RNAi-mediated downregulation of MMP-2 activates the extrinsic apoptotic pathway in human glioma xenograft cells.
RNAi-mediated downregulation of MMP-2 activates the extrinsic apoptotic pathway in human glioma xenograft cells.
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DOI：
10.3892/ijo_00000399
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发表时间：
2009-10
影响因子：
5.2
通讯作者：
Rao JS
中科院分区：
文献类型：
--
作者：
Gondi CS;Talluri L;Dinh DH;Gujrati M;Rao JS
恶性神经胶质瘤的特征在于通常涉及正常脑组织破坏的侵袭性和浸润性行为。治疗浸润性神经胶质瘤的策略，如化疗和基因治疗，在很大程度上仍然不成功。神经胶质瘤的浸润性质可以在很大程度上归因于蛋白酶，其包括丝氨酸、金属和半胱氨酸蛋白酶。我们以前的工作和其他人强烈建议之间的关系uPAR，MMP-9和MMP-2的表达，这种关系通常是神经胶质瘤的浸润性表型的指示。在本研究中，我们已经证明，RNA干扰介导的MMP-2的下调诱导4910人胶质瘤异种移植细胞系的凋亡。使用蛋白质印迹分析，我们观察到MMP-2下调的细胞中caspase-8水平增加，而TRADD和TRAF-2水平降低。此外，MMP-2下调的细胞中NIK水平增加。为了确定AIF和IκB-α的核定位，我们分析了MMP-2下调细胞的胞浆和核组分中AIF、IκB-α和p-IκB-α的水平。Western blot分析显示，MMP-2下调导致AIF向核内移位，并抑制p-IκB-α的核定位。为了证实AIF的参与，我们进行了FACS分析，以使用Mito-PT方法确定线粒体膜的完整性。流式细胞仪分析表明，MMP-2的下调引起线粒体细胞膜的崩溃。AIF的免疫定位显示，在MMP-2下调的细胞中，AIF易位到细胞核，从而能够诱导细胞凋亡。RT-PCR分析显示caspase-8过表达57倍，而p73下调28倍。凋亡的证据通过TUNEL测定和通过DAPI染色的核碎裂的可视化来确定。总之，从我们的研究结果可以明显看出，MMP-2下调诱导caspase-8和AIF介导的细胞凋亡，因此，显示出胶质瘤治疗的潜力。
Malignant gliomas are characterized by invasive and infiltrative behavior that generally involves the destruction of normal brain tissue. Strategies to treat infiltrating gliomas, such as chemotherapy and gene therapy, have remained largely unsuccessful. The infiltrative nature of gliomas can be attributed largely to proteases, which include serine, metallo and cysteine proteases. Our previous work and that of others strongly suggest a relationship between the expression of uPAR, MMP-9, and MMP-2; this relationship is generally indicative of the infiltrative phenotype of gliomas. In the present study, we have demonstrated that the RNAi-mediated downregulation of MMP-2 induces apoptosis in the 4910 human glioma xenograft cell line. Using western blot analysis, we observed that caspase-8 levels increased in MMP-2-downregulated cells whereas TRADD and TRAF-2 levels decreased. Further, NIK levels increased in MMP-2-downregulated cells. To determine the nuclear localization of AIF and IκB-α, we analyzed the levels of AIF, IκB-α and p-IκB-α in the cytosolic and nuclear fractions of MMP-2-downregulated cells. Western blot analysis revealed that MMP-2 downregulation resulted in the translocation of AIF to the nucleus and also inhibited the nuclear localization of p-IκB-α. To confirm the involvement of AIF, we performed FACS analysis to determine the integrity of the mitochondrial membrane using the Mito-PT method. FACS analysis showed that the downregulation of MMP-2 caused a collapse in the mitochondrial cell membrane. Immunolocalization of AIF revealed that in MMP-2-downregulated cells, AIF translocates to the nucleus, thereby enabling the induction of apoptosis. RT-PCR analysis revealed that caspase-8 was overexpressed 57-fold, whereas p73 was downregulated 28-fold. Evidence of apoptosis was determined by TUNEL assay and visualization of nuclear fragmentation by DAPI staining. In summary, it is evident from our results that MMP-2 downregulation induces caspase-8 and AIF-mediated apoptosis and, as such, shows potential for glioma therapy.
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影响因子：
11.4
作者：
Mori, H;Tomari, T;Seiki, M
通讯作者：
Seiki, M
影响因子：
8
作者：
Suenaga, N;Mori, H;Seiki, M
通讯作者：
Seiki, M
影响因子：
8
作者：
Mohanam, S;Chandrasekar, N;Rao, JS
通讯作者：
Rao, JS
影响因子：
64.8
作者：
Susin, SA;Lorenzo, HK;Kroemer, G
通讯作者：
Kroemer, G
DOI：
10.2741/1016
发表时间：
2003-01-01
期刊：
FRONTIERS IN BIOSCIENCE
影响因子：
--
作者：
Nakada, M;Okada, Y;Yamashita, J
通讯作者：
Yamashita, J