005419:Flavonoids / Q000737:chemistry* / Q000494:pharmacology* / Q000627:therapeutic use; D056945:Hep G2 Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism*
C457499:2-chloro-5-nitrobenzanilide; C552860:8-bromo-7-methoxychrysin; D000813:Anilides; D000970:Antineoplastic Agents; D000975:Antioxidants; D005419:Flavonoids; D017382:Reactive Oxygen Species; C043561:chrysin; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3; D000111:Acetylcysteine