Arecoline <i>N</i>‑Oxide Upregulates Caspase‑8 Expression in Oral Hyperplastic Lesions of Mice

Areca nut is strongly associated with oral squamous cell carcinoma (OSCC) occurrence. Arecoline <i>N</i>-oxide (ANO), a metabolite of the areca alkaloid arecoline, exhibits an oral fibrotic effect in NOD/SCID mice. Caspase-8, a cysteine protease encoded by the <i>CASP8</i> gene, is a central mediator in the extrinsic apoptotic pathway via death receptors. Deregulation of caspase-8 in OSCC has been reported. This study investigates the regulation of caspase-8 in ANO-induced oral squamous epithelial hyperplasia that represents the initial highly proliferative stage of oral carcinogenesis. <i>CASP8</i> somatic mutations were identified from whole-exome sequencing of OSCC samples. Immunohistochemical staining showed upregulation of caspase-8 in ANO-induced hyperplasia of both NOD-SCID and C57BL/6 mice. Levels of expression of CASP8, APAF-1, BAX, and BAD increased in ANO-treated DOK cells. Co-localization of increased caspase-8 and PCNA levels was detected in ANO-induced hyperplastic lesions, whereas no co-localization among γ-H2A.X, caspase-3, and upregulated caspase-8 was observed. The findings indicate that upregulation of caspase-8 is involved in cell proliferation rather than apoptosis during the initial stage of ANO-mediated oral tumorigenesis

Baseline sample characteristics (N = 162,682).

<p>*Missing data excluded.</p><p>Baseline sample characteristics (N = 162,682).</p

Cox proportional hazards regression analyses of factors associated with suicide mortality.

<p>Note. HR = hazard ratio; CI = confidence interval; AHR = adjusted hazard ratio</p><p>^aAdjusted for gender, age, SHS exposure and the highest education of parents,</p><p>^bAdjusted for gender, age, the highest education of parents, SHS exposure and cigarette smoking,</p><p>^cAdjusted for gender, age, the highest education of parents, SHS exposure, cigarette smoking, the lifetime asthma, and allergic rhinitis,</p><p>^dAdjusted for gender, age, the highest education of parents, SHS exposure, cigarette smoking, the lifetime asthma, allergic rhinitis, and alcoholic drinking</p><p>Cox proportional hazards regression analyses of factors associated with suicide mortality.</p

Cause-specific mortality by smoking status (N = 162,098).

<p>^a. PYs: person-years</p><p>^b. incidence: number per 100,000 person-years.</p><p>The difference of incidence in specific causes of deaths between smoker and non-smoker group were calculated using Wilcox (Gehan) Statistic by survival life table analysis.</p><p>Cause-specific mortality by smoking status (N = 162,098).</p

STROBE style flow chart of the cohort in southern Taiwan included in analysis.

<p>STROBE style flow chart of the cohort in southern Taiwan included in analysis.</p

Fibrotic Effects of Arecoline <i>N</i>‑Oxide in Oral Potentially Malignant Disorders

The metabolites of environmental chemicals play key roles in carcinogenesis. Areca nut is strongly associated with the development of oral potentially malignant disorders (OPMD) or cancer. The main alkaloid in the areca nut is arecoline, which is highly cytotoxic and genotoxic. Arecoline <i>N</i>-oxide, a metabolite of areca nut alkaloids, which has been identified in animal urine, has been shown to induce mutagenicity in bacteria. In this study, it was found that its protein adduct could be detected in oral keratinocytes treated with areca nut extract. Increased collagen expression and severity of squamous hyperplasia were observed in arecoline <i>N</i>-oxide treated mice. In cultured oral fibroblasts, arecoline <i>N</i>-oxide showed stronger effects on the increase of fibrotic related genes including <i>TGF-beta1</i>, <i>S100A4</i>, <i>MMP-9</i>, <i>IL-6</i>, and <i>fibronectin</i> and a decrease of <i>E-cadherin</i> as compared with arecoline. Finally, arecoline <i>N</i>-oxide stimulation effectively increased the DNA damage marker, gamma-H2A.X, both in vitro and in vivo. Taken together, these results indicate that arecoline <i>N</i>-oxide shows a high potential for the induction of OPMD

Associations between asthma, betel chewing, plasma markers and respiratory function in male participants (n = 147).

<p>Arecoline, arecaidine, IgE, hs-CRP, leptin, TGF-β1 and eotaxin-1 levels were logarithmically transformed before statistical testing to meet the assumption of a normal distribution.</p>a<p>Least significant difference (LSD) multiple comparisons were performed to compare plasma markers and pulmonary function between current and non-current betel chewing in males with asthma and controls, respectively.</p>b<p>General linear regression was used to assess the associations between plasma markers and the four groups of male cases and controls with and without betel chewing by adjusting for age, BMI and smoking.</p

In cultured gingival fibroblast cells, the levels of eotaxin-1 were measured by ELISA in supernatants from cells treated with different levels of arecoline (0, 10, 25, 100, and 200 μg/ml for 24 h).

<p>The levels of eotaxin-1 (mean±SD) were analyzed by one-way ANOVA and Bonferroni multiple correction tests in which the P values are adjusted by multiplying by 10. * p = 0.03301 for the pretreatment of arecoline at 100 μg/ml induced significant elevation of eotaxin-1 levels (1489±78 pg/ml) compared with the pretreatment of arecoline at 0 μg/ml (1044±95 pg/ml) under TNF-alpha and IL-4 stimulation. Arecoline, at a concentration of 200 μg/ml, increased cytotoxicity by more than 45% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091889#pone.0091889-Chiang1" target="_blank">[22]</a>. The stimulated groups indicate that the fibroblast cells were treated with IL-4 (50 ng/ml) and TNF-alpha (100 ng/ml) for 72 hours. The baseline groups are fibroblast cells that were treated with arecoline alone, not stimulated with IL-4 and TNF-alpha. Arecoline alone at any tested concentrations from 0 to 200 μg/ml cannot induce detectable eotaxin-1 release (0±0 pg/ml for any tested dose of arecoline).</p

Logistic regression analyses of the adjusted associations between betel chewing and asthma.

1<p>Logistic regression adjusted for age, gender, BMI and smoking.</p>2<p>Logistic regression adjusted for age, gender, BMI, smoking and log eotaxin-1 levels (345 cases and 389 controls were randomly selected to measure eotaxin-1 levels).</p>3<p>The mediation effect of eotaxin-1 was 50.5% of the total effect of betel chewing on asthma being mediated through this pathway.</p

Characteristics of the case and control participants.

<p>Total IgE levels were logarithmically transformed before statistical testing to meet the assumption of a normal distribution.</p