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.

In vitro cytotoxicity test is an initial step to identify the harmful effects of new dental materials. Aim of this study was to develop a stable human cell line derived from normal gingival fibroblasts (hNOF) and to assess its feasibility in in vitro cytotoxicity testing. Immortalized human gingival fibroblasts (hTERT-hNOF) were successfully established with human telomerase reverse transcriptase gene transfection, preserving its phenotypical characteristics, replicative potential and biological properties. Utilizing standard cytotoxicity test modeling and dental materials, hTERT-hNOF were evaluated for their feasibility in cytotoxicity testing, compared with hNOF and L929 cells. Similar pattern of cytotoxic response was observed among hNOF, hTERT-hNOF and L929 cells. Cytotoxicity response of hTERT-hNOF was significantly similar to hNOF, moreover hTERT-hNOF and hNOF were found to be more sensitive towards the tested dental materials compared to L929 cells. This study suggested that hTERT-hNOF is an effective cytotoxic test model for dental materials.

BackgroundOral 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.MethodsThirty‐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, β‐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.ResultsThe expression of Ki67, cyclin D1, c‐Met, IMP3, and β‐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.ConclusionThe 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.

Careful consideration on interpreting the results for cytotoxicity evaluation of ZOE and ZONE cements is needed when different cells are used.