NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7
NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7
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DOI：
10.1186/s12943-015-0379-0
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发表时间：
2015-06-27
期刊：
影响因子：
37.3
通讯作者：
Kim, Jung-Ae
中科院分区：
文献类型：
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作者：
Banskota, Suhrid;Regmi, Sushil C.;Kim, Jung-Ae
背景资料：虽然基质金属蛋白酶（MMP）-7表达与人结肠癌细胞转移潜能增加相关，但侵袭表型的潜在分子机制仍不清楚。本研究探讨了膜NADPH氧化酶（NOX）和AMP活化蛋白激酶（AMPK）对结肠癌细胞MMP-7表达和侵袭表型变化的调节作用。用光泽精化学发光法测定全细胞和蛋白提取物中超氧阴离子的产生（NADPH氧化酶活性）和细胞内活性氧物质（ROS），通过使用2 '，7'-二氯荧光素二乙酸酯（DCF-DA）的荧光显微镜观察。定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹分别用于测量mRNA和蛋白质水平。siRNA转染用于评估参与癌症侵袭的基因，其通过Matrigel transwell侵袭测定来鉴定。结果：在基础培养条件下，侵袭性较低的人结肠癌细胞（HT 29和Caco-2）MMP-7表达较低，而NOX 1表达和AMPK磷酸化水平较高。用12-O-十四烷酰基佛波醇-13-乙酸酯（TPA）处理HT 29和Caco-2细胞诱导了侵袭性表型应答沿着着ROS产生和NOX 2和MMP-7表达的相应增加以及AMPK磷酸化的减少，这类似于高度侵袭性人结肠癌细胞（SW 620和HCT 116）的基础条件。此外，在不同浓度的外源性过氧化氢处理的HT 29细胞中，观察到AMPK磷酸化与NOX 2和MMP-7表达之间的逆调节。TPA诱导的HT 29细胞侵袭表型被Vit处理所消除。E、DPI、夹竹桃素和NOX 2 siRNA，但不含NOX 1 siRNA，表明NOX 2衍生的ROS产生诱导了侵袭性表型。AP-1、NF-κ B和MAPK抑制TPA诱导的MMP-7表达（ERK、p38和JNK）抑制剂，而TPA诱导的NOX 2及其调节剂p47 phox和p67 phox的表达被p38和NF-κ B抑制剂阻断。结肠癌细胞中从NOX 1到NOX 2的分子开关诱导ROS产生，随后通过失活AMPK增强MMP-7表达，其另外抑制刺激诱导的ROS和NOX 2基因表达的自动调节。
Background: Although matrix metalloproteinase (MMP)-7 expression is correlated with increased metastatic potential in human colon cancer cells, the underlying molecular mechanism of invasive phenotype remains unknown. In the current study, we investigated the regulatory effects of membrane NADPH oxidase (NOX) and AMP activated protein kinase (AMPK) on MMP-7 expression and invasive phenotype change in colon cancer cells.Methods: Production of superoxide anion was measured by lucigenin chemiluminescence assay using whole cells and protein extracts (NADPH oxidase activity), and intracellular reactive oxygen species (ROS) by fluorescence microscopy using 2',7'-dichlorofluorescein diacetate (DCF-DA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Luciferase reporter assay was performed to identify transcription factors linked to gene expression.Results: Under basal conditions, less invasive human colon cancer cells (HT29 and Caco-2) showed low MMP-7 expression but high NOX1 expression and AMPK phosphorylation. Treatment of HT29 and Caco-2 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced an invasive phenotype response along with corresponding increases in ROS production and NOX2 and MMP-7 expression as well as reduced AMPK phosphorylation, which resemble basal conditions of highly invasive human colon cancer cells (SW620 and HCT116). In addition, inverse regulation between AMPK phosphorylation and NOX2 and MMP-7 expression was observed in HT29 cells treated with different concentrations of exogenous hydrogen peroxide. TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit. E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype. TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-kappa B, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-kappa B inhibitors.Conclusions: Molecular switch from NOX1 to NOX2 in colon cancer cells induces ROS production and subsequently enhances MMP-7 expression by deactivating AMPK, which otherwise inhibits stimulus-induced autoregulation of ROS and NOX2 gene expression.