Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression.
Matrix metalloproteinases (MMPs) are thought to play crucial roles in tumor invasion and metastasis. Because we have shown that EBV latent membrane protein 1 (LMP1) enhances MMP-9 expression by activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 (T. Yoshizaki, et al., Proc. Natl. Acad. Sci. USA, 95: 3621-3626, 1998), we therefore tested whether up-regulation of MMP-9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP-9 expression in tumors grown in nude mice were also tested. C33A cells stably expressing LMP1 had increased expression of MMP-9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells (P < 0.03) and suppressed both the LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced MMP-9 promoter activity in CAT reporter assays (P < 0.01). Endogenous MMP-2 levels were unaffected by either drug. Both drugs repressed the CAT activity of the truncated MMP-9 promoter construct, which only contained a binding site for AP-1, to the basal level (P < 0.05). Moreover, EMSA indicated that the effects of the salicylates were through the inhibition of not only NF-kappaB but also AP-1 binding activity. Inhibitory effect of salicylates could be reversed by p50/p65 subunits of NF-kappaB or c-Jun overexpression. The inhibitory effect of aspirin on NF-kappaB activity was attributable to the inhibition of IkappaB kinase activity. Finally, tumors derived from C33A cells stably expressing LMP1 grown in nude mice showed enhanced MMP-9 levels compared with tumors derived from vector-transfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.
Murono
S
S
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599-7295, USA.
Yoshizaki
T
T
Sato
H
H
Takeshita
H
H
Furukawa
M
M
Pagano
J S
JS
D000818:Animals; D001241:Aspirin / Q000494:pharmacology*; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012980:Sodium Salicylate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000494:pharmacology*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D061965:Matrix Metalloproteinase Inhibitors; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014763:Viral Matrix Proteins; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D020780:Matrix Metalloproteinase 9; D001241:Aspirin; D012980:Sodium Salicylate