Implication of cytokines released from gingival fibroblasts exposed by areca nut extract in carcinogenesis

Abstract

The mechanism that cancers develop in Oral Submucous Fibrosis (OSF) has not been proven yet. Besides the direct cytotoxic and genotoxic effects of Areca nut (AN) on oral epithelium, indirect effects have also been identified, one of which is about the role of gingival fibroblasts influenced by AN. In this study, we mainly focused on the role of secretory molecules produced by the gingival fibroblasts upon AN extract (ANE) exposure.We hypothesized that ANE could stimulate submucosal fibroblasts to secrete inflammatory cytokines continuously, which would cause a genotoxic effect to the OSF epithelium, and which may contribute to the malignant transformation of OSF.Therefore as a start, cytokine antibody array was utilized to identify the cytokines secreted by ANE-stimulated fibroblasts. Water extract of AN was used in concentrations of 30 μg/ml and 40 μg/ml in in vitro studies. Normal human gingival fibroblasts (hNOF) and immortalized human gingival fibroblasts (hTERT-hNOF) were used in in vitro ANE stimulation. Cytokine antibody array data wasixconfirmed using ELISA, RT-PCR and immunofluorescence studies. The histologic sections of OSF patients were also used for immunohistochemical analysis of cytokine expressions.hNOF and hTERT–hNOF were treated with ANE for short-term and hTERT-hNOF for long-term to study their cytokine secretion pattern and were analyzed by ELISA and immunofluorescence. As hNOF and hTERT-hNOF showed similar response pattern to ANE treatment, hTERT-hNOF were selected to expose for long term stimulation with ANE. Long-term ANE treated hTERT-hNOF were tested for senescence by β- galactosidase staining.After identified the cytokines secreted from ANE-stimulated fibroblasts, to find their effect on oral epithelium, immortalized human oral keretinocytes (IHOK) were utilized. Namely GRO-α, IL-6, and IL-8 were used to stimulate IHOK. Cytokine

effect on IHOK proliferation was measured by MTT assay. This study also investigated whether these cytokines could cause DNA damage in IHOK. Phospho-Histone H2A.X staining was used to detect DNA damage in a form of DNA double strand breaks (DSB) and FITC conjugated 8-oxo-guanine (8-oxoG) probing was used to detect the oxidative DNA damage. Tissue samples of OSF patients were also subjected to analyze for DNA DSB and oxidative DNA damage. In addition, H2DCFDA dye was used to detect reactive oxygen species (ROS) in IHOK by cytokine treatment. FACS analysis was used to test the cell cycle status and wound healing assay was used to test the cell motility in IHOK, after cytokine treatment.Finally to find out whether these cytokines could induce epithelial-mesenchymal transition (EMT) in IHOK, immunocytochemistry staining for EMT related molecules were carried out together with morphological examinations.Cytokine antibody array showed that ANE treatment increased GRO-α, IL-6, and IL-8 expressions in both hNOF and hTERT-hNOF. Angiogenin expression was reduced in both hNOF and hTERT-hNOF after ANE treatment. These results were confirmed using ELISA and immunofluorescence. OSF patients’ tissues were also showed increased GRO-α, IL-6, IL-8 and reduced angiogenin expressions which were compatible to the in vitro results. Long term stimulation of hTERT-hNOF with ANE also showed increased GRO-α, IL-6, and IL-8 secretion. 8wks of ANE exposure made hTERT-hNOF to become senescence which was confirmed by β- galactosidase staining.MTT assay showed the cytokines GRO-α, IL-6, IL-8 have no significant effect on the proliferation of IHOK. Phospho-Histone H2A.X staining clearly showed that GRO-α, IL-6, and IL-8 individually and in combination caused DNA damage; DNA double strand breaks (DSB) precisely. Patients’ tissues of OSF were also positive for Phospho-Histone H2A.X staining showing positive foci in an around the basal layer. Furthermore, FITC conjugated 8-oxo-guanine (8-oxoG) probing detected these cytokines causing oxidative DNA damage in IHOK. Oxidative DNA damage was detected in the cells in and around the basal cell layer of the OSF patients’ tissues as well. In addition, H2DCFDA dye detected GRO-α, IL-6, and IL-8 individually and in combination generates reactive oxygen species (ROS) in IHOK. FACS analysis revealed these cytokines causing increased cell aneuploidy in IHOK. Moreover these cytokines increased cell motility in IHOK and induced EMT changes as well.To sum up, the present study attempted to identify the role of fibroblasts in carcinogenesis of OSF by investigating the cytokines produced by the gingival fibroblasts upon ANE stimulation. Results figured out that GRO-α, IL-6 and IL-8 were capable of causing DNA damage in IHOK and especially the oxidative DNA damage was evident. Increased ROS production by GRO-α, IL-6, and IL-8 could cause the DNA damage in IHOK. Studies involved using OSF patients’ tissues supported our in vitro findings showing OSF tissues have elevated expressions of GRO-α, IL-6, and IL-8 and moreover, OSF tissues were positive for DNA damage and oxidative DNA damage markers as well. Furthermore long-term ANE stimulated hTERT-hNOF became senescent .This may be the senescence associated secretory phenotype (SASP) which has been extensively described in the literature, which contributes to the epithelial malignancies via producing secretory molecules such as cytokines and chemokines.GRO-α, IL-6 and IL-8 were capable of causing increased cell aneuploidy, increased cell motility and EMT changes in IHOK which are hallmarks of carcinogenesis.This study clarified one aspect of the indirect effect of AN in the carcinogenic transformation of oral epithelium with special reference to the cytokines secreted from submucosal fibroblasts. Our approach of this study could be further elaborated to elucidate the role of inflammatory cytokines in the carcinogenesis of OSF.