Research Paper Volume 11, Issue 23 pp 11624—11639

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

Mei-Chi Chang 1, 2, *, , Yu-Hwa Pan 2, *, , Hsyueh-Liang Wu 3, , Yi-Jie Lu 4, , Wan-Chuen Liao 5, , Chien-Yang Yeh 5, , Jang-Jaer Lee 5, , Jiiang-Huei Jeng 5, ,

  • 1 Chang-Gung University of Science and Technology, Kwei-Shan, Taoyuan, Taiwan
  • 2 Department of Dentistry, Chang Gung Memorial Hospital, Taipei, Taiwan
  • 3 Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan
  • 4 Graduate Institute of Oral Biology, National Taiwan University Medical College, Taipei, Taiwan
  • 5 School of Dentistry, National Taiwan University Medical College, and Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan
* Equal contribution

received: October 17, 2019 ; accepted: November 20, 2019 ; published: December 12, 2019 ;
How to Cite

Copyright © 2019 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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.


ADAM17: a disintegrin and metalloprotease domain 17; ANE: areca nut extract; BQ: betel quid; Chk: checkpoint kinase; COX: cyclooxygenase; DMEM: Dulbecco’s modified eagle’s medium; DMSO: dimethylsulfoxide; EGFR: epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; HC: hydroxychavicol; GK: gingival keratinocyte; GM-CSF: granulocyte macrophage-colony stimulating factor; IL-1β: interleukin-1β; MAPK: mitogen-activated protein kinase; MMP-9: matrix metalloproteinase-9; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide; NAC: N-acetyl-cysteine; OSCC: oral squamous cell carcinoma; OSF: oral submucous fibrosis; PBL: piper betle leaf; PBS: phosphate buffered saline; PGE2: prostaglandin E2; ROS: reactive oxygen species; RT-PCR: reverse transcription-polymerase chain reaction; TAK1: transforming growth factor activated protein kinase 1; TCIPA: tumor cell induced platelet aggregation; TGF-β: transforming growth factor-β; TIMP-1: tissue inhibitor metalloproteinase-1; TNF-α: tumor necrosis factor-α.