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In Taiwan, the combination of betel quid chewing, alcohol consumption, and smoking habits increases oral cancer risk by 123-fold compared to persons without these habits. Lymphocyte populations in patients may potentially affect the malignant transformation of oral precancer. Methods: A total of 28 patients with oral precancer from our previous cohort were enrolled in this study, and their personal information and oral habits were documented. Their lymphocyte populations (CD4+, CD8+, CD19+, and CD56+) and activation markers (CD25 and CD69) were determined by flow cytometry from 1999 to 2004. After follow up till December 2014, data of patients with/without malignant transformation were recorded, and the relation between oral habits and percentage of initial lymphocyte markers was evaluated using the Student t test and Fisher's exact test. Results: Ten precancer patients developed oral squamous cell carcinoma with a mean period of malignant transformation of 6.8 ± 2.1 years. Patients with malignant transformation had a mean age of 48.4 ± 5.0 years (n = 10), relatively more than that of patients without malignant transformation (41.6 ± 6.3 years, n = 18) (p

AimsChewing of betel quid (BQ) increases the risk of oral cancer and oral submucous fibrosis (OSF), possibly by BQ-induced toxicity and induction of inflammatory response in oral mucosa.MethodsPrimary gingival keratinocytes (GK cells) were exposed to areca nut (AN) components with/without inhibitors. Cytotoxicity was measured by 3-(4,5-dimethyl- thiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. mRNA and protein expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. PGE2/PGF2α production was measured by enzyme-linked immunosorbent assays.ResultsAreca nut extract (ANE) stimulated PGE2/PGF2α production, and upregulated the expression of cyclooxygenase-2 (COX-2), cytochrome P450 1A1 (CYP1A1) and hemeoxygenase-1 (HO-1), but inhibited expression of keratin 5/14, cyclinB1 and cdc25C in GK cells. ANE also activated epidermal growth factor receptor (EGFR), Src and Ras signaling pathways. ANE-induced COX-2, keratin 5, keratin 14 and cdc25C expression as well as PGE2 production were differentially regulated by α-naphthoflavone (a CYP 1A1/1A2 inhibitor), PD153035 (EGFR inhibitor), pp2 (Src inhibitor), and manumycin A (a Ras inhibitor). ANE-induced PGE2 production was suppressed by piper betle leaf (PBL) extract and hydroxychavicol (two major BQ components), dicoumarol (aNad(p)hQuinone Oxidoreductase--NQO1 inhibitor) and curcumin. ANE-induced cytotoxicity was inhibited by catalase and enhanced by dicoumarol, suggesting that AN components may contribute to the pathogenesis of OSF and oral cancer via induction of aberrant differentiation, cytotoxicity, COX-2 expression, and PGE2/PGF2α production.ConclusionsCYP4501A1, reactive oxygen species (ROS), EGFR, Src and Ras signaling pathways could all play a role in ANE-induced pathogenesis of oral cancer. Addition of PBL into BQ and curcumin consumption could inhibit the ANE-induced inflammatory response.

The article presents a study which investigates areca nut (AN) extract (ANE)-induced micronuclei (MN) formation and aneuploidy in Chinese hamster ovary cells (CHO-K1), and the role of reactive oxygen especies (ROS) in ANE-induced carcigenesis. The results of the study indicate that AN components might contribute to carcinogenesis via an increase in MN frequency. It also notes that incubation of CHO-K1 cells with ANE for hours caused cell cycle arrests at G2/M phase leading to cytokinesis failure, which is associated with oxidative stress, and accumulation of hyperploid/aneuploid cells.

Betel quid (BQ) chewing is an etiologic factor of oral cancer and submucus fibrosis (OSF). Keratinocyte inflammation is crucial for the pathogenesis of cancer and tissue fibrosis. We found that areca nut (AN) extract (100–400 µg/ml) induced PGE2 production by KB cells by 2.34- to 23.1-fold and also TNF-α production by gingival keratinocytes (GK). Arecoline (0.2–1.2 mM) elevated PGE2 production by KB cells by 2.5- to 6.1-fold. AN extract (200–400 µg/ml) also induced IL-6 production by GK (7.5- to 8.4-fold) and KB cells. In contrast, arecoline (0.1–1.2 mM) suppressed IL-6 production by GK and KB cells, with 42–81 and 41–63% inhibition, respectively. A 48 h exposure of GK to 800–1200 µg/ml AN extract led to 37–69% cell death. Arecoline cytotoxicity to GK was noted at concentrations of 0.8–1.2 mM, which led to 28–38% cell death. AN extract (400–800 µg/ml) induced Cox-2 and IL-6 mRNA expression and also COX-2 protein production by KB cells. IL-6 (5–100 ng/ml) suppressed GK growth by 20–33%, but enhanced oral fibroblast (OMF) and KB cell growth. PGE2 (0.05–5 µg/ml) and anti-IL-6 antibody (ab) (50–1000 ng/ml) showed little effect on GK and KB cell growth. Incubation of GK and KB cells with aspirin, anti-IL-6 ab and anti-TNF-α ab showed little effect on arecoline- and AN-induced cytotoxicity, cell cycle arrest and apoptosis. Exposure to anti-TNF-α ab slightly affected arecoline- and AN-modulated PGE2 and IL-6 production by GK and KB cells. Arecoline- and AN-conditioned medium decreased phytohemagglutinin-mediated CD4+ and CD8+ T cell activation. These results indicate that BQ chewing contributes to the pathogenesis of cancer and OSF by impairing T cell activation and by induction of PGE2, TNF-α and IL-6 production, which affect oral mucosal inflammation and growth of OMF and oral epithelial cells. [ABSTRACT FROM AUTHOR]

BETEL QUID CHEWING IS a popular oral habit in India, South Africa, and many Southeast Asian countries. The effects of areca nut IAN) extract on the growth, attachment. and protein synthesis of healthy human gingival fibroblasts (GF) were investigated to determine why betel quid (BQ) chewers have higher prevalence of periodontal disease than non-chewers. Twenty-four hour exposure of human GF to AN extract (> 200 µg/ml) in culture led to the formation of numerous intracellular vacuoles. As analyzed by modified MTT [3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-tetrazolium bromide] assay, AN extract significantly suppressed the growth of GF over 5 days of incubation in a dose-dependent manner. At concentrations of 50 and 300 µg/ml, AN extract suppressed the growth of GF with 30% and 57% (P < 0.05), respectively. AN extract also significantly suppressed the synthesis of [³H]proline incorporation into trichloroacetic acid (TCA) precipitated proteins. At concentrations of 200, 400, and 600 µg/ ml, AN extract suppressed the protein synthesis with 33%, 58%, and 63% of inhibition (P < 0.05), respectively. Preincubation of cells in a medium containing AN extract for 2 hours inhibits the subsequent attachment of cultured GF to type 1 collagen at the 50% inhibitory concentration (IC50) which is about 720 to 798 µg/ml. Considering the frequent consumption of BQ throughout the day, impairment of sequential fibroblast functions by BQ ingredients is a potential mechanism through which BQ chewing exert a deleterious effect to the gingival tissues. [ABSTRACT FROM AUTHOR]

Because betel quid (BQ) chewing has been linked to a higher prevalence of periodontal diseases, the pathobiological effects of arecoline, a main alkaloid found in areca nut, were investigated in cultured human gingival fibroblasts. At concentrations higher than 0.4 mM, arecoline inhibits cell attachment, cell spreading and cell migration in a dose-dependent manner. These inhibitory effects were associated with intracellular depletion of glutathione (GSH). At concentrations of 0.4 mM and 1 mM, arecoline depleted about 26% and 45% of GSH after 2 h incubation. Exposure of cells to arecoline at concentrations lower than 0.4 mM for 2 h showed no significant decrease in either cell viability or intracellular GSH. However, incubation of cells for 24 h in 1 mM arecoline decreased the cell numbers to only 35% of those in the untreated control. Arecoline also decreased cell growth and collagen synthesis in a dose-dependent manner. Because of repeated and long-term exposure to arecoline, BQ chewers could be more susceptible to periodontal damage and less responsive to new attachment procedures. [ABSTRACT FROM AUTHOR]