Expression of Human Papillomavirus and the p16 Gene in Oral Potentially Malignant Disorders (OPMD): a Comparative Study With Oral Squamous Cell Carcinoma.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) of the tongue is increasing in the younger population without traditional risk habits that lead researchers to find other related factors such as diet and viruses, especially human papillomavirus (HPV). It is noteworthy that many OSCCs develop from oral potentially malignant disorders (OPMDs). Correct diagnosis and timely management of OPMDs may help to prevent malignant transformation, and therefore it is worth seeing the involvement of HPV in OPMDs and oral cancers, as the preventive and curative measures in HPV-induced cancer types are different from the conventional types of OPMDs and OSCCs. Therefore, the main objective of this study was to identify a relationship between HPV and p16 in OPMDs and compare it with OSCC. METHODS: This study was conducted on 83 cases of known OSCCs and OPMDs (oral submucous fibrosis, leukoplakia, and oral lichen planus). Assays, such as polymerized chain reaction (PCR) and reverse transcription-PCR, were carried out for HPV and p16 . The results were compared with clinical information and with the literature. The results were analyzed using SPSS 16.0 for windows. RESULTS: P16 expression was mostly seen in males than in female patients. Out of 21 cases of keratosis with dysplasia, 19% expressed p16 . Of 26 oral lichen planus patients, 29% showed the p16 gene with immunohistochemistry. Interestingly, a high percentage of OSF cases expressed p16 (48.27%). Minimal expression was observed in OSCC (6.25%). HPV DNA was detected in 2.4% of the total sample. Both p16 and HPV were detected in a single case of OSCC. OPMDs expressed a significant amount of the p16 gene by immunohistochemistry and reverse transcription-PCR technique when compared with malignant lesions, suggesting a possible inactivation of the p16 gene. HPV and p16 are mostly negative in our OSCC sample, exhibiting low prevalence. CONCLUSIONS: OPMDs expressed a significant amount of the p16 gene when compared with malignant lesions, suggesting a possible inactivation of the p16 gene. Although OSF expressed p16 , HPV was not detected, suggesting that over-expression could be independent of HPV. OSCC shows low HPV prevalence.

Carcinogenic Effects of Areca Nut and Its Metabolites: A Review of the Experimental Evidence.

Oral cancers (OC) are among the most frequent malignancies encountered in Southeast Asia, primarily due to the prevalent habit of betel quid (BQ) and smokeless tobacco use in this region. Areca nut (AN), the primary ingredient in BQ, contains several alkaloids, including arecoline, arecaidine, guvacoline, and guvacine. These have been associated with both the AN abuse liability and carcinogenicity. Additionally, variations in AN alkaloid levels could lead to differences in the addictiveness and carcinogenic potential across various AN-containing products. Recent studies based on animal models and in vitro experiments show cellular and molecular effects induced by AN. These comprise promoting epithelial-mesenchymal transition, autophagy initiation, tissue hypoxia, genotoxicity, cytotoxicity, and cell death. Further, clinical research endorses these undesired harmful effects in humans. Oral submucosal fibrosis, a potentially malignant disease of the oral cavity, is predominantly reported from the geographical areas of the globe where AN is habitually chewed. OC in chronic AN users presents a more aggressive phenotype, such as resistance to anti-cancer drugs. The available evidence on the carcinogenicity of AN based on the findings reported in the recently published experimental studies is discussed in the present review.

The Protective Effect of Antioxidants in Areca Nut Extract-Induced Oral Carcinogenesis.

OBJECTIVE: Oral submucous fibrosis (OSF) is the premalignant disorder associated with fibrosis and epithelial atrophy. Areca Nut (AN) is the most significant risk factors for OSF. However, the molecular mechanism behind AN induced OSF remains unclear, and there exists no effective treatment for the malignant disorder. We aimed to investigate whether AN-extract causes epithelial-mesenchymal transition (EMT) in oral keratinocytes, and evaluated the therapeutic potential of antioxidants. METHODS: The HPV16 E6/E7-transfected immortalized human oral keratinocytes (IHOK) were employed in the present study. For the preparation of AN-extract, dried AN was dissolved in distilled water overnight. The solution was centrifuged and the supernatant was collected for further use. For the determination of change in cytokine levels, ELISA was performed. To investigate EMT-related protein expression and phenotype, immunoblot and immunofluorescence were performed. RESULTS: Among tumor-promoting cytokines (Gro-α, IL-6 and IL-8), IL-6 was remarkably increased by AN in IHOK. AN-extract induced EMT phenotypes, such as cell elongation, up-regulation of vimentin and snail. After treatment with neutralizing antibody of IL-6, AN-induced snail expression was reduced remarkably. Collectively, AN-extract induced IL-6 expression and mediated EMT. The use of antioxidants (EGCG, glutathione and NAC) significantly reduced IL-6 expression in AN-treated IHOK. Also, AN-decreased E-cadherin and increased vimentin were reversed by antioxidants, indicating that the effectiveness of antioxidants in inhibiting IL-6-induced EMT by AN. CONCLUSION: AN promotes EMT and antioxidants interrupt AN-induced-EMT in oral keratinocytes. Consequently, it is proposed that antioxidants could prevent AN-induced carcinogenesis and function as a prototype for developing therapeutic interventions of OSF.
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Identification of a combined biomarker for malignant transformation in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic progressive disease of the oral cavity that is considered a common potentially malignant disorder in South Asia. Areca nut chewing is the main etiological factor, but its carcinogenic mechanism has yet to be proven. The purpose of this study was to identify the useful biomarkers in predicting high-risk patients with OSF. METHODS: Thirty-six cases of OSF and six cases of normal oral mucosa (NOM) were used for this study. Immunohistochemical staining was performed for Ki67, cyclin D1, p16, p53, ß-catenin, c-Jun, c-Met, and insulin-like growth factor II mRNA-binding protein 3 (IMP3). The expression patterns of NOM served as guidelines for the scoring system. RESULTS: The expression of Ki67, cyclin D1, c-Met, IMP3, and ß-catenin showed a significant difference between OSF and NOM samples. The combined biomarkers of Ki67 and p16 showed significantly different expression between the transformation and non-transformation groups. With discriminant analysis, we proposed a noble formula and cutoff value for predicting high-risk patients with OSF. CONCLUSION: The notable biomarkers in our present study were Ki67 and p16 showing significantly different expression levels between the transformation and non-transformation groups. With the identification of high-risk patients with OSF, we can expect to develop more intensive treatment modalities, leading to the reduction in cancer transformation rate from OSF.

Effect of Areca Nut on Helicobacter pylori-Induced Gastric Diseases in Mice.

Areca nut (AN) chewing is a habit in many countries in Central, Southern, and Southeast Asia. It is strongly associated with the occurrence of oral, pharyngeal, and esophageal cancer as well as systemic inflammation. However, the association between AN intake and the development of gastric lesions has not yet been identified. The aim of this study was to investigate the effect of AN on gastric diseases using a mouse model for Helicobacter pylori infection. We studied four groups of mice: those fed a normal diet (ND), those fed a diet containing 2.5% AN (AD), those fed ND and infected with H. pylori PMSS1 strain (ND/HP), and those fed AD and infected with H. pylori PMSS1 strain (AD/HP). Food intake and body weight were monitored weekly during the experiments. At 10 weeks, the mice were sacrificed, and the stomach weight, H. pylori colonization, and gastric inflammation were evaluated. The stomach weight had increased significantly in the ND/HP and AD/HP groups along with increases in H. pylori colonization; however, there was no significant difference between these two groups with respect to stomach weight and colonization. On histological grading, mononuclear cell infiltration was severer in the AD/HP group than in the ND/HP group. These data suggest that chronic gastric inflammation was aggravated by AN treatment in the mice with H. pylori-induced gastric lesions. Furthermore, as previously suggested, this animal model is useful to determine the effect of potential carcinogens on gastric lesions induced by H. pylori infection.

Areca nut exposure increases secretion of tumor-promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes.

Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)-exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT-hNOF) was used. We found that the levels of GRO-α, IL-6 and IL-8 increased in AN-exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN-exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8-oxoG FITC-conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN-exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine-triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.

A distinct role for interleukin-6 as a major mediator of cellular adjustment to an altered culture condition.

Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial-mesenchymal transition (EMT)/ mesenchymal-epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7-transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL-6) from IHOK. We found IL-6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL-6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions.