MMP-2 siRNA induced Fas/CD95-mediated extrinsic II apoptotic pathway in the A549 lung adenocarcinoma cell line

Abstract

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 proapoptotic 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.

Trials, tribulations and speculation! Report from the 7th Biennial Hatter Cardiovascular Institute Workshop

“…However, more recently, MMPs have been demonstrated to have intracellular targets -cytoskeletal [2,3], contractile [4,5] and potentially also in terms of cell survival and cell death pathways [6,7]. Robert Bell and Derek Yellon presented data demonstrating that not only does MMP inhibition at reperfusion attenuate infarction in both in-vitro and in-vivo preparations, but the protection observed is additional to that seen following targeted deletion of the cyclophillin-D component of the mPTP.…”

Multicentric osteolysis with nodulosis, arthritis, and cardiac defect syndrome: loss of MMP2 leads to increased apoptosis with alteration of apoptotic regulators and caspases and embryonic lethality

“…The Fas-FasL pathway was the primary candidate pathway because of its known strong effects on osteoblasts 7 and because it has been shown to be affected by MMP2 loss in a number of other cell types. [8][9][10][11] Furthermore, activation of both caspase-8 and caspase-9 in our siMMP2 SaOS2 clones demonstrated involvement of both the extrinsic and intrinsic apoptotic pathways, a hallmark of Fas-mediated apoptosis. 11 We therefore examined the levels of Fas expressed by MMP2-deficient osteoblasts.…”

“…[8][9][10][11] Furthermore, activation of both caspase-8 and caspase-9 in our siMMP2 SaOS2 clones demonstrated involvement of both the extrinsic and intrinsic apoptotic pathways, a hallmark of Fas-mediated apoptosis. 11 We therefore examined the levels of Fas expressed by MMP2-deficient osteoblasts. Fas protein levels were found to be increased between two-and six-fold in siMMP2A and siMMP2B, respectively (Figure 3).…”

Tissue Inhibitor of Metalloproteinase 3 Suppresses Tumor Angiogenesis in Matrix Metalloproteinase 2–Down-regulated Lung Cancer

“…Titers were obtained for the viruses used in this work are Ad-SV (7.6 10 11 pfu/mL) and Ad-MMP-2-Si (5.0 10 11 ). The specificity of this virus to inhibit MMP-2 was documented in our previous studies (21,22). The amount of infective adenoviral vector per cell (pfu per cell) in culture medium was expressed as multiplicity of infection (MOI).…”

“…The samples were incubated in TUNEL reaction mixture in a humidified atmosphere at 37jC for 1 h in the dark. For tissue sections, after TUNEL, immunohistochemical analysis was performed using anti-CD31 antibody as described previously (22). Images were captured with an Olympus BX 60 research fluorescence microscope attached to a charge-coupled device (CCD) camera, and cells were counted.…”