Departments of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
PLoS One. 2011 May 4;6(5):e19341. doi: 10.1371/journal.pone.0019341.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
CONCLUSION/SIGNIFICANCE: 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.
延长细胞存活的细胞凋亡的消除对于癌症进展是至关重要的。在我们的先前研究中，我们显示了 MMP-2 的下调诱导了癌细胞系的凋亡。在这里，我们试图研究 MMP-2 耗竭如何导致神经胶质瘤异种细胞系凋亡的确切分子机制。
方法/主要发现：MMP-2siRNA(pM)对 MMP-2 的转录抑制诱导了人神经胶质瘤异种细胞 4910 和 5310 中的凋亡，伴有 PARP、caspase-8 和 -3 的裂解。Western 印迹和细胞因子阵列显示，与 mock 和 pSV 对照相比，TNF-α 的细胞和分泌水平显著降低，同时伴随 TNFR1、TRADD、TRAF2、RIP、IKKβ 和 pIκBα 表达水平降低，导致 pM 处理细胞中 p65 磷酸化和核易位受到抑制。此外，MMP-2 抑制导致 Fas-L、Fas 和 FADD 表达水平升高，同时 p38 和 JNK 磷酸化增加。JNK-活性测定显示 pM 转染细胞中 JNK 的激活延长。用 SB203580 特异性抑制 p38 没有显示出任何效果，而用 SP600125 抑制 JNK 磷酸化显著逆转了 pM 诱导的 PARP、caspase-8 和 -3 的裂解，表明 JNK 在 pM 诱导的细胞死亡中起重要作用。rhMMP-2 的补充通过显著提高 TNF-α、TRADD、IKKβ 和 pIκBα 的表达并降低 FADD、Fas-L 和磷酸化 JNK 水平来对抗 pM 的作用。EMSA 分析表明，rhMMP-2 处理显著逆转了 pM 抑制的 NF-κB 活性，挽救了 pM 诱导的细胞死亡。体内研究表明，pM 处理减少了颅内肿瘤的生长，免疫组织化学分析显示，pM 处理肿瘤切片中磷酸化 p65 减少，磷酸化 JNK 增加，与 TUNEL 阳性凋亡细胞增加相关。
结论/意义：总之，我们的研究表明 MMP-2 在调节 TNF-α 介导的神经胶质瘤异种细胞系中 NF-κB 激活和 Fas 介导的 JNK 介导的凋亡中起作用。
