Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

How Each Component of Betel Quid Is Involved in Oral Carcinogenesis: Mutual Interactions and Synergistic Effects with Other Carcinogens-a Review Article.

PURPOSE OF REVIEW: The roles of the components of betel quid in oral carcinogenesis remain unclear. The purpose of the present review is to highlight the effect of each component of betel quid and to discuss the synergistic effects of other carcinogens along with betel quid in the development of oral cancer in habitual betel quid chewers. RECENT FINDINGS: Betel quid may synergistically participate in carcinogenesis by disrupting the compositions of oral microbiota, accompanied by endotoxins secretion and reactive oxygen species (ROS) production. Microbiome dysbiosis mediated by synergistic effects of betel quid chewing, smoking, and alcohol drinking is possibly linked to oral carcinogenesis, which is firstly discussed in this report. Betel quid and other carcinogenic components, mainly contribute to downregulate the antioxidant proteins and lead to the induction of ROS. The elimination of ROS may prove most effective chemoprevention for betel quid-mediated oral carcinogenesis.

Alteration of oral flora in betel quid chewers in Sri Lanka.

BACKGROUND: Betel quid chewing is known as a crucial risk factor for oral diseases such as periodontal diseases, oral cancer, and precancerous lesions in Southeast Asian countries. Although abnormal oral bacterial flora may be linked to betel quid related-oral diseases such as oral cancer, precancerous lesions, and periodontal diseases, little information is available on alterations of their oral flora thus far. To identify these alterations, we analyzed the oral flora in betel quid chewers (BQC) and non-chewers (NC) in Sri Lanka. METHODS: Samples obtained from buccal swabs of BQC and NC were analyzed with a next generation sequencer. Data were processed and analyzed using the QIIME software package. Mann-Whitney U test and Permutational multivariate analysis of variance were used for statistical analyses. P values < 0.05 were considered to be statistically significant. RESULTS: In BQC, the proportion of periodontal pathogens including Actinomyces, Tannerella, and Prevotella was higher than that in NC (P < 0.05), while the proportion of cariogenic pathogens including Streptococcus, Lautropia, and Actinobacillus was lower than that in NC (P < 0.05). A statistically significant difference in Shannon index and PD Whole tree was observed between BQC and NC (P < 0.05). PCoA analysis detected different clusters in BQC and NC (P < 0.05). CONCLUSION: The results suggested that betel quid chewing significantly altered oral flora. Adequate oral health care may help prevent BQC from developing bacterial pathogen-related oral diseases.

DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing-a possible predictive biomarker for malignant transformation.

BACKGROUND: DNA hypermethylation of tumor suppressor genes is observed in precancerous lesions and oral cancer of individuals with the habits of betel quid (BQ) chewing. SIRT1 has been identified as playing a role in the maintenance of epithelial integrity, and its alteration is often related to carcinogenesis. However, the methylation and transcription status of SIRT1 in patients with BQ chewing-related oral cancer has not been investigated. We examined the methylation status of SIRT1 in paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC) obtained from BQ chewing and non-chewing patients and in tissue samples from healthy control subjects. In addition, we examined whether the hypermethylation of SIRT1 followed by its transcriptional downregulation in the human gingival epithelial cells could be caused by arecoline, a major component of BQ. Furthermore, we investigated the methylation status of SIRT1 in smear samples of macroscopically healthy buccal mucosa from subjects with a habit of BQ chewing. RESULTS: SIRT1 was significantly hypermethylated in tissue samples of OSCC from BQ chewers and non-chewers than in oral mucosa from healthy control subjects. Results also showed that the hypermethylation level of SIRT1 was significantly higher in OSCC of patients with BQ chewing habits than in those of non-chewing habits (p < 0.05). Our in vitro model showed that hypermethylation is followed by downregulation of the transcriptional level of SIRT1 (p < 0.05). The methylation levels of SIRT1 in the smear samples obtained from BQ chewing individuals were significantly higher than those in the samples obtained from individuals that did not chew BQ. The duration of BQ chewing habits was correlated positively to the frequency of SIRT1 hypermethylation (p < 0.05). CONCLUSIONS: Our results suggest that DNA hypermethylation of SIRT1 is involved in the occurrence of oral cancer in BQ chewing patients and that hypermethylation in the oral mucosa of BQ chewers could be a predictive marker for the occurrence of malignant transformation. This is the first report that showed DNA hypermethylation in clinically healthy oral epithelium of BQ chewers. Our study shows evidence that DNA hypermethylation may be an early event of oral carcinogenesis prior to observable clinical changes.

Upregulated expression of MMP-9 in gingival epithelial cells induced by prolonged stimulation with arecoline.

Betel quid chewing is implicated in the high prevalence of oral cancer in Southeast Asian countries. One of the major components of betel quid is arecoline. In the present study, in order to characterize the association between chronic arecoline stimulation and carcinogenesis the expression level of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA in human gingival epithelial progenitor cells (HGEPs) stimulated with arecoline was assessed. The HGEPs were alternated between 3 days of incubation with arecoline (50 µg/ml), and 3 days without arecoline, for up to 30 days. The expression levels of the MMPs and TIMPs in the cells stimulated with arecoline were evaluated by reverse transcription-quantitative polymerase chain reaction at 18 and 30 days. The expression of MMP-9 mRNA in the experimental group was significantly increased compared with in the control group (P<0.01). No significant differences in the expression of MMP-2, TIMP-1 or TIMP-2 mRNA were observed between the experimental and control groups. Using an MMP-9 activity assay, the levels of MMP-9 activity in the experimental group were demonstrated to be significantly higher than in the control group (P<0.05). To investigate associated cellular signaling pathways, PDTC [a nuclear factor (NF)-κB/inhibitor of NF-κB (IκB) inhibitor], PD98059 [a mitogen-activated protein kinase kinase (MAPKK)1 and MAPKK2 inhibitor], SB203580 (a p38 MAPK inhibitor) and 5,15-DPP [a signal transduction and activator of transcription (STAT) 3 inhibitor] were used. All inhibitors decreased the extent of MMP-9 upregulation induced by stimulation with arecoline. Based on the data, it is hypothesized that MMP-9 activity may be involved in the pathological alterations of oral epithelium induced by betel quid chewing, and that the NF-κB/IκB, MAPK, p38 MAPK and STAT3 signaling pathways may be involved in the production of MMP-9 induced by betel quid chewing.

CYP2A6 gene deletion reduces oral cancer risk in betel quid chewers in Sri Lanka.

We investigated the relationship between inter-individual difference in CYP2A6 genotype and susceptibility to oral cancer among habitual betel quid chewers in a Sri Lanka population. A total of 286 subjects showing oral malignant or premalignant lesions and 135 control subjects with no lesions were analyzed. The frequency of homozygotes for CYP2A6*4C mutation, a gene deletion type of polymorphism, was significantly lower in the case subjects than the controls. The odds ratio (OR) of the group homozygous for the deletion was significantly lower and calculated to be 0.14 (95% CI; 0.03-0.72). In the allelic base analysis, there was also a significant decrease in the OR of the deletion allele. Our data suggest that deficient CYP2A6 activity due to genetic polymorphism reduces oral cancer risk in betel quid chewers.