Journal Article

Ripe areca nut extract induces G<sub>1</sub> phase arrests and senescence-associated phenotypes in normal human oral keratinocyte

Ssu-Yi Lu, Kuo-Wei Chang, Chung-Ji Liu, Yu-Hsin Tseng, Hsuan-Hsuan Lu, Suz-Ying Lee and Shu-Chun Lin

in Carcinogenesis

Volume 27, issue 6, pages 1273-1284
Published in print June 2006 | ISSN: 0143-3334
Published online February 2006 | e-ISSN: 1460-2180 | DOI:
Ripe areca nut extract induces G1 phase arrests and senescence-associated phenotypes in normal human oral keratinocyte

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Around 200–600 million Asians chew areca (also called betel), which contains a mixture of areca nut and other ingredients. Epidemiological evidences indicated that areca use is tightly linked to oral carcinogenesis. This study investigated the effects of ripe areca nut extract (ANE) on cultured normal human oral keratinocyte (NHOK). Acute subtoxic ANE treatment inhibited DNA synthesis and induced cell cycle arrest at G1 phase in early passage (<4th passage) cells. This was accompanied by a slight increase in the sub-G1 cellular fraction. O(6)-Methylguanine-DNA methyltransferase (MGMT), Hsp27 and p38MAPK was upregulated. p16 and p21 were remarkably upregulated early and declined afterwards. In contrast, the increase of dephosphorylated Rb seemed to be secondary to the episodes of p16 and p21 upregulation. To simulate the chronic areca exposure in vivo, constant ANE treatment in serial NHOK culture was performed. It resulted in a significant decrease in the population doubling, increase in senescence-associated β-galactosidase (SA-β-Gal) and decrease in cell proliferation in NHOK of late passages (≥4th passage). Induction of senescence-associated phenotypes, G2/M accumulation and genomic instability following long-term ANE treatment were also observed in a low-grade oral carcinoma cell. ANE-treated NHOK also had a higher nuclear factor-κB (NF-κB) fraction and a lower cytosolic IκBα level relative to the control in late passages. Moreover, electrophoretic mobility shift assay (EMSA) indicated that ANE treatment shifted the NF-κB complex from high mobility position to lower mobility position in late-passaged NHOK. ANE treatment also upregulated IL-6 and cyclooxygenase-2 (COX-2) mRNA expressions in late-passaged NHOK. In summary, our findings suggest that ANE induces the cell cycle arrest at G1/S phase and the occurrence of senescence-associated phenotypes of NHOK. The upregulation of p38MAPK, p16, p21, NF-κB, IL-6 and COX-2 are likely to participate in the control of these impacts.

Keywords: ANE, areca nut extract; BrdU, 5-bromo-2′-deoxyuridine; COX, cyclooxygenase; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated protein kinase; IκBα, inhibitory subunit of NF-κB α subunits; IL-1α, interleukin-1α; IL-6, interleukin-6; MAPK, mitogen-activated protein kinase; MGMT, O(6)-methylguanine-DNA methyltransferase; NF-κB, nuclear factor κB; NHOK, normal human oral keratinocyte; OSCC, oral squamous cell carcinoma; PDL, population doubling level; SA-β-Gal, senescence-associated β-galactosidase; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Journal Article.  8047 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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