Abstract
Abstract Oral Submucous Fibrosis (OSF) is a premalignant fibrotic disease occurred due to chewing habit of Areca nut consumption. We hypothesize that Areca nut exposure induces submucosal fibroblasts to secrete cytokines, by which they contribute to the malignant transformation of overlying epithelium through DNA damage. This study aims to find out these cytokines and to elucidate their role in carcinogenesis in OSF. For this study, normal gingival fibroblasts (hNOF), immortalized gingival fibroblast (hTERT-hNOF), and immortalized oral keratinocytes (IHOK) were utilized. Aqueous Areca nut extract (ANE) was used to stimulate hNOF and hTERT-hNOF. Conditioned media from ANE treated fibroblasts were screened using a cytokine antibody array. ELISA, RT-PCR and immunofluorescence were used for further analysis of the secretory cytokines. For evaluating DNA damage of IHOK by cytokine treatment, phospho-Histone H2A.X and 8-oxodG were utilized by immunofluorescence staining and FACS. DNA aneuploidy was measured by FACS. ROS production by cytokine treatment was also detected by immunofluorescence staining and FACS using H2DCFDA. Morphologic change of IHOK by long-term treatment of cytokines was also observed. Human tissues obtained from OSF patients were assessed by immunohistochemistry for the cytokine expressions and the detection of DNA damage. Increased expressions of GRO-α, IL-6, and IL-8 were found in ANE-stimulated hNOF and hTERT-hNOF. Long term ANE exposure of hTERT-hNOF caused senescent features, showing increased secretion of GRO-α, IL-6, and IL-8. Cytokine (GRO-α, IL-6, IL-8) treated IHOK caused increased aneuploid cell population. It also showed these cytokines produce ROS in IHOK causing oxidative DNA damage as well as DNA double strand breaks. Treatment of Glutathione and neutralizing antibodies reduced DNA damage in IHOK. OSF tissues also were evident of cytokine expression, oxidative DNA damage and DNA double strand breaks in the epithelium. Cytokines induced high motility in IHOK and long-term treatment of cytokines induced epithelial mesenchymal transition (EMT) in IHOK. Taken together, ANE stimulates gingival fibroblasts to secrete increase GRO-α, IL-6, and IL-8, by which they caused oxidative DNA damage via ROS production in IHOK. In addition, these cytokines induced DNA aneuploidy and EMT in IHOK. These results may provide insights to understand a contributory mechanism responsible for malignant transformation of OSF epithelium and to develop novel preventive modalities inhibiting their transforming routes. This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0094028) and (2010-0029702) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5471. doi:1538-7445.AM2012-5471
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