Suppression of MMP-2 attenuates TNF-α induced NF-κB activation and leads to JNK mediated cell death in glioma.
Suppression of MMP-2 attenuates TNF-α induced NF-κB activation and leads to JNK mediated cell death in glioma.
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DOI：
10.1371/journal.pone.0019341
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发表时间：
2011-05-04
期刊：
影响因子：
3.7
通讯作者：
Rao JS
中科院分区：
文献类型：
--
作者：
Kesanakurti D;Chetty C;Bhoopathi P;Lakka SS;Gorantla B;Tsung AJ;Rao JS
消除细胞凋亡以延长细胞存活在癌症进展中是必不可少的。在我们以前的研究中，我们发现MMP-2下调可诱导癌细胞凋亡。在这里，我们试图研究MMP-2耗竭如何导致胶质瘤异种移植细胞系凋亡的确切分子机制。MMP-2siRNA（pM）抑制MMP-2转录诱导人胶质瘤异种移植细胞4910和5310中与PARP、半胱天冬酶-8和-3切割相关的凋亡。Western印迹和细胞因子阵列显示，与模拟和pSV对照相比，PM处理的细胞中TNF-α的细胞和分泌水平显著降低，同时TNFR 1、TRADD、TRAF 2、RIP、IKKβ和pIκBα表达水平降低，导致p65磷酸化和核转位受到抑制。此外，MMP-2抑制导致Fas-L、Fas和FADD表达水平升高沿着p38和JNK磷酸化增加。JNK活性测定显示在pM转染的细胞中JNK活化延长。用SB 203580特异性抑制p38没有显示出任何效果，而用SP 600125抑制JNK磷酸化显著逆转PM诱导的PARP、半胱天冬酶-8和-3的切割，证明JNK在PM诱导的细胞死亡中的重要作用。rhMMP-2通过显著升高TNF-α、TRADD、IKKβ和pIκBα表达，降低FADD、Fas-L和磷酸化JNK水平，抵消pM的作用。EMSA分析表明，rhMMP-2处理可显著逆转pM抑制的NF-κB活性，从而使细胞免于pM诱导的细胞死亡。在体内研究表明，PM治疗减少颅内肿瘤的生长和免疫组织化学分析表明，减少磷酸化p65和增强磷酸化JNK水平，与增加TUNEL阳性凋亡细胞在PM处理的肿瘤切片。总之，我们的研究表明MMP-2在调节TNF-α介导的NF-κB激活和Fas介导的JNK介导的胶质瘤异种移植细胞凋亡中的作用。
Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo.
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