8-溴-7-甲氧基白杨素诱导肝癌细胞凋亡涉及 ROS 和 JNK。
8-Bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK.
机构信息
Laboratory of Medicine Engineering, Medical College, Hunan Normal University, Changsha 410013, Hunan Province, China.
出版信息
World J Gastroenterol. 2010 Jul 21;16(27):3385-93. doi: 10.3748/wjg.v16.i27.3385.
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 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.
目的
研究 8-溴-7-甲氧基白杨素（BrMC）的促凋亡活性是否涉及人肝癌细胞（HCC）中活性氧（ROS）的生成和 c-Jun N 端激酶（JNK）的激活。
方法
体外培养 HepG2、Bel-7402 和 L-02 细胞系，用碘化丙啶（PI）染色后通过流式细胞术（FCM）评估 BrMC 的促凋亡作用，用酶联免疫吸附测定（ELISA）检测 caspase-3 活性，以及用 DNA 琼脂糖凝胶电泳检测。用二氯二氢荧光素二乙酸酯（DCHF-DA）探针标记后通过 FCM 评估 ROS 的产生。通过 Western 印迹分析 JNK 和 c-Jun 蛋白的磷酸化水平。
结果
PI 染色后的 FCM 显示，随着 BrMC 浓度的增加，亚 G1 细胞群的比例呈剂量依赖性增加（P<0.05），在 10 μmol/L BrMC 处理 48 h 后，HepG2 细胞达到 39.0%±2.8%。与阳性对照化合物白杨素（HepG2 细胞 16.2%±1.6%，Bel-7402 细胞 11.0%±1.3%）相比，BrMC 对 HepG2 和 Bel-7402 细胞（分别为 32.1%±2.6%和 29.3%±2.3%）的作用更为有效，与 5-氟尿嘧啶（HepG2 细胞 33.0%±2.1%，Bel-7402 细胞 29.3%±2.3%）在 10 μmol/L 时的作用相似。BrMC 对人胚肝 L-02 细胞几乎没有影响，亚 G1 细胞群的比例为 5.4%±1.8%。用 BrMC 处理 HepG2 细胞 48 h 也增加了 caspase-3 的活性，呈浓度依赖性。caspase-3 特异性抑制剂 z-DEVD-fmk 可阻止 caspase-3 的激活。用 BrMC 处理 10 μmol/L 48 h 导致 DNA 梯形成。用 BrMC（10 μmol/L）处理细胞可使 HepG2 细胞中 DCHF-DA 的平均荧光强度从 0 小时的 7.2±1.12 增加到 3 小时的 79.8±3.9 和 6 小时的 89.7±4.7。BrMC 不影响 L-02 细胞中 ROS 的产生。在用 N-乙酰半胱氨酸（10 mmol/L）预处理的 HepG2 细胞中，BrMC 处理未能诱导细胞死亡和 caspase-3 激活。此外，在 BrMC（2.5、5.0、10.0 μmol/L）处理 HepG2 细胞 12 h 时，观察到 JNK 的激活。JNK 的激活在处理后 12 小时达到峰值，这种激活持续到 24 小时。用 BrMC 处理细胞 12 h 后，N-乙酰半胱氨酸和 SP600125 预处理抑制了磷酸化 JNK 和 c-Jun 蛋白的表达，但 GW9662 没有作用。SP600125 显著减少了 BrMC 诱导的 HepG2 细胞死亡和 caspase-3 激活。N-乙酰半胱氨酸和 GW9662 也减轻了 BrMC 处理的 HepG2 细胞的细胞死亡和 caspase-3 激活的诱导。
结论
BrMC 通过 ROS 的产生和持续的 JNK 激活诱导 HCC 细胞凋亡。
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