Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes.
OBJECTIVE
Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular basis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.


METHODS
Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.


RESULTS
Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase. Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation.


CONCLUSIONS
Our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India.
G S
GS
Thangjam
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Kondaiah
D001052:Apoferritins; D001115:Arecoline / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005954:Glucosephosphate Dehydrogenase / Q000187:drug effects; D005721:Glutamate-Cysteine Ligase / Q000187:drug effects; D005980:Glutathione Reductase / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000187:drug effects; D006801:Humans; D053582:Interleukin-1alpha / Q000096:biosynthesis; D015603:Keratinocytes / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000032:analysis; D011976:Receptors, Muscarinic / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects
D018679:Cholinergic Agonists; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D053582:Interleukin-1alpha; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D011976:Receptors, Muscarinic; D001115:Arecoline; D001052:Apoferritins; D005954:Glucosephosphate Dehydrogenase; D051547:Heme Oxygenase-1; D005980:Glutathione Reductase; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005721:Glutamate-Cysteine Ligase