RNAi-mediated downregulation of MMP-2 activates the extrinsic apoptotic pathway in human glioma xenograft cells.
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.
Gondi
Christopher S
CS
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.
Talluri
Lavanya
L
Dinh
Dzung H
DH
Gujrati
Meena
M
Rao
Jasti S
JS
D021581:Active Transport, Cell Nucleus; D017209:Apoptosis / Q000235:genetics; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D005434:Flow Cytometry; D015316:Genetic Therapy; D005910:Glioma / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial; D000072000:NF-KappaB Inhibitor alpha; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053199:TNF Receptor-Associated Death Domain Protein / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000097782:NF-kappaB-Inducing Kinase
D051033:Apoptosis Inducing Factor; D053181:Caspase 8; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D020778:Matrix Metalloproteinase 2; D000072000:NF-KappaB Inhibitor alpha; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D053199:TNF Receptor-Associated Death Domain Protein; D047992:TNF Receptor-Associated Factor 2; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; D000097782:NF-kappaB-Inducing Kinase; C494829:AIFM1 protein, human; C000606204:NFKBIA protein, human; C000606460:TP73 protein, human; C505530:CASP8 protein, human; C522361:MMP2 protein, human