Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro

Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 μg/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0–200 μg/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 μg/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 μg/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P<0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy. Received: 31 August 1998 / Accepted: 3 November 1998     

Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas

Oral Diseases (2012) 18 , 713–719 Objectives: Heat shock protein (HSP) 27 is a low‐molecular‐weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. Methods: Forty‐eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin‐3 gallate (EGCG), glutathione precursor N‐acetyl‐l ‐cysteine (NAC), cyclooxygenase‐2 inhibitor NS‐398, HSP inhibitor quercetin, extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose‐ and time‐dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline‐induced HSP27 expression (P < 0.05). Conclusions: Heat shock protein 27 expression is significantly elevated in areca quid chewing‐associated OSCCs. Arecoline‐induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.     

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.     

Heat shock protein 47 expression in oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

J Oral Pathol Med (2010) 40 : 390–396 Background: Heat shock protein 47 (HSP47) is a product of CBP2 gene located at chromosome 11q13.5, a region frequently amplified in human cancers. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP47 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP47 expression. Methods: Thirty‐two OSCC specimens and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line OC2 cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, cyclooxygenase‐2 inhibitor NS‐398, and tyrosine kinase inhibitor herbimycin A were added to find the possible regulatory mechanisms. Results: HSP47 expression was significantly higher in OSCC specimens than normal epithelium (P < 0.05). No significant difference in HSP47 expression was observed with respect to age, sex, T category, stage, and differentiation (P > 0.05). The lower HSP47 expression was associated with lymph node metastasis (P = 0.015). Arecoline was found to elevate HSP47 expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, PD98059, LY294002, NS398, and herbimycin A markedly inhibited the arecoline‐induced HSP47 expression (P < 0.05). Conclusion: Our findings demonstrated that HSP47 expression is significantly upregulated in areca quid chewing‐associated OSCCs. HSP47 could be used clinically as a marker for lymph node metastasis of oral carcinogenesis. In addition, arecoline‐induced HSP47 expression was downregulated by NAC, PD98059, LY294002, NS398, and herbimycin A.     

Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro

BACKGROUND, aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. METHOD: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. RESULTS: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. CONCLUSION: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.     

Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes

Betel quid (BQ) chewing has a strong correlation with oral leukoplakia, submucous fibrosis and oral cancer. For elucidation of its pathogenesis, we investigated the effects of areca nut (AN) and inflorescence piper betle (IPB) extracts and arecoline on the growth, total DNA synthesis (TDS) and unscheduled DNA synthesis (UDS) of cultured human gingival keratinocytes (GK). Arecoline and AN extract suppressed the growth of GK over 5 days of incubation in a dose-dependent fashion. At concentrations of 100, 200 and 400 μg/ml, AN extract suppressed the growth of GK by 31%, 46% and 90%, respectively. The IPB extracts exerted less inhibitory effect on the growth of GK. IPB extract (200–400 μg/ml) decreased cell numbers by 20–40% over 5 days of incubation. Moreover, at a concentration of 0.1, 0.2 and 0.4 mM, arecoline suppressed cell growth by 44%, 77% and 96%, respectively. However, only AN extract induced TDS and UDS in cultured GK within 6 h of exposure. Induction of UDS by AN extract was concomitant with the presence of apparent intracellular vaculoization. Arecoline was also toxic to GK, but did not induce intracellular vacuolization. At a concentration range of 200–1600 μ/ml, AN extract induced TDS by 2.1- to 6.5-fold. Furthermore, at a concentration of 400–1600 μ/ml, AN extract elevated the UDS by 2.4- to 5.5-fold more than that of untreated control. On the contrary, IPB extract (200–1600 μ/ml) and arecoline (0.2–1.6 mM) inhibited the TDS and UDS of GK to a different extent. Simultaneous exposure of confluent GK to AN extract, IPB extract and arecoline for 1 to 5 days led to different degrees of cytotoxicity that was dose-and time-dependent. These results indicate that AN, IPB and arecoline take part in the pathogenesis of BQ chewing-related oral mucosal lesions, possibly through both genotoxic and non-genotoxic mechanisms.     

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

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.     

Is workplace screening for potentially malignant oral disorders feasible in India?

J Oral Pathol Med (2010) 39 : 672–676 Background: Because of delays in diagnosis, oral cancer usually presents for therapy at a late stage. Patients are unaware of having lesions as they are mostly asymptomatic and physicians generally do not examine the mouth sufficiently. People in rural areas or are underserved may not frequently visit the dentist who can easily pick up these lesions early. Screening programs are useful in that regard. Such programs in general are conducted by either inviting people to come to a screening center or by health care workers visiting the individual households. However, those who work during the day may not visit screening centers or be at home during the day of the screening by a visiting health care worker. Workplace screening overcomes these challenges. Methods: To assess the feasibility of a screening program to detect potentially pre‐malignant oral disorders in a workplace in India, clinically visible mucosal lesions were compared with the clinical photographs of the same lesions assessed by an expert. Role of smoking, alcohol, and chewing betel quid and tobacco in the etiology of those lesions were assessed. Results: Sixty‐nine percent of the eligible subjects participated in the screening (n = 1613). Prevalence of leukoplakia was 5%. Bidi (OR = 35.6), and cigarette smoking (OR = 22.8), alcohol (OR = 17.6), and tobacco and areca nut chewing (OR = 7.5), were significantly associated with leukoplakia and erythroplakia (all P < 0.05). Conclusions: Conduction of a screening program by valid visual inspection to detect potentially malignant oral disorders within a workplace is not only feasible but also effective.     

Areca nut extract lowers the permeability of vaginal mucosa to reduced arecoline and arecaidine

Areca nut chewing has been implicated in the development of oral cancer and oral submucous fibrosis. Arecoline and arecaidine, which are alkaloids present in the areca nut, are thought to play a major role in the development of adverse effects resulting from this chewing habit. Because these alkaloids appear to be associated with the development of the above diseases, we determined their diffusion kinetics through human vaginal mucosa in the presence and absence of a 1% areca nut extract. Seven clinically healthy vaginal mucosa specimens (mean patient age+/-standard deviation, 52+/-13 years; age range, 38-74 years) were obtained during surgery. In vitro flux values of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. The flux values across vaginal mucosa of r-arecoline and r-arecaidine were decreased in the presence of 1% areca nut extract. For r-arecoline, these flux values were significantly lower statistically when compared to those obtained in the absence of areca nut extract. These findings concur with results previously obtained for water, where the astringent action of the tannins present in the areca nut extract was thought to alter the barrier properties of the epithelium, resulting in decreased permeability.     

Inhibition of the migration, attachment, spreading, growth and collagen synthesis of human gingival fibroblasts by arecoline, a major areca alkaloid, in vitro

Because betel quid (BQ) chewing has been linked to a higher prevalence of periodontal diseases, the pathobiological effects of arecoline, a main alkaloid found in areca nut, were investigated in cultured human gingival fibroblasts. At concentrations higher than 0.4 mM, arecoline inhibits cell attachment, cell spreading and cell migration in a dose-dependent manner. These inhibitory effects were associated with intracellular depletion of glutathione (GSH). At concentrations of 0.4 mM and 1 mM. arecoline depleted about 26% and 45% of GSH after 2 h incubation. Exposure of cells to areeoline at concentrations lower than 0.4 mM for 2 h showed no significant decrease in either cell viability or intracellular GSH. However, incubation of cells for 24 h in 1 mM arecoline decreased the cell numbers to only 35% of those in the untreated control. Arecoline also decreased cell growth and collagen synthesis in a dose-dependent manner. Because of repeated and long-term exposure to arecoline. BQ chewers could be more susceptible to periodontal damage and less responsive to new attachment procedures.     

Observations on the effect of areca nut extracts on oral fibroblast proliferation

The effects of aqueous extracts of raw, baked and boiled areca nuts were tested on cultured human buccal mucosa fibroblasts. Cells were exposed to extract concentrations of 0, 50, 100, 150, 300 and 500 μg/ml. The arecoline and arecaidine content was determined in the extracts with HPLC and raw nut contained 5.5% in m. baked nut 6.6% mm and boiled nut 7.1% m/m. Extract concentrations of 50 to 150 μg/ml inhibited cell growth in a concentration-dependent manner but did not lead to total cell death during a 7 day period. However, total cell death did occur with concentrations of 300 and 500 μg/ml. It is concluded that areca nut extract is toxic to cultured fibroblasts and inhibits their proliferation in a concentration-dependent manner.     

Areca nut extracts reduce the intracellular reactive oxygen species and release of myeloperoxidase by human polymorphonuclear leukocytes

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes (PMN) represent the first line of host defense. Areca nut extract inhibits the bactericidal activity of, and the release of superoxide anion (O2- ) by, PMN. This study investigated the effects of areca nut extract on the intracellular production of reactive oxygen species (ROS) and on the extracellular release of lysosomal enzyme, myeloperoxidase (MPO), by PMN. The effects of arecoline, a principal component of areca nut, were also examined. MATERIAL AND METHODS: Human PMN were treated with various concentrations of areca nut extract or arecoline followed by treatment with Hanks' balanced salt solution, with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). The viability of PMN was determined using propidium iodide staining and flow cytometry. The presence of intracellular ROS was determined using 2',7'-dichlorofluorescin diacetate and fluorometry. MPO release was determined using a substrate assay. RESULTS: Areca nut extract (25 and 50 microg/ml) significantly decreased the viability of PMN. The intracellular levels of ROS and the extracellular release of MPO were induced in PMN by CB/fMLP. Exposure of PMN to areca nut extract (up to 25 microg/ml) or to arecoline (up to 2 mg/ml) did not directly affect the levels of ROS and MPO activity. However, under conditions that did not affect the viability of PMN, the ability of CB/fMLP to trigger production of intracellular ROS and release of MPO in human PMN was significantly suppressed by areca nut extract and arecoline. CONCLUSION: Areca nut impaired the activation of PMN by CB/fMLP that might decrease the effectiveness of PMN in the host defense. Alternatively, exposure of PMN to areca nut extract could decrease the capacity of PMN to damage tissues.     

槟榔碱预处理后的Hacat细胞及人口腔黏膜成纤维细胞中AngⅡ及AT1R蛋白的表达研究

目的:通过检测血管紧张素Ⅱ(AngⅡ)及血管紧张素Ⅱ-1型受体(AT1R)在不同浓度槟榔碱预处理后的Hacat细胞及人口腔黏膜成纤维细胞(OMFB)中的表达与分布情况,了解槟榔碱对这两种细胞表达AngⅡ及AT1R的影响,探讨口腔黏膜下纤维性变(OSF)可能的发病机制。方法:采用免疫细胞化学SABC法,分别测定AngⅡ及AT1R兔抗人多克隆抗体在Hacat细胞和OMFB及分别在20μg/mL、40μg/mL、80μg/mL槟榔碱预处理后的这两种细胞中的表达与分布情况。结果:①0～80μg/mL槟榔碱预处理后Hacat细胞中AngⅡ蛋白均有表达,80μg/mL预处理组阳性细胞数高于其余3组,差别有统计学意义(P<0.01)。②对照组及20μg/mL槟榔碱干预组Hacat细胞中未见明显AT1R蛋白表达,40μg/mL槟榔碱干预组细胞中AT1R蛋白弱阳性表达,80μg/mL槟榔碱干预组细胞AT1R蛋白呈阳性表达,表达有显著性差异(P<0.01)。结论:一定浓度的槟榔碱可以诱导Hacat细胞中AngⅡ及AT1R蛋白的表达升高,且二者的表达多位于发生了形态学变化的细胞上,推测AngⅡ及AT1R可能参与了槟榔碱的致OSF进程。     

槟榔致癌物质与口腔癌

槟榔为一级致癌物,咀嚼槟榔引起口腔癌缘于槟榔中的槟榔碱（ARC）、槟榔鞣质、槟榔特异性亚硝胺（ASNA）和活性氧（ROS）等具有细胞毒性、遗传毒性、致突变性和致癌性。ARC可诱导口腔成纤维细胞、角质形成细胞和人脐静脉内皮细胞程序性死亡。槟榔鞣质有否遗传毒性和致突变性至今仍有争议,不同类型的短期筛选试验结果差异很大,但含鞣质的槟榔多酚是槟榔的主要致癌成分。3-甲基亚硝氨基内醛可诱发人颊黏膜角质形成细胞的DNA链断裂和DNA蛋白交联。3-甲基亚硝氨基丙腈为强致癌剂,可诱发试验动物肿瘤,靶器官包括鼻腔、食管、舌等。槟榔咀嚼过程中可产生大量的ROS,造成DNA氧化性损伤和激活癌基因的方式促使癌症的发生。相对分子质量为3．0×10^4-10．0×10^4的槟榔提取物组分中一种新发现的蛋白聚糖通过增加胞内ROS水平及一系列信号级联放大,上调口腔癌细胞低氧诱导因子-1d的表达,最终诱导细胞自噬。细胞自噬有利于保护癌细胞免遭ARC诱导的程序性细胞死亡,促进口腔癌的发展。槟榔提取物还可能通过ROS增强舌鳞状上皮细胞癌细胞株刺激血小板聚集的效应,从而促进舌癌转移。     

Arecoline and oral keratinocytes may affect the collagen metabolism of fibroblasts

Background:  The characteristic of oral submucous fibrosis (OSF) is related with the disturbance of synthesis and degradation of extracellular matrix. Arecoline, the areca nut (betel nut) component of betel quid, plays a major role in pathogenesis of OSF. But the exact mechanism how arecoline influences the collagen metabolism is unclear.
Methods:  Oral keratinocytes and fibroblasts were cocultured and keratinocytes were pre-treated by arecoline. Fibroblasts alone, fibroblasts stimulated by arecoline, fibroblasts cocultured with keratinocytes and fibroblasts cocultured with keratinocyte pre-treated by arecoline were included as the four groups in the present study. The concentration of collagen, the content and activity of matrix metalloproteinase (MMP) and the concentration of tissue inhibitor of metalloproteinase (TIMP) were assessed.
Results:  The collagen production of fibroblasts decreased when cocultured with keratinocytes; when cocultured with keratinocytes pre-treated by arecoline, fibroblasts produced more soluble collagen than non-pretreated coculture group. MMP-9 was produced only in coculture groups. There was no significant difference in the two coculture groups. The activation ratio of pro-MMP-2 in arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than that of non-coculture groups, but no significant difference existed in the two coculture groups. TIMP-1 produced by arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than those by the other three groups.
Conclusion:  TIMP-1 and the interaction of oral keratinocytes and fibroblasts play important role in pathogenesis of OSF.     

Salivary osmolality and hydration status in children with cerebral palsy

J Oral Pathol Med (2011) 40 : 582–586 Background: Unstimulated whole salivary parameters have been identified as potential markers of hydration status. Reduced salivary flow rate and increased salivary osmolality have been shown to be useful to identify dehydration, even when minimal loss of body water occurs. This study aimed to evaluate whether unstimulated salivary flow rate and salivary osmolality from individuals with cerebral palsy correlate with plasma and urine osmolality. Methods: Thirty‐five male and female children, aged 9–13 years old, diagnosed with cerebral palsy were compared to 27 nondisabled children (10–12 years old). Unstimulated whole saliva was collected under slight suction and salivary flow rate (ml/min) was calculated. Plasma without venostasis and urine were also collected. Salivary, plasma and urine osmolality were measured using a freezing point depression osmometer. Results: Cerebral palsy children presented a reduction in salivary flow rate (50%) compared to the control group (P < 0.01). Moreover, an increase in salivary (50%), plasma (3%), and urine osmolality (20%) was also observed in the cerebral palsy children compared to the control group (P < 0.01). Salivary flow rate was negatively correlated with the salivary, plasma and urine osmolality (P < 0.01). Salivary osmolality correlated positively with plasma and urine osmolality (P < 0.01). Conclusion: Cerebral palsy children seem to present impaired adequate hydration status. Since the possible hypohydration condition may be reflected in saliva fluid, which could compromise the protective function exerted by saliva, the earlier this condition is identified the greater the chances of administering preventive measures. Moreover, salivary osmolality is a reliable parameter that reflects changes in plasma and urine.