Prolonged exposure to arecoline arrested human KB epithelial cell growth: regulatory mechanisms of cell cycle and apoptosis.
Arecoline, the main areca alkaloid in betel quid (BQ), is reported to have cytotoxic, genotoxic, and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucous fibrosis, leukoplakia, and oral cancer. To clarify the role of arecoline in BQ-induced carcinogenesis, primary human gingival keratinocyes (GK) and human KB epithelial cells were used for studying the molecular mechanisms of arecoline-mediated cell cycle deregulation for comparison. After 24 h of exposure, arecoline (0.2-0.8 mM) inhibited KB cell growth in a dose- and time-dependent manner with a reduction in cell number by 27-37 and 37-58%, respectively, as determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. Incubation of KB cells with arecoline (0.1-0.4 mM) caused late-S and G2/M phases' cell cycle arrest. Western blot analysis revealed that arecoline induced cyclin Bl, Wee 1, and phosphorylated cdc2 protein levels whereas it declined p21 protein expression in KB cancer cells. Nevertheless, arecoline induced p21, but decreased cdc2 and cyclin B1 protein levels in GK. We demonstrated that higher concentrations of arecoline (0.2-1.2 mM) induced both cell necrosis and apoptosis as detected by DNA fragmentation and Annexin V-PI staining after long-term (48 h) treatment. Our results suggest that differential regulation of S and/or G2/M cell cycle-related proteins in the GK and KB cells play a crucial role in different stages of BQ-mediated carcinogenesis.
Lee
Po-Hsuen
PH
Laboratory of Dental Pharmacology & Toxicology, Department of Dentistry, National Taiwan University Hospital, National Taiwan University Medical College, No. 1, Chang-Te Street, Taipei 100, Taiwan.
Chang
Mei-Chi
MC
Chang
Wen-Hui
WH
Wang
Tong-Mei
TM
Wang
Ying-Jen
YJ
Hahn
Liang-Jiunn
LJ
Ho
Yuan-Soon
YS
Lin
Chuan-Yu
CY
Jeng
Jiiang-Huei
JH
D017209:Apoptosis / Q000187:drug effects*; D001115:Arecoline / Q000633:toxicity*; D017610:Calcium Compounds; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation; D006801:Humans; D007624:KB Cells; D010087:Oxides; D031701:Piper; D010936:Plant Extracts; D012235:Rhodamines / Q000378:metabolism
D017610:Calcium Compounds; D018797:Cell Cycle Proteins; D010087:Oxides; D010936:Plant Extracts; D012235:Rhodamines; C495400:lime-piper betel quid; C022027:lissamine rhodamine B; D001115:Arecoline