去甲斑蝥素通过调节 NF-κB 活性转录抑制 MMP-9 表达对肝癌的抗转移作用。

Antimetastatic effects of norcantharidin on hepatocellular carcinoma by transcriptional inhibition of MMP-9 through modulation of NF-kB activity.

机构信息

Department of Emergency Medicine, School of Medicine, Chung Shan Medical University, Taichung, Taiwan.

出版信息

PLoS One. 2012;7(2):e31055. doi: 10.1371/journal.pone.0031055. Epub 2012 Feb 7.

BACKGROUND

The rate of morbidity and mortality of hepatocellular carcinoma (HCC) in Taiwan has not lessened because of difficulty in treating tumor metastasis. Norcantharidin (NCTD) is currently used as an anticancer drug for hepatoma, breast cancer, and colorectal adenocarcinoma. NCTD possesses various biological anticancer activities, including apoptosis. However, detailed effects and molecular mechanisms of NCTD on metastasis are unclear. Thus, HCC cells were subjected to treatment with NCTD and then analyzed to determine the effects of NCTD on cell metastasis.

METHODOLOGY/PRINCIPAL FINDINGS: Modified Boyden chamber assays revealed that NCTD treatment inhibited cell migration and invasion capacities of HCC cells substantially. Results of zymography and western blotting showed that activities and protein levels of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (u-PA) were inhibited by NCTD. Western blot analysis showed that NCTD inhibits phosphorylation of ERK1/2. Testing of mRNA level, quantitative real-time PCR, and promoter assays evaluated the inhibitory effects of NCTD on MMP-9 and u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay for analyzing the genomic DNA sequences bound to these proteins was reactive to the transcription protein nuclear factor (NF)-kappaB, which was inhibited by NCTD. The expression of NF-kappa B was measured by western blot analysis, which revealed decreased nuclear-factor DNA-binding activity after NCTD treatment.

CONCLUSIONS

NCTD inhibited MMP-9 and u-PA expression through the phosphorylation of ERK1/2 and NF-kappaB signaling pathway which serves as a powerful chemopreventive agent in HCC cell metastasis.

背景

由于肿瘤转移治疗困难，台湾的肝细胞癌（HCC）发病率和死亡率并未降低。去甲斑蝥素（NCTD）目前被用作肝癌、乳腺癌和结直肠腺癌的抗癌药物。NCTD 具有多种生物抗癌活性，包括凋亡。然而，NCTD 对转移的详细作用和分子机制尚不清楚。因此，用 NCTD 处理 HCC 细胞，然后分析 NCTD 对细胞转移的影响。

方法/主要发现：改良 Boyden 室测定显示，NCTD 处理显著抑制 HCC 细胞的迁移和侵袭能力。明胶酶谱和 Western blot 结果表明，NCTD 抑制基质金属蛋白酶-9（MMP-9）和尿激酶纤溶酶原激活物（u-PA）的活性和蛋白水平。Western blot 分析表明，NCTD 抑制 ERK1/2 的磷酸化。通过检测 mRNA 水平、实时定量 PCR 和启动子分析，评估 NCTD 对 HCC 细胞中 MMP-9 和 u-PA 表达的抑制作用。用于分析与这些蛋白结合的基因组 DNA 序列的染色质免疫沉淀（ChIP）测定对转录蛋白核因子（NF）-kappaB 有反应，NF-kappaB 被 NCTD 抑制。NF-kappaB 的表达通过 Western blot 分析进行测量，结果显示 NCTD 处理后核因子 DNA 结合活性降低。