RNAi-mediated downregulation of MMP-2 activates the extrinsic apoptotic pathway in human glioma xenograft cells

Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.
International Journal of Oncology (Impact Factor: 3.03). 11/2009; 35(4):851-9. DOI: 10.3892/ijo_00000399
Source: PubMed


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 IkappaBalpha, we analyzed the levels of AIF, IkappaBalpha and pIkappaBalpha 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 pIkappaBalpha. To confirm the involvement of AIF, we performed FACS analysis to determine the integrity of the mitochondrial membrane using the MitoPT 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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Available from: Dzung Dinh, Jul 09, 2015
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    • "TUNEL and caspase-3 activity assays demonstrated that apoptosis was significantly reduced in mmp1(RNAi) animals, suggesting that MMP1 functions as a positive regulator of apoptosis in planarians. Apoptotic evasion is a common strategy to increase cell number in tumors [100], [101], [102]. Preventing abnormal proliferation might provide anti-tumorigenic protection in these plastic animals. "
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    ABSTRACT: Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.
    PLoS ONE 02/2013; 8(2):e55649. DOI:10.1371/journal.pone.0055649 · 3.23 Impact Factor
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    • "The decreased TRADD-TRAF2 interaction might lead to the inhibition of downstream IKKβ mediated NF-κB activation. These results are in corroboration with the previous report on MMP-2 depletion induced decrease in TRADD in 4910 cells [32]. FADD was originally identified as an important molecule for Fas-mediated apoptosis and is an important component in DISC. "
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    ABSTRACT: 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.
    PLoS ONE 05/2011; 6(5):e19341. DOI:10.1371/journal.pone.0019341 · 3.23 Impact Factor
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    • "TUNEL assay was performed with adherent cells [40] and paraffin-embedded tissue sections [60] using In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Indianapolis, IL). After transfection with SV, pU, PC, or pCU, cells were fixed with buffered formaldehyde (pH 7.4) and incubated with a reaction mixture containing biotin-dUTP and terminal deoxynucleotidyl transferase for 1 hr. "
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    ABSTRACT: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
    PLoS ONE 10/2010; 5(10):e13731. DOI:10.1371/journal.pone.0013731 · 3.23 Impact Factor
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