Activation of TGF-β Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.     

Slug mediates myofibroblastic differentiation to promote fibrogenesis in buccal mucosa

Epithelial–mesenchymal transition (EMT) has been implicated in fibrogenesis and carcinogenesis; however, the exact role of EMT-inducer Slug in the progression of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we showed that the expression of Slug was upregulated in OSF tissues and associated with various myofibroblast markers. After silence of Slug in fibrotic buccal mucosal fibroblasts (fBMFs), the elevated myofibroblast activities and fibrosis markers were all downregulated. Our data revealed that arecoline, an areca nut alkaloid, increased the expression of Slug in normal BMFs, and inhibition of Slug successfully prevented the arecoline-induced myofibroblast activation. Additionally, overexpression of Slug in BMFs stimulated the activities of myofibroblasts, indicating that upregulation of Slug by arecoline contributes to the myofibroblast transdifferentiation. Most importantly, Slug was able to bind to the E-box of type I collagen, leading to increased expression of type I collagen. Altogether, this study demonstrated the abnormal elevation of Slug in OSF and its significance in arecoline-induced fibrogenesis. Moreover, downregulation of Slug could be a potential target for OSF remedy via suppression of myofibroblast activities and type I collagen.     

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro‐proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro‐proliferative effects of ANW on fibroblasts are dependent on insulin‐like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC₅₀ of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki‐67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.     

Arecoline N-oxide regulates oral squamous cell carcinoma development through NOTCH1 and FAT1 expressions

Areca nut has been evaluated as a group I carcinogen to humans. However, the exact compounds of areca nut causing oral cancer remain unproven. Previous findings from our lab revealed that arecoline N-oxide (ANO), a metabolite of arecoline, exhibits an oral fibrotic effect in immune-deficient NOD/SCID mice. The aim of this study is to investigate the oral potentially malignant disorders (OPMD) inductive activity between areca-alkaloid arecoline and its metabolite ANO in C57BL/6 mice. Our findings show that ANO showed higher activity in inducing hyperplasia with leukoplakia and collagen deposition in C57BL/6 mice compared with the arecoline treated groups. Importantly, immunohistochemical studies showed significant upregulation of NOTCH1, HES1, FAT1, PCNA, and Ki67 expressions in the pathological hyperplastic part. In addition, in vitro studies showed that upregulation of NOTCH1 and FAT1 expressions in ANO treated HGF-1 and DOK cell models. We found that NOTCH1 regulates TP53 expression from NOTCH1 knockdown oral cancer cells. The DNA damage was significantly increased after arecoline and ANO treatment. Further, we found that arecoline-induced H2AX expression was regulated by FMO3. Altogether, our findings show that ANO exhibited higher toxicity in OPMD activity and play a significant role in the induction of areca nut mediated oral tumorigenesis.     

Angiotensin (1–7) inhibits arecoline-induced migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome activation

Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1–7) (Ang-(1–7)) protects against fibrosis by counteracting angiotensin II (Ang-II) via the Mas receptor. However, the effects of Ang-(1–7) on OSF remain unknown. NOD-like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang-(1–7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang-II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang-(1–7) improved arecoline-induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin-converting enzyme (ACE)/Ang-II/AT1R axis and NLRP3 inflammasome/interleukin-1β axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA-ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase-1 blocker VX-765. Ang-(1–7) shifted the balance of RAS toward the ACE2/Ang-(1–7)/Mas axis, inhibited arecoline-induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang-(1–7) attenuates arecoline-induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts.     

miR‐200b ameliorates myofibroblast transdifferentiation in precancerous oral submucous fibrosis through targeting ZEB2

Oral submucous fibrosis (OSF) is a progressive scarring disease. MicroRNA‐200b (miR‐200b) has been reported as a tumour suppressor, but its role in the precancerous OSF remains unknown. In this study, we investigated the impact of miR‐200b on myofibroblastic differentiation activity. Arecoline is a major areca nut alkaloid and has been employed to induce the elevated myofibroblast activity in human buccal mucosal fibroblasts (BMFs). Treatment of arecoline in BMFs dose‐dependently reduced gene expression of miR‐200b, which corresponded with the decreased expression of miR‐200b in fBMFs. The arecoline‐induced myofibroblast activities were abolished by overexpression of miR‐200b in BMFs, and the same results were observed in fBMFs. In addition, α‐SMA was inhibited by an increase in miR‐200b. We further demonstrated that miR‐200b‐mediated decrease in ZEB2 led to down‐regulation of α‐SMA, vimentin. Loss of miR‐200b resulted in enhanced collagen contraction and migration capabilities, and knockdown of ZEB2 reversed these phenomena. Lastly, we showed the expression of miR‐200b was significantly less and ZEB2 was markedly higher in OSF tissues. These results suggested that down‐regulation of miR‐200b may contribute to the pathogenesis of areca quid‐associated OSF through the regulation of ZEB2 and myofibroblast hallmarks.     

Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells

Background  

Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells.     

Betel nut extract and arecoline block insulin signaling and lipid storage in 3T3-L1 adipocytes

According to several population-based studies, betel nut chewing is associated with metabolic syndrome and diabetes in British South Asians and Taiwanese. However, the underlying molecular mechanism is not yet clear. Arecoline is an alkaloid-type natural product found in betel nuts. Our aim was to clarify the influence of betel nut extract and arecoline on lipid accumulation and insulin signaling in adipocytes. We found that betel nut extract and arecoline blocked lipid storage in 3T3-L1 adipocytes. The possible mechanism may function by inhibiting the expression of the insulin receptor, glucose transporter-4, fatty acid synthase, and the lipid droplet proteins perilipin and adipophilin. In addition, betel nut extract and arecoline increased the basal level of IRS-1 serine³⁰⁷ phosphorylation and decreased insulin-stimulated IRS-1 tyrosine, Akt, and PI3 kinase phosphorylation. In conclusion, betel nut extract and arecoline have diabetogenic potential on adipocytes that may result in insulin resistance and diabetes at least in part via the obstruction of insulin signaling and the blockage of lipid storage.     

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.     

Arecoline induces TNF-alpha production and Zonula Occludens-1 redistribution in mouse Sertoli TM4 cells

Background

Arecoline, a major alkaloid in Areca nut has the ability to induce oxidative stress. The effect of Areca nut, arecoline on reducing sperm quality and quantity were documented previously using several animal models. Junction disruption by down-regulation of the junction-adhesive protein via oxidative stress is an important route mediating abnormal spermatogenesis. Therefore, in this present study, we investigated the functional role of arecoline on junctional proteins.

Results

To analyze direct effects of arecoline on testis cells, confluent mouse testicular Sertoli cell line TM4 was exposed to arecoline. Arecoline decreased insoluble zonula occludens-1 (ZO-1) protein expression in TM4 cells, however, arecoline treatment increased TNF-alpha production in both TM4 and monocytic THP1 cells. In addition, ERK1/2 inhibitor PD98059 reversed arecoline effects on TNF-alpha and ZO-1.

Conclusions

Arecoline increases the production of TNF-alpha and induces protein redistribution of ZO-1. All these results explain the role of arecoline in male reproductive dysfunction, besides its cytotoxic induction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0093-z) contains supplementary material, which is available to authorized users.     

Nicotinic Activity of Arecoline,the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity

Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6–10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline''s activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts.     

Autophagy induction by a natural ingredient of areca nut

We recently identified an autophagy-inducing areca nut ingredient (AIAI) in the partially purified 30-100 kDa fraction of areca nut extract (ANE), designated as ANE 30-100K. Before disintegration, most ANE 30-100K-treated cells exhibit rounding morphology, cytoplasmic clearance, and nuclear shrinkage, distinct from arecoline- and cisplatin-induced cellular apoptosis. This unique death pattern is verified to be autophagy by LC3-I cleavage, acidic vesicles and autophagic vacuoles. As analyzed by Molish’s Test, Selinowaff’s Test, and thin-layer chromatography, most of the ANE 30-100K constituents are carbohydrates, whereas the protein content of this fraction is less than 1% as assessed by Protein Assay reagent. The cytotoxicity of ANE 30-100K is further shown to be sensitive to cellulase and proteinase K digestion suggesting AIAI in ANE 30-100K to be a proteoglycan (or glycoprotein). Thus, although ANE contains apoptosis-inducing ingredients such as arecoline, it predominantly triggers autophagic cell death by this natural AIAI.

Addendum to: Liu SY, Lin MH, Hsu YR, Shih YY, Chiang WF, Lee CH, Chou TH, Liu YC. Arecoline and the 30-100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: A pilot study. J Biomed Sci 2008; doi:10.1007/s11373-008-9273-8.     

Areca (betel) nut chewing practices of adults and health behaviors of their children in the Freely Associated States,Micronesia: Findings from the Children’s Healthy Living (CHL) Program

BackgroundChewing areca (betel) nut has been deemed carcinogenic. The practice has become a public health concern in Micronesia. The Children's Healthy Living (CHL) Program included an areca (betel) nut questionnaire in a survey of household characteristics in the Freely Associated States (FAS). This paper describes areca (betel) nut chewing practices of adults and the health behaviors of their children.MethodsA cross-section of 1200 children (2–8 year-olds) and their caregivers in Chuuk, Kosrae, Pohnpei, Republic of Palau, Republic of the Marshall Islands (RMI), and Yap were recruited. Socio-demographics, adult areca (betel) nut chewing practices, and other health behaviors of children and adults were assessed. Child anthropometric measurements were collected to estimate weight status.ResultsThe FAS areca (betel) nut chewing prevalence was 42%, ranging from 3% (RMI) to 94% (Yap). Among chewers, 84% added tobacco, 97% added slaked lime, 85% added betel leaf, and 24% mixed the components with alcohol. Among FAS children, 95% practiced daily teeth-brushing and 53% visited the dentist annually. Compared to non-chewing households, areca (betel) nut chewing households were more likely to have very young children enrolled, more highly educated adults, and members that used tobacco and alcohol.ConclusionThe FAS areca (betel) nut chewing prevalence (42%) is above the world prevalence of 10–20%, with wide variability across the islands. The oral health findings in this study may inform future oral cancer prevention programs or policies. Regular monitoring of areca (betel) nut use is needed to measure the impact of such programs or policies.     

Epidemiology of areca (betel) nut use in the mariana islands: Findings from the University of Guam/University of Hawai`i cancer center partnership program

BackgroundAreca (betel) nut is considered a Group 1 human carcinogen shown to be associated with other chronic diseases in addition to cancer. This paper describes the areca (betel) nut chewing trend in Guam, and health behaviors of chewers in Guam and Saipan.MethodsThe areca (betel) nut module in the Guam Behavioral Risk Factor Surveillance Survey was used to calculate the 5-year (2011–2015) chewing trend. To assess the association between areca (betel) nut chewing and health risks in the Mariana Islands, a cross-section of 300 chewers, ≥18 years old, were recruited from households in Guam and Saipan. Self-reported socio-demographics, oral health behaviors, chronic disease status, diet, and physical activity were collected. Anthropometry was measured. Only areca (betel) nut-specific and demographic information were collected from youth chewers in the household.ResultsThe 5-year areca (betel) nut chewing prevalence in Guam was 11% and increased among Non-Chamorros, primarily other Micronesians, from 2011 (7%) to 2015 (13%). In the household survey, most adult chewers (46%) preferred areca nut with betel leaf, slaked lime, and tobacco. Most youth chewers (48%) preferred areca nut only. Common adult chronic conditions included diabetes (14%), hypertension (26%), and obesity (58%).ConclusionThe 5-year areca (betel) nut chewing prevalence in Guam is comparable to the world estimate (10–20%), though rising among Non-Chamorros. Adult and youth chewers may be at an increased risk for oral cancer. Adult chewers have an increased risk of other chronic health conditions. Cancer prevention and intervention strategies should incorporate all aspects of health.     

The induction of prostaglandin E2 production, interleukin-6 production, cell cycle arrest, and cytotoxicity in primary oral keratinocytes and KB cancer cells by areca nut ingredients is differentially regulated by MEK/ERK activation

There are about 200-600 million betel quid (BQ) chewers in the world. BQ chewing is one of the major risk factor of hepatocarcinoma, oropharyngeal, and esophagus cancers in Taiwan, India, and Southeast Asian countries. Thus, the precise molecular mechanisms deserve investigation. We used cultured primary keratinocytes and KB cells, RT-PCR, flow cytometry, Western blotting, and ELISA to evaluate whether alterations in early gene expression is crucial in the carcinogenic processes of BQ. We observed the induction of c-Fos mRNA expression in human gingival keratinocyte (GK) and KB carcinoma cells by areca nut (AN) extract and arecoline. A maximal increment in c-fos gene expression was shown at about 30 min after challenge. AN extract (100-800 microg/ml) and arecoline (0.1-0.8 mM) also stimulated ERK1/ERK2 phosphorylation with a maximal stimulation at 5-10 min of exposure. Pretreatment by U0126 (30 microM), a MEK inhibitor, markedly inhibited the c-Fos, cyclooxygenase-2 (COX-2), and IL-6 mRNA expression of the KB epithelial cells. In addition, U0126 and PD98059 (50 microM) also decreased AN extract- and arecoline-associated PGE2 and IL-6 production in GK and KB cells. However, U0126 by itself arrested the cells in G0/G1 phase, but was not able to prevent AN- and arecoline-induced cell death or apoptosis. In contrast, U0126 enhanced the AN-induced apoptosis of KB cells. AN ingredients thus play a significant role in the pathogenesis of oropharyngeal cancer by activation of MEK1/ERK/c-Fos pathway, which promotes keratinocyte inflammation, cell survival, and affects cell cycle progression.