TAK1 Mediates Areca Nut-induced MMP-9 and ICAM-1 Expression/Secretion of Cells

Objectives: There are about 600 million betel quid (BQ) chewer distributed around India, Taiwan and other Southeast Asian countries. BQ chewing increases the risk of oral cancer, possibly due to stimulation of metalloproteinase-9 (MMP-9) and intercellular cell adhesion molecule-1 (ICAM-1) of oral mucosal cells. Areca nut extract (ANE), as a major BQ component, is shown to stimulate MMP-9 production of gingival keratinocytes (GK). However, the role of TGF-β1-activated kinase-1 (TAK1) in mediating these events is not clear.
Methods: Human GK and SAS oral cancer epithelial cells were exposed to AN extract (ANE) or arecoline with/without pretreatment and co-incubation with 5z-7oxozeaenol (a TAK1 inhibitor) or siRNA of TAK1. Cytotoxicity was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. MMP-9 and ICAM-1 mRNA expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-9 and ICAM-1 protein secretion was determined by enzyme-linked immunosorbent assays. ANOVA and Tukey test were used for statistical analysis. Moreover, healthy oral mucosa and oral cancer tissues were used for immunohistochemical staining study of MMP-9 expression and analyzed by t-test.
Results: ANE and arecoline showed little cytotoxicity at tested concentrations. ANE, but not arecoline, stimulated MMP-9 and ICAM-1 mRNA expression and protein secretion in both GK and SAS cancer cells. ANE-induced MMP-9 and ICAM-1 expression and secretion of cells can be attenuated by 5z-7oxozeaenol. Transfection of siTAK1 prevented the ANE-induced ICAM-1 expression and secretion. Moreover, OSCC tissues (n=29) expressed higher level of MMP-9 protein relative to healthy oral mucosa (n=26) (p<0.05).
Conclusions: These results indicate that MMP-9 and ICAM-1 play important roles in oral carcinogenesis. AN may be involved in oral carcinogenesis by stimulation of MMP-9 and ICAM-1 expression/secretion. These events are associated with TAK1 signaling. Inhibition of TAK1, MMP-9 and ICAM-1 may be useful for targeting therapy for prevention and treatment of oral cancer.