Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.     

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation

Objectives

Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation.

Materials

Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2′, 7′-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-l-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms.

Results

TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05).

Conclusions

Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation.

Clinical relevance

TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

     

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Specific inhibition of cyclooxygenase-2 results in inhibition of proliferation of oral cancer cell lines via suppression of prostaglandin E2 production

Prostaglandins (PGs) are known to play important roles in the proliferation of various types of cancer cells. PGs are produced by the action of cyclooxygenase (COX) enzymes, and two forms of COX, COX-1 and COX-2, have been described. Previous studies have demonstrated that overexpression of COX-2 is associated with colon carcinogenesis, tumor invasion and metastatic potential of colon cancer. In this study, the role of COX-2 on proliferation of squamous cell carcinoma cell lines was investigated. NS-398, a selective COX-2 inhibitor, inhibited proliferation of NA cells, a squamous cell caricinoma cell line that constitutively expresses COX-2 mRNA. NS-398 suppressed the spontaneous production of PGE2 by NA cells, and the antiproliferative effect of NS-398 was abolished by addition of PGE2. Similar results were obtained from experiments using COX-2 antisense oligonucleotide. These results suggest that specific inhibition of COX-2 inhibits proliferation of cancer cells expressing COX-2 mRNA via suppression of PGE2 production.     

Therapeutic Interventions in Oral Submucous Fibrosis: An Experimental and Clinical Study

Introduction

Oral submucous fibrosis (OSF) is a chronic debilitating disease and premalignant condition of the oral cavity and is a serious public health issue in India and many parts of the world. The treatment is still elusive and empirical because of poorly understood etiopathogenesis, which is believed to be multifactorial including areca nut chewing, ingestion of chillies, genetic and immunologic processes, nutritional deficiencies, and many others. The present investigations was focused to understand the possible therapeutic interventions of anti-OSF agents in arecoline induced experimental in vitro model of OSF and clinical application of these anti-OSF agents in the restoration of various grade of the disease.

Materials and Methods

The 127 subjects were selected from patients who visited the OPD of Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, K.G. Medical University, Lucknow. Further the subjects were divided in two groups on the basis of clinical examination. Group-1 subjects showed presence of fibrosis bands in the labial and/or buccal mucosa, loss of elasticity, difficulty to open the mouth and had a habit chewing areca-nut in some form. Group-2 subjects had no habit of chewing areca-nut, were apparently healthy with no mucosal disorder. The samples were collected and were immediately transported to Indian Institute of Toxicology Research, Lucknow, for isolation and cultivation of primary cultures of mucosal fibroblast cells. Then isolation and cultivation of oral mucosal fibroblast, identification of non-cytotoxic doses of arecoline, real time PCR, immunocytochemistry, cytokine determination in culture cells, western blot analyses, functional activity of collagenase, lysyl oxidase enzyme activity, collagen beads assay, cyclooxygenase (COX-2) expression analysis was done.

Results and Conclusions

This study, shows that the reduction of phagocytic cells was strongly related to the arecoline levels in fibroblast culture when we exposed arecoline to normal oral mucosal cells (NOMC) and cells from OSF patient. An enhancement of phagocytic cells was observed following the pre exposure of cells to 1 μM dexamethasone, a glucocorticoids, In this study, histologic evidence is presented which supports the finding that COX-2 expression is upregulated in OSF specimens compared to normal oral submucosal cells. Strong immunostaining for COX-2 was detected in arecoline exposed NOMC and cells from OSF patient. Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes. The number of phagocytic cells and phagocytic activity in cultured human oral fibroblasts from OSF site was lower than the fibroblasts from the normal regions of the same person.     

口腔黏膜下纤维性变中MMP-2基因的表达及意义

目的：检测口腔黏膜下纤维性变中基质金属蛋白酶-2（MMP-2）的表达并探讨其病理意义.方法：抽提11例口腔黏膜下纤维化组织和10例正常口腔黏膜组织的总RNA,通过逆转录聚合酶链反应（PF-PCR）检测MMP-2mRNA在口腔黏膜下纤维性变患者颊黏膜中的表达并与正常口腔黏膜进行比较.结果：口腔黏膜下纤维性变患者颊黏膜组织中MMP-2 mRNA表达高于正常颊黏膜（p＜0.05）.结论：MMP-2基因的表达与口腔黏膜下纤维性变组织重塑过程密切相关.     

EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts

Objectives

Transforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) and cyclooxygenase-2 (COX-2) were found to overexpress in OSF. The aim of this study was to investigate the molecular mechanism underlying the TGFβ-induced CCN2 expressions in human buccal mucosal fibroblasts (BMFs) to identify the potential targets for drug intervention or chemoprevention of OSF.

Materials and methods

TGFβ-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in BMFs.

Results

TGFβ1 stimulated CCN2 synthesis in BMFs. Pretreatment with c-Jun NH₂-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and activin receptor-like kinase 5 (ALK5) inhibitor SB431542 significantly reduced TGFβ1-induced CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) completely blocked TGFβ1-induced CCN2 synthesis by inhibiting the phosphorylation of JNK and p38 MAPK. Prostaglandin E₂ (PGE₂) inhibited the TGFβ1-induced CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs.

Conclusions

The TGFβ1-induced CCN2 synthesis in BMFs could be mediated by the ALK5, JNK, and p38 MAPK pathways. EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in BMFs.

Clinical relevance

The exceptional signal transduction pathways of TGFβ1-induced CCN2 production in BMFs contribute to the resistance of PGE₂ downregulation of CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. EGCG may serve as a useful agent in controlling OSF.     

Tyrosine sulphation of CXCR4 induces the migration of fibroblast in OSF

Oral submucous fibrosis (OSF) caused by areca nut chewing is a prevalent fibrotic disease in Asia-Pacific countries. Arecoline-induced migration of fibroblasts (FBs) plays a vital role in the development of OSF. However, the specific molecular mechanisms involved remain unclear. Many studies have shown that tyrosine sulphation of chemokines can influence cell migration. Herein, we demonstrated that arecoline stimulates tyrosine sulphation of the chemokine receptor 4 (CXCR4) through the tyrosylprotein sulphotransferase-1 (TPST-1) to enhance the migration ability of FBs. Moreover, by RNA-Seq analysis, we found that the most significantly altered pathway was the EGFR pathway after the arecoline stimulation for FBs. After the knockdown of arecoline-induced EGFR expression, the tyrosine sulphation of CXCR4 was significantly decreased by the inhibition of TPST-1 induction. Finally, in human OSF specimens, TPST-1 expression was directly correlated with the expression of CXCR4. These data indicate that the arecoline-induced tyrosine sulphation of CXCR4, which is regulated by TPST-1, might be a potential mechanism that contributes to FB migration in OSF.     

Cytotoxic and non-genotoxic effects of arecoline on human buccal fibroblasts in vitro

Chang YC, Tai KW, Cheng MH, Chou LSS, Chou MY: Cytotoxic and non-genotoxic effects of arecoline on human buccal fibroblasts in vitro. J Oral Pathol Med 1998; 27: 68–71. © Munksgaard, 1998.
Betel quid chewing has been linked to oral submucous fibrosis and oral cancer. Cytotoxicity and genotoxicity assays were used to investigate the pathobiologi-cal effects of arecoline on cultured human buccal fibroblasts. Arecoline increased double-stranded polynucleic acid at the concentration of 0.1 to 10 μg/ml in a concentration-dependent manner. At a concentration higher than 50 μg/ml, arecoline was cytotoxic to cultured fibroblasts and the cytotoxicity was dose-dependent. No genotoxicity for arecoline was found even at a concentration of 400 μg/ml. On the other hand, 600 μg/ml glutathione (GSH) and 200 μg/ml glycyrrhizin could prevent the arecoline-induced cytotoxicity. These results indicate that arecoline is a cytotoxic agent and no genotoxicity was found to human buccal fibroblasts. Furthermore, increasing consumption of GSH- and glycyrrhizin-rich foods may reduce the oral diseases associated with betel quid chewing.     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

槟榔碱及机械刺激构建大鼠口腔黏膜下纤维化模型

目的 利用槟榔碱和机械刺激构建Sprague-Dawley（SD）大鼠口腔黏膜下纤维化（OSF）模型。方法 采用两因素析因实验设计将48只大鼠分为8个组,每组6只。分别用不同浓度（0、0.5、2、8 mg·mL ^-1）槟榔碱涂擦及机械刺激 (有或无毛刷涂擦)。处理16周后测量开口度;取局部颊黏膜行苏木精-伊红（HE）染色观察病理变化;并检测组织内Ⅲ型胶原、转化生长因子-β1（TGF-β1）和γ干扰素（IFN-γ）的表达情况。结果 2和8 mg·mL ^-1（中、高）浓度槟榔碱处理16周,颊黏膜出现了典型的OSF病理特征;开口度显著减小并且Ⅲ型胶原、TGF-β1表达显著增加（P<0.05）。机械刺激虽可导致黏膜Ⅲ型胶原、TGF-β1和IFN-γ表达增高（P<0.05）,但无病理学改变,开口度改变不显著。结论 中、高浓度槟榔碱有致OSF作用;机械刺激无法导致大鼠OSF的发生。     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

NS-398诱导舌鳞癌细胞凋亡及其对E2F-1蛋白表达的影响

目的观察选择性环氧化酶-2(COX-2)抑制剂NS-398诱导舌鳞癌Tca8113细胞凋亡及其对细胞核转录因子E2F-1蛋白表达的影响。方法NS-398作用于舌癌Tca8113细胞,噻唑蓝法检测细胞生长抑制情况,流式细胞仪检测细胞周期及其凋亡率的改变,放射免疫法测定细胞上清液中前列腺素E2(PGE2)含量,Western blot法检测细胞中COX-2和E2F-1蛋白表达的变化。结果NS-398可以抑制Tca8113细胞的增殖,其抑制作用随着时间的延长和药物浓度的增加而增强。NS-398(50μmol/L)可引起Tca8113细胞G0/G1期细胞的逐渐增加,S期和G2/M期细胞比例数减少,并随着时间的延长细胞上清液中PGE2含量降低,细胞中COX-2和E2F-1蛋白表达下调。结论NS-398可以抑制Tca8113细胞的增殖,阻断细胞生长停滞于G0/G1期;该效应可能与其诱导细胞凋亡和降低细胞产生前列腺素E2有关;E2F-1蛋白可能参与了这些过程。     

Involvement of cyclooxygenase-2 in interleukin-1alpha-induced prostaglandin production by human periodontal ligament cells.

BACKGROUND: Human periodontal ligament (PDL) cells produce prostaglandin (PG) E2 in response to proinflammatory cytokines. However, the mechanism of PGE2 production is not well understood. The purpose of the present study was to investigate the involvement of cyclooxygenase (COX)-1 and COX-2 in PGE2 production by PDL cells stimulated with a proinflammatory cytokine, interleukin-1alpha (IL-1alpha), and to examine the regulation of PGE2 production by cell-cell interaction of human gingival keratinocytes and PDL cells. METHODS: The levels of PGE2 in the culture media of PDL cells stimulated with IL-1alpha or culture media of human gingival keratinocytes were determined by an enzyme-linked immunosorbent assay. Expression of COX-1 and -2 mRNA and protein was studied by Northern blot analysis and Western blot analysis, respectively. RESULTS: IL-1alpha-stimulated PDL cells produced PGE2 in a time-dependent manner. Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE2 production by the IL-1alpha-stimulated cells. COX-2 mRNA was detected after IL-1alpha stimulation, although it was not detected in unstimulated cells. There was no difference in expression of COX-1 mRNA between unstimulated cells and IL-la-stimulated cells. Expression of COX-2 protein in IL-1alpha-stimulated cells was increased, compared with that in unstimulated cells, whereas COX-1 protein expression was almost the same in both the cells. Treatment of IL-1alpha-stimulated PDL cells with dexamethasone, known to inhibit COX-2 expression, prevented PGE2 production and COX-2 mRNA expression. Addition of the culture media of human gingival keratinocytes to PDL cells increased PGE2 production. The PGE2 production was depressed by treatment of the cells with IL-1 receptor antagonist and anti- IL-1alpha antibody, not with anti-IL-1beta antibody. The PGE2 production was also inhibited by treatment with NS-398 and dexamethasone. CONCLUSIONS: We suggest that PDL cells stimulated with IL-1alpha produce PGE2 through de novo synthesis of COX-2 and that the cell interaction of gingival keratinocytes and PDL cells controls COX-2 expression and PGE2 production via IL-1alpha or 1alpha IL-la-like factor(s). Selective COX-2 inhibitors, which have the advantage of reduced gastric toxicity, may provide a useful approach to treatment of periodontal disease.     

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.