Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2

Background:  Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis.
Methods:  Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2.
Results:  Chronic subtoxic (<10 μg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated β-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody.
Conclusions:  This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers.     

Areca nut extract upregulates vimentin by activating PI3K/AKT signaling in oral carcinoma

J Oral Pathol Med (2011) 40 : 160–166 Background: Areca nut is a group I carcinogen. Areca nut extract (ANE) is known to activate signaling pathways in oral epithelial cells. Activation of the serine/threonine protein kinase AKT/pKB (AKT) signaling pathway is known to be important during the neoplastic process. Vimentin is a mesenchymal intermediate filament and a regulator of tumor progression. This study investigated the impact of ANE on PI3K/AKT activation during vimentin expression. Materials and methods: Oral carcinoma cells were treated with ANE to explore the signaling changes underlying vimentin expression. Oral carcinoma tissues were subjected to immunohistochemical analysis to study the implications that vimentin expression has on patient survival. Results: After ANE treatment, the OECM‐1 and Fadu cells developed a fibroblastoid morphology and there was an increase in vimentin expression. The treatment also induced the phosphorylation of AKT and glycogen synthase kinase 3β in OECM‐1 cells. Blockage of phosphatidylinositol 3‐kinase (PI3K)/AKT signaling attenuated vimentin expression when it was induced by ANE. However, it did not affect ANE‐mediated extracellular signal‐regulated kinase (ERK) activation or cyclooxygenase 2 (COX‐2) upregulation. Oral carcinoma tissue samples were found to have significantly higher levels of vimentin and pAKT expression than their controls. Tumors exhibiting no vimentin expression and weak AKT phosphorylation were found to be associated with better survival than groups with high levels of expression. Conclusion: Our results imply that PI3K/AKT activation and vimentin expression are important pathogenic cascades in areca‐associated oral carcinogenesis.     

槟榔提取物抑制人类口腔黏膜成纤维细胞生长的实验研究

目的通过比较正常口腔黏膜和口腔黏膜下纤维性变(OSF)组织中成纤维细胞(FB)增殖差异、检测槟榔提取物(ANE)对成纤维细胞增殖的影响,来探讨OSF的发病机理.方法对人类口腔黏膜成纤维细胞进行分离培养,然后用四唑盐(MTT)比色试验法检测OSF患者和正常人口腔黏膜FB增殖状况,并且观察ANE对FB增殖的影响.结果表示增殖水平的OD值在OSF-FB为0.254±0.045,高于NM-FB的OD值0.236±0.012(P<0.05),ANE以浓度-效应依赖关系抑制FB增殖.结论 OSF-FB细胞增殖率较NM-FB高;ANE对口腔黏膜FB有细胞毒作用,提示槟榔及其有效成分不完全是通过直接刺激FB增殖而诱发OSF.     

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。     

Development of an in vivo mouse model to study oral submucous fibrosis

BACKGROUND: Epidemiological data have shown an association of areca nut chewing with oral submucous fibrosis (OSF). Experimental evidence to confirm this has been limited. Fibrosis-promoting activity of areca nut was tested in an animal model. METHOD: Buccal mucosa of a group of 20 female BALB/c strain mice, 10-12 weeks of age, was treated twice daily 6 days per week with topical application of aqueous areca nut extracts for 300-600 days. A control group (n = 20) was treated with 50 mM NaCl. The influence of areca nut on the oral epithelium and connective tissue was recorded semiquantitatively by light microscopy. RESULTS: The areca nut-treated oral epithelium showed progressive changes in epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of connective tissue, focal infiltration of inflammatory cells and muscle atrophy. On killing after 600 days of treatment, the scores on cellularity, inflammation and muscle atrophy were significantly different to the control group (P = 0.03). CONCLUSION: The study provides further evidence that areca nut contributes to the development of OSF in treated animals. The model has the potential to test synergism of areca nut with other carcinogens and any therapeutic interventions.     

槟榔提取物抑制人类口腔粘膜角朊细胞生长的实验研究

目的：建立人类口腔粘膜上皮角朊细胞体外原代培养方法：探讨口腔粘膜下纤维性变患者上皮厚度改变的机理;方法;先对人类口腔粘膜上皮角朊细胞进行分离,培养和鉴定,然后用四唑盐比色试验检测ＯＳＦ患者和正常人口腔粘膜上皮ＫＣ增殖状况,并且观察槟榔提取物对ＫＣ增殖的影响。     

Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis

Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.     

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.     

槟榔提取物对内皮细胞分泌一氧化氮的影响

目的 :探讨口腔黏膜下纤维性变 (OSF)可能的发病机理。方法 :用槟榔提取物干预体外培养的内皮细胞 ,收集培养上清 ,用ELISA方法检测内皮细胞分泌一氧化氮 (NO)的浓度。结果 :槟榔提取物能抑制体外培养的内皮细胞分泌NO。结论 :OSF的发生可能与槟榔引起的内皮细胞合成NO减少有关。     

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.     

Molecular and cellular cues of diet‐associated oral carcinogenesis—with an emphasis on areca‐nut‐induced oral cancer development

In modern times, potent dietary carcinogens are key contributors for neoplastic development. For oral squamous cell carcinoma (OSCC), one of the leading cancer types in developing countries, main oncogenic inducers/enhancers, including areca nut chewing, tobacco smoking, and alcohol consumption, were shown to promote cancer initiation/progression. Over decades, studies from different laboratories have identified underlying cellular and molecular mechanisms for carcinogen‐induced OSCC. In this review, we will give an overview of where we are in understanding potential oral carcinogenic factors stimulated OSCC tumorigenesis, especially those associated with areca nut chewing in Asians, aiming to provide future scope of possible interception.     

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.     

Establishment of OC3 oral carcinoma cell line and identification of NF-κB activation responses to areca nut extract

BACKGROUND: Cell lines derived from oral squamous cell carcinoma (OSCC) exposed to variable etiological factors can bestow advantages in understanding the molecular and cellular alterations pertaining to environmental impacts. Most OSCC cell lines have been established from smoker patients or areca chewing/smoker patients, carrying the genomic alterations in p53. METHODS: A new cell line, oral carcinoma 3 (OC3), was established from an OSCC in a long-term areca (betel) chewer who does not smoke. Cellular and molecular features of OC3 were determined by variable assays. RESULTS: The cultured monolayer cells were mainly polygonal and had the expression of cytokeratin 14. The chromosomal analysis using comparative genomic hybridization has revealed the gain in chromosomes 1q, 5q, and 8q, the loss in 4q, 6p, and 8p as well as the gain of entire chromosome 20. Loss of heterozygosity and instability in multiple microsatellite markers in chromosome 4q were also noted. OC3 cells bear wild-type p53 coding sequence and have a high level of p53 expression. Its p21 expression was similar to that in normal human oral keratinocyte (NHOK). Interestingly, activation of nuclear factor kappa B (NF-kappa B) in OC3 cells following the treatment of areca nut extract was observed. CONCLUSION: OC3 cell line could be valuable in understanding the genetic impairments and phenotypic changes associated with areca in oral keratinocyte.     

槟榔提取物刺激口腔黏膜角朊细胞培养上清对成纤维细胞增殖活性的影响

目的：探讨槟榔提取物刺激口腔黏膜角朊细胞在黏膜下纤维性变发病中的作用。方法：采用不同浓度槟榔提取液刺激体外培养的角朊细胞,取细胞培养上清,MTT法观察细胞培养上清对成纤维细胞增殖的影响。结果：一定浓度槟榔提取液刺激的角朊细胞培养上清能促进成纤维细胞增殖;细胞培养上清对成纤维细胞增殖的促进作用存在个体差异。结论：槟榔成分可能通过改变口腔黏膜角朊细胞的活性而导致口腔黏膜下纤维性变的发生。     

Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis

Background

In humans, areca nut induces oral submucous fibrosis (OSMF), a potentially malignant disorder, characterised by the deposition of collagen in the lamina propria. This study aimed to determine whether OSMF-like characteristics develop in a rabbit model following treatment with areca nut and lime.

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.     

Molecular pathways regulated by areca nut in the etiopathogenesis of oral submucous fibrosis

Many oral mucosal lesions are due to substance abuse, such as tobacco and areca nut, amongst others. There is considerable evidence that oral lesions/disorders such as some leukoplakias, most erythroplakias, and submucous fibrosis have malignant potential, with a conversion rate of 5%‐10% over a 10‐year period. There have been several reports on possible biomarkers that predict malignant conversion of the oral lesions associated with these disorders. Management of these is mostly surgical removal of the lesion followed by observation, and in some cases treatment by antioxidants and anti‐inflammatory agents. Oral submucous fibrosis is due to excessive deposition of extracellular matrix in the connective tissue plus, particularly, collagens. The deposition of collagen leads to stiffness of the affected regions and results in difficulty in mouth opening. Areca nut chewing is proposed as the most probable etiological factor in the manifestation of oral submucous fibrosis. Several studies suggest involvement of proinflammatory cytokines, dysregulated by areca nut, in the development of the disease. Amongst these, transforming growth factor‐β is in the forefront, which is also shown to be involved in fibrosis of other organs. This review addresses the molecular mechanisms involved in oral submucous fibrosis development and provides a model for the regulation of transforming growth factor‐β by areca nut. It provides an exemplar of the role of modern molecular techniques in the study of oral disease.