Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and –independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin

Background: There are 200-600 million betel quid (BQ) chewers in the world. BQ increases oral cancer risk. Matrix metalloproteinase-9 (MMP-9) is responsible for matrix degradation, cancer invasion and metastasis. Whether areca nut extract (ANE), a BQ component, stimulates MMP-9 secretion, and the related signaling pathways awaits investigation. Results: ANE (but not arecoline) stimulated MMP-9 production of gingival keratinocytes and SAS cancer epithelial cells. ANE stimulated TGF-β1, p-Smad2, and p-TAK1 protein expression. ANE-induced MMP-9 production/expression in SAS cells can be attenuated by SB431542 (ALK5/Smad2 inhibitor), 5Z-7-Oxozeaenol (TAK1 inhibitor), catalase, PD153035 (EGFR tyrosine kinase inhibitor), AG490 (JAK inhibitor), U0126 (MEK/ERK inhibitor), LY294002 (PI3K/Akt inhibitor), betel leaf (PBL) extract, and hydroxychavicol (HC, a PBL component), and melatonin, but not by aspirin. Conclusions: AN components contribute to oral carcinogenesis by stimulating MMP-9 secretion, thus enhancing tumor invasion/metastasis. These events are related to reactive oxygen species, TGF-β1, Smad2-dependent and –independent signaling, but not COX. These signaling molecules can be biomarkers of BQ carcinogenesis. PBL, HC and melatonin and other targeting therapy can be used for oral cancer treatment. Methods: ANE-induced MMP-9 expression/secretion of oral epithelial cells and related TGF-β1, Smad-dependent and –independent signaling were studied by MTT assay, RT-PCR, western blotting, immunofluorescent staining, and ELISA.