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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.

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.

Human β-defensin-2 (hBD-2) is a type of epithelial antimicrobial peptide. The expression level of hBD-2 mRNA is lower in oral carcinoma cells (OCCs) than in healthy oral epithelium. Yet, it is still unknown how hBD-2 expression is downregulated in OCCs. The present study investigated DNA hypermethylation of hBD-2 in OCCs and the effect of the demethylation and increased expression of hBD-2 on cell proliferation and invasion. Six different types of oral carcinoma cell lines (OSC-19, BSC-OF, SAS, HSC-2, HSC-4 and HSY) and normal oral keratinocytes (NOKs) were used. The expression levels of hBD-2 in all OCCs were significantly lower than that in the NOKs. Treatment with DNA methyltransferase inhibitor, 5-aza-dC, at the concentration of 50 µM significantly induced upregulation of expression of hBD-2 in the OCCs. Using methylation-specific PCR, DNA hypermethylation was observed in all OCCs. These results suggest that DNA hypermethylation is, at least in part, involved in the decreased expression of hBD-2 in OCCs. We examined the effect of 5-aza-dC on the cell proliferation and invasive ability of OCCs. The cell invasion assays showed that the number of OCCs treated with 5-aza-dC on the filters was significantly lower than that of the controls. We examined whether increased expression of hBD-2 generated by gene transfection inhibited the proliferation and invasion of SAS cells. The number of SAS cells exhibiting increased expression of hBD-2 on the filters in the invasion assay were significantly lower on day 7 when compared with the control. hBD-2 may function as a tumor suppressor. Increased expression of hBD-2 induced by demethylation or increased expression generated by gene transfection may be useful therapeutic methods for oral carcinoma.

Background/purpose: Epigenetic alterations such as DNA methylation and histone acetylation are described as changes in the pattern of gene expression not involving the DNA sequence. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis has been shown to inhibit osteoblastic cell differentiation. We examined whether DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL)., Methods: The HPDL cells were incubated with LPS derived from P. gingivalis at a concentration of 10 μg/ml for 24 h. The cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza). Untreated cells were used as a control. Cell viability was determined using cell proliferation reagent. DNA methyltransferase (DNMT1) and runt-related transcription factor 2 (RUNX2) mRNAs were evaluated by quantitative polymerase chain reaction (RT-PCR). Analysis of RUNX2 DNA methylation was performed using quantitative methylation-specific PCR., Results: The expression level of RUNX2 was significantly lower in the cells stimulated with LPS than the controls. The presence of 5Aza increased the expression of RUNX2 in cells stimulated with LPS. The expression levels of DNMT1 mRNA in the cells stimulated with LPS were significantly higher than in the control. The presence of 5Aza completely abolished the upregulated expression of DNMT1 in cells stimulated with LPS. The methylation of DNA at 0.1 kb and -1.9 kb in the cells stimulated with LPS was significantly higher than the control., Conclusion: The results indicate that DNA hypermethylation may be involved in the inhibitory effect of LPS on osteoblastic differentiation in HPDL., (Copyright © 2012. Published by Elsevier B.V.)

Objective and Background: Heated tobacco products have recently become commercially available. These products, as well as combustible cigarettes, produce aerosols; the risk of various diseases associated with heated tobacco products may be the same or higher than that with combustible cigarettes. In this study, we examined the effect of Ploom TECH+ extract on gingival epithelial cells. Methods: Tobacco leaves from Ploom TECH+ tobacco capsules and water were mixed and heated; the supernatant subsequently collected was the heated tobacco product (HTP; control: HTP not added). Normal human gingival epithelial progenitors were cultured alternately with or without HTP for a total of 1 month. Subsequently, RNA, DNA, and proteins were isolated from these samples and comprehensively analyzed using RNA sequencing (RNA‐seq), reduced representation bisulfite sequencing (RRBS), and western blotting, respectively. Results: RNA‐seq revealed that 284 genes showed a twofold increase and 145 genes showed a twofold decrease in gene expression. A heat map showed genetic differences between the control and HTP groups. A principal component analysis plot showed a clear genetic distribution between the control and HTP. Gene Ontology (GO) analysis showed that genes related to seven GO terms, including cornification and keratinization, were induced by long‐term HTP stimulation. By contrast, GO pathways with a significant decrease in component expression were not detected. RRBS revealed that CpG island methylation increased more than twofold in 158 genes and decreased to less than twofold in 171 genes. Methylation of these CpG islands was not correlated with changes in gene expression levels. HTP treatment increased S100A7 expression. Conclusion: Long‐term HTP stimulation affected epithelial differentiation and keratinization of gingival epithelial cells. Thus, habitual use of Ploom TECH+ may be a risk factor for tobacco‐related oral mucosal diseases. [ABSTRACT FROM AUTHOR]

Objectives: Epigenetic phenomena are changes in gene expression not involving the DNA sequence. DNA methylation is a major occurrence underlying epigenetic changes in human cells. Although aberrant DNA methylation is well documented in malignant lesions, limited information has been shown on the involvement of DNA methylation in oral lichen planus and oral lichenoid lesions (OLP). The present study aimed to investigate DNA methylation of E‐cadherin and p16 in OLP, and compare the findings with those in non‐inflamed gingiva (Non), radicular cyst (RC), and oral squamous cell carcinoma (SCC). Materials and methods: Paraffin‐embedded surgical biopsy specimens were sliced, DNA was extracted, bisulfite treatment was applied, and methylation‐specific polymerase chain reaction was performed. Immunohistochemistry was performed to observe the relative expression patterns of these genes. Results: E‐cadherin was hypermethylated in OLP (p < 0.01), SCC (p < 0.01), and RC (p < 0.05), when compared with Non; DNA hypermethylation was confirmed in OLP and SCC when compared to Non and RC. Hypermethylation of p16ink4a was observed only in SCC (p < 0.01). Conclusion: DNA methylation levels of E‐cadherin and p16ink4a were significantly higher in OLP than in normal tissues, and may be associated with the pathogenesis and progression of the disease. [ABSTRACT FROM AUTHOR]

Background Epigenetics involves alterations in gene expression that do not involve modifications in the DNA sequence, the memory of which can be passed down to the next generation in somatic cells. DNA methylation is an example of a mechanism that produces epigenetic changes. The purpose of this review is to summarize recent publications on DNA methylation in oral infections and inflammatory diseases, and to discuss its potential as a cause of disease and as a therapeutic target. Highlight Several types of oral bacteria and viruses may lead to DNA hypermethylation in oral tissues. Aberrant DNA hypermethylation is observed in oral inflammatory diseases, including chronic periodontitis, lichen planus, and radicular cysts. Conclusion Since epigenetic modifications are reversible, aberrant DNA methylation is a possible therapeutic target for such diseases. However, little is known about the epigenetics in oral inflammatory diseases, and further investigation is needed to elucidate the underlying mechanisms before epigenetic therapy can be used to treat oral inflammatory diseases. [ABSTRACT FROM AUTHOR]