MMP-2 siRNA induced Fas/CD95-mediated extrinsic II apoptotic pathway in the A549 lung adenocarcinoma cell line.
We have previously reported that the downregulation of MMP-2 by adenovirus-mediated delivery of MMP-2 siRNA (Ad-MMP-2) reduced spheroid invasion and angiogenesis in vitro, and, metastasis and tumor growth in vivo. In this study, we investigated the mechanism of Ad-MMP-2-mediated growth inhibition in vitro and in vivo. Ad-MMP-2 infection led to the induction of apoptosis as determined by TUNEL assay, Annexin-V staining and PARP-1 cleavage in a dose-dependent manner in A549 cells. Ad-MMP-2 decreased the content of the antiapoptotic members of the Bcl-2 family proteins (Bcl-2 and Bcl-xL) and increased the content of the pro-apoptotic members of the Bcl-2 family (Bax and Bcl-xS) as determined by immunoblotting analysis. Furthermore, Ad-MMP-2-mediated apoptosis was accompanied by increase in truncated Bid, release of cytochrome c and the activation of caspase-8, -9 and -3. Immunoblot analysis showed that Ad-MMP-2 infection caused upregulation of Fas/Fas-L and FADD, and Anti-Fas-L antibody reversed Ad-MMP-2-induced apoptosis. Tissue inhibitor of metalloproteinases (TIMP)-3, an endogenous inhibitor of MMP-2, which cleaves Fas-L and activates the Fas/Fas-L inducing apoptotic pathway, was increased in Ad-MMP-2-treated cells. Adenovirus-mediated expression of MMP-2 siRNA in human lung xenografts in vivo resulted in increased immunostaining of Fas, Fas-L, cleaved Bid and TIMP-3. This is the first report, to our knowledge, showing that MMP-2 inhibition upregulates TIMP-3 levels, which in turn, promotes apoptosis in lung cancer.
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Program of Cancer Biology, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA.
Bhoopathi
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Lakka
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Rao
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D000230:Adenocarcinoma / Q000201:enzymology*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053222:Fas Ligand Protein / Q000032:analysis; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000032:analysis / Q000378:metabolism*
C505533:FADD protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D061965:Matrix Metalloproteinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D019717:Tissue Inhibitor of Metalloproteinase-3; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D020778:Matrix Metalloproteinase 2