Areca Nut Alkaloid Research Articles

Oxidative Stress, Oxidative Damage, and Cell Apoptosis: Toxicity Induced by Arecoline in Caenorhabditis elegans and Screening of Mitigating Agents.

As the areca nut market is expanding, there is a growing concern regarding areca nut toxicity. Areca nut alkaloids are the major risky components in betel nuts, and their toxic effects are not fully understood. Here, we investigated the parental and transgenerational toxicity of varied doses of areca nut alkaloids in Caenorhabditis elegans. The results showed that the minimal effective concentration of arecoline is 0.2-0.4 mM. First, arecoline exhibited transgenerational toxicity on the worms' longevity, oviposition, and reproduction. Second, the redox homeostasis of C. elegans was markedly altered under exposure to 0.2-0.4 mM arecoline. The mitochondrial membrane potential was thereafter impaired, which was also associated with the induction of apoptosis. Moreover, antioxidant treatments such as lycopene could significantly ameliorate the toxic effects caused by arecoline. In conclusion, arecoline enhances the ROS levels, inducing neurotoxicity, developmental toxicity, and reproductive toxicity in C. elegans through dysregulated oxidative stress, cell apoptosis, and DNA damage-related gene expression. Therefore, the drug-induced production of reactive oxygen species (ROS) may be crucial for its toxic effects, which could be mitigated by antioxidants.

Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged −7.09–11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Oxidative Stress and Endoplasmic Reticulum Stress Contributes to Arecoline and Its Secondary Metabolites-Induced Dyskinesia in Zebrafish Embryos.

Areca nut has been listed as one of the most addictive substances, along with tobacco, alcohol and caffeine. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on embryonic development and motor behavior are rarely addressed in zebrafish embryo-larvae. Herein, we investigated the effects of exposure to three alkaloids (arecoline and secondary metabolites-arecaidine and arecoline N-oxide) on the developmental parameters, locomotive behavior, oxidative stress and transcriptome of zebrafish embryos. Zebrafish embryos exposed to different concentrations (0, 0.1, 1, 10, 100 and 1000 μM) of arecoline, arecaidine and arecoline N-oxide showed no changes in mortality and hatchability rates, but the malformation rate of zebrafish larvae was significantly increased in a dose-dependent manner and accompanied by changes in body length. Moreover, the swimming activity of zebrafish larvae decreased, which may be due to the increase in reactive oxygen species and the imbalance between oxidation and antioxidation. Meanwhile, transcriptome analysis showed that endoplasmic reticulum stress and the apoptosis p53 signaling pathway were significantly enriched after exposure to arecoline and arecoline N-oxide. However, arecaidine exposure focuses on protein synthesis and transport. These findings provide an important reference for risk assessment and early warning of areca nut alkaloid exposure.

Carcinogenic Effects of Areca Nut and Its Metabolites: A Review of the Experimental Evidence.

Oral cancers (OC) are among the most frequent malignancies encountered in Southeast Asia, primarily due to the prevalent habit of betel quid (BQ) and smokeless tobacco use in this region. Areca nut (AN), the primary ingredient in BQ, contains several alkaloids, including arecoline, arecaidine, guvacoline, and guvacine. These have been associated with both the AN abuse liability and carcinogenicity. Additionally, variations in AN alkaloid levels could lead to differences in the addictiveness and carcinogenic potential across various AN-containing products. Recent studies based on animal models and in vitro experiments show cellular and molecular effects induced by AN. These comprise promoting epithelial-mesenchymal transition, autophagy initiation, tissue hypoxia, genotoxicity, cytotoxicity, and cell death. Further, clinical research endorses these undesired harmful effects in humans. Oral submucosal fibrosis, a potentially malignant disease of the oral cavity, is predominantly reported from the geographical areas of the globe where AN is habitually chewed. OC in chronic AN users presents a more aggressive phenotype, such as resistance to anti-cancer drugs. The available evidence on the carcinogenicity of AN based on the findings reported in the recently published experimental studies is discussed in the present review.

Comparison of the psychoactive activity of four primary Areca nut alkaloids in zebrafish by behavioral approach and molecular docking

Areca palm nut (Areca catechu) has been listed as one of the most addictive substances, along with tobacco, alcohol, and caffeine. It belongs to the family Arecaceae and is widely used in Asia. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on behaviors are rarely to be addressed in zebrafish. Therefore, this study aims to compare the psychoactive and potential adverse effects of four primary alkaloids (arecoline, arecaidine, guvacine, and guvacoline) isolated from areca nut on zebrafish. We found that four alkaloids induced hyperactivity-like behaviors in zebrafish larvae. Cooperating the results with the previous study, molecular docking scores suggested these alkaloids might bind to multiple muscarinic acetylcholine receptors (mAChRs), and various best binding modes were shown. According to the adult zebrafish behavioral test, arecoline was found to slightly increase the locomotor activity and caused tightening shoaling formations of adult zebrafish. Meanwhile, zebrafish exposed to arecaidine have reduced aggressiveness and conspecific social interaction. Similar to arecaidine, guvacoline treatment also caused abnormalities in zebrafish social behaviors. Furthermore, the fish displayed abnormal exploratory behaviors after being exposed to guvacoline. Interestingly, altered fear response behaviors were only displayed by guvacine-treated fish besides their lower locomotor activity. Based on the results of molecular docking, we hypothesize that the behavior alterations might be a consequence of the interaction between alkaloids and multiple mAChRs in the nervous system. In summary, our study found that each alkaloid specifically affects adult zebrafish behaviors.

Potential health risk of areca nut consumption: Hazardous effect of toxic alkaloids and aflatoxins on human digestive system

Contemporarily, there has been a growing consumption rate of areca nut (AN) products worldwide, despite the fact that both fresh and processed AN contain various hazardous ingredients, including toxic alkaloids and carcinogenetic aflatoxins. However, there is a dearth of toxicity and potential cancer risk information regarding toxic alkaloids and aflatoxins via consuming AN products. The present study conducted a comprehensive assessment of the combined hazardous effects of AN alkaloids and aflatoxins towards human digestive system, by methods of HPLC analysis, cell study and in vitro digestive system study. The results revealed a synergetic effect of arecoline and aflatoxins was on human gingival normal fibroblast cell of HGF-1 and a proliferation effect on human tongue squamous carcinoma cell of CAL-27. Specifically, the residual arecoline was as high as 91.08 μg·ml−1 in oral phase and 72.41 μg·ml−1 in gastric phase, which could be an evidence of oral cancer. More importantly, 25.93 % of AN products were contaminated with aflatoxins and the maximum value was three times the MRLs. Under these circumstances, the cytotoxic and MOE values raised a considerable health concern in terms of malignancy risk for children that consume processed AN product, especially compared to scenarios that involve adults and/or fresh AN samples. This study would give rise to a better understanding of the hazards associated with AN alkaloids and aflatoxins towards digestive system, and thus to predict the potential carcinogenic risk of AN products.

The molecular mechanisms underlying arecoline-induced cardiac fibrosis in rats.

The areca nut is one of the most commonly consumed psychoactive substances worldwide, with an estimated consumption by approximately 10% of the world’s population, especially in some regions of South Asia, East Africa, and the tropical Pacific. Arecoline, the major areca nut alkaloid, has been classified as carcinogenic to humans as it adversely affects various organs, including the brain, heart, lungs, gastrointestinal tract, and reproductive organs. Earlier studies have established a link between areca nut chewing and cardiac arrhythmias, and yet research pertaining to the mechanisms underlying cardiotoxicity caused by arecoline is still preliminary. The main purpose of this study is to test the hypothesis that arecoline causes cardiac fibrosis through transforming growth factor-β (TGF-β)/Smad-mediated signaling pathways. Male Wistar rats were injected intraperitoneally with low (5 mg/kg/day) or high (50 mg/kg/day) doses of arecoline for 3 weeks. Results from Masson’s trichrome staining indicated that arecoline could induce cardiac fibrosis through collagen accumulation. Western blot analysis showed that TGF-β and p-Smad2/3 protein expression levels were markedly higher in the arecoline-injected rat hearts than in those of the control rats. Moreover, arecoline upregulated other fibrotic-related proteins, including SP1-mediated connective tissue growth factor expression. Tissue-type plasminogen activator and its inhibitor, plasminogen activator inhibitor, and matrix metalloproteinase (MMP) 9 were upregulated, and the inhibitor of MMP9 was downregulated. This study provides novel insight into the molecular mechanisms underlying arecoline-induced cardiac fibrosis. Taken together, the areca nut is a harmful substance, and the detrimental effects of arecoline on the heart are similar to that caused by oral submucous fibrosis.

Rapid green analytical methodology for simultaneous biomonitoring of five toxic areca nut alkaloids using UHPLC-MS/MS for predicting health hazardous risks

Areca nut (AN) is a fundamental component of betel quid (BQ), an addictive and carcinogenic mixture chewed by hundreds of millions of people in India-Asia-Pacific. Chewing of BQ is associated with oral cancers due to specific carcinogenic alkaloids (arecaidine, guvacine, guvacoline, arecoline, N-Nitrosoguvacoline) in AN. To predict the hazardous health risks of short and long-term chewing of BQ, it is crucial to identify five toxic AN alkaloids in saliva and urine of BQ chewers. This study reports a green analytical methodology comprising in-syringe assisted vortex-induced salt-enhanced liquid-liquid microextraction coupled with ultra-HPLC-MS/MS for simultaneous biomonitoring of five AN alkaloids in saliva and urine. The analytical method validation results exhibited good linearities between 0.05 and 1000 ng mL−1 with r2 > 0.9930. The detection and quantification limits were between 0.01 and 1.5 and 0.05–5 ng mL−1. Relative recoveries ranged between 87.9% and 110.1% with RSD < 9.1% for saliva samples, 81.5–115.1% with RSD < 9.7% for urine samples. The results indicated the successful identification and real-time monitoring of concentrations of five target AN alkaloids in saliva and urine of BQ chewers and demonstrated the utility of this technique as an efficient analytical protocol for routine biomonitoring of levels of toxic AN alkaloids from BQ chewers and to predict the exposure level and its harmful health risk.

LINC00084/miR-204/ZEB1 Axis Mediates Myofibroblastic Differentiation Activity in Fibrotic Buccal Mucosa Fibroblasts: Therapeutic Target for Oral Submucous Fibrosis.

Oral submucosal fibrosis (OSF) is a precancerous condition in the oral cavity and areca nut consumption has been regarded as one of the etiologic factors implicated in the development of OSF via persistent activation of buccal mucosal fibroblasts (BMFs). It has been previously reported that an epithelial to mesenchymal transition (EMT) factor, ZEB1, mediated the areca nut-associated myofibroblast transdifferentiation. In the current study, we aimed to elucidate how areca nut affected non-coding RNAs and the subsequent myofibroblast activation via ZEB1. We found that long non-coding RNA LINC00084 was elicited in the BMFs treated with arecoline, a major alkaloid of areca nut, and silencing LINC00084 prevented the arecoline-induced activities (such as collagen gel contraction, migration, and wound healing capacities). The upregulation of LINC00084 was also observed in the OSF tissues and fibrotic BMFs (fBMFs), and positively correlated with several fibrosis factors. Moreover, we showed knockdown of LINC00084 markedly suppressed the myofibroblast features in fBMFs, including myofibroblast phenotypes and marker expression. The results from the luciferase reporter assay confirmed that LINC00084 acted as a sponge of miR-204 and miR-204 inhibited ZEB1 by directly interacting with it. Altogether, these findings suggested that the constant irritation of arecoline may result in upregulation of LINC00084 in BMFs, which increased the ZEB1 expression by sequestering miR-204 to induce myofibroblast transdifferentiation.

Arecoline induces epithelial-mesenchymal transformation and promotes metastasis of oral cancer by SAA1 expression.

Arecoline, the main alkaloid of areca nut, is well known for its role in inducing submucosal fibrosis and oral squamous cell carcinoma (OSCC), however the mechanism remains unclear. The aim of this study was to establish an arecoline‐induced epithelial‐mesenchymal transformation (EMT) model of OSCC cells and to investigate the underlying mechanisms. CAL33 and UM2 cells were induced with arecoline to establish an EMT cell model and perform RNA‐sequence screening. Luminex multiplex cytokine assays, western blot, and RT‐qPCR were used to investigate the EMT mechanism. Arecoline at a concentration of 160 μg/ml was used to induce EMT in OSCC cells, which was confirmed using morphological analysis, transwell assays, and EMT marker detection. RNA‐sequence screening and Luminex multiplex cytokine assays showed that many inflammatory cytokines (such as serum amyloid A1 [SAA1], interleukin [IL]‐6, IL‐36G, chemokine [CCL]2, and CCL20) were significantly altered during arecoline‐induced EMT. Of these cytokines, SAA1 was the most highly upregulated. SAA1 overexpression induced EMT and promoted the migration and invasion of CAL33 cells, while SAA1 knockdown attenuated arecoline‐induced EMT. Moreover, arecoline enhanced cervical lymph node metastasis in an orthotopic xenograft model of the tongue established using BALB/c nude mice. Our findings revealed that arecoline induced EMT and enhanced the metastatic capability of OSCC by the regulation of inflammatory cytokine secretion, especially that of SAA1. Our study provides a basis for understanding the mechanism of OSCC metastasis and suggests possible therapeutic targets to prevent the occurrence and development of OSCC associated with areca nut chewing.

Serum aflatoxin B1 antibody titer, percent hemolysis and transaminases in oral submucous fibrosis.

Introduction:Areca nut is deeply rooted sociocultural habit in India. Areca nut reported to be infested by fungi during the field and storage conditions. Areca nut alkaloids, nitrosamines, tobacco and aflatoxin are cytotoxic, immunotoxic to red blood cell and epithelial cell. Hence, the present study was conducted to assess the serum aflatoxin B1 (AFB1) antibody titer, percent hemolysis and transaminases in oral submucous fibrosis (OSMF) patients.Materials and Methods:In this study, 128 participants of which 88 were suffering from OSMF. Twenty participants were areca nut habitual without OSMF (habitual control) and 20 participants without any habit (healthy control). For the detection of AFB1 antibody titer, AFB1 antigen (Sigma) A6636 from Aspergillus flavus was used. Percent hemolysis was estimated as per the procedure described by Mathuria and Verma. Serum Glutamic oxalo acetic transaminase (SGOT) and Serum Glutamic pyruvic transaminase (SGPT) were estimated by the optimized ultraviolet method using the enzyme-linked immunosorbent assay kit.Results:Mean SGOT, SGPT, percent hemolysis and AFB1 antibody titer were significantly higher in participants with OSMF than the habitual and healthy controls. AFB1 antibody titer and % hemolysis in both OSMF and habitual without OSMF were showed significant correlation, i.e., increased AFB1 antibody titer with increased % hemolysis.Conclusions:A study result demonstrates that aflatoxin causes increase in serum transaminases which is indicative of liver damage in OSMF. The combined toxic effects of areca nut alkaloids, tobacco and AFB1 on red blood cell (RBC) cell wall might be responsible for increased percent hemolysis in OSMF and habitual control.

Genetic toxicology and toxicokinetics of arecoline and related areca nut compounds: an updated review.

Areca nut (AN) is consumed by more than 600 million of individuals, particularly in some regions of South Asia, East Africa, and tropical Pacific, being classified as carcinogenic to humans. The most popular way of exposure consists of chewing a mixture of AN with betel leaf, slaked lime, and other ingredients that may also contain tobacco named betel quid (BQ). Arecoline is the principal active compound of AN, and, therefore, has been systematically studied over the years in several in vitro and in vivo genotoxicity endpoints. However, much of this information is dispersed, justifying the interest of an updated and comprehensive review article on this topic. In this sense, it is thus pertinent to describe and integrate the genetic toxicology data available as well as to address key toxicokinetics aspects of arecoline. This review also provides information on the effects induced by arecoline metabolites and related compounds, including other major AN alkaloids and nitrosation derivatives. The complexity of the chemicals involved renders this issue a challenge in genetic toxicology. Overall, positive results in several endpoints have been reported, some of them suggesting a key role for arecoline metabolites. Nevertheless, some negative genotoxicity findings for this alkaloid in short-term assays have also been reported in the literature. Finally, this article also collates information on the potential mechanisms of arecoline-induced genotoxicity, and suggests further approaches to tackle this important toxicological issue.

Effects of melatonin to arecoline-induced reactive oxygen species production and DNA damage in oral squamous cell carcinoma

Arecoline, the major alkaloid of areca nut, is known to induce reactive oxygen species (ROS) and DNA damage during oral cancer progression. This study aim to evaluate whether melatonin, an antioxidant, supported or repressed the arecoline-induced carcinogenesis phenotypes in oral squamous cell carcinoma (OSCC). The cytotoxicity of arecoline or melatonin treatment alone and their co-treatment in the OSCC cell line OEC-M1 were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle, cell death, and total ROS production were analyzed using flow cytometer. The protein expression was determined using western blot analysis. The genotoxicity and mutation rate were determined using micronucleus assay and hypoxanthine phosphoribosyl transferase (HPRT) forward mutation assay, respectively, in CHO-K1 cells. The ataxia telangiectasia mutated (ATM) promoter activity and DNA repair ability were determined through reporter assay. The result showed that both the arecoline and melatonin induced ROS production and antioxidant enzymes expression. Melatonin treatment enhanced arecoline-induced ROS production, cytotoxicity, G2/M phase arrest, and cell apoptosis in OSCC cells. On the other hand, melatonin treatment activated DNA repair activity to reverse arecoline-induced DNA damage and mutation. These results indicated that melatonin is a potential chemopreventive agent for betel quid chewers to prevent OSCC initiation and progression.

Positive Feedback Loop of SNAIL-IL-6 Mediates Myofibroblastic Differentiation Activity in Buccual Mucosa Fibroblasts: Therapeutic Target for Oral Submucous Fibrosis

Background: Oral submucosal fibrosis is a premalignant disorder of the oral cavity, and areca nut chewing habit has been known as a major etiologic factor. Various studies have revealed that areca nut induced epithelial to mesenchymal transition (EMT) and activation of buccal mucosal fibroblasts (BMFs), but the detailed mechanism has not been fully understood. Methods: The expression of Snail was assessed in the OSF tissues and BMFs derived from OSF specimens (fBMFs). We examined the functional role of Snail in myofibroblast activation, and used the bioinformatics software to predict the interacting factor of Snail and studied their impact on myofibroblast activities. Findings: The upregulation of Snail in OSF samples and fBMFs may result from constant irritation of arecoline, a major alkaloid of areca nut. The elevation of Snail triggered myofibroblast activation and was crucial to the transdifferentiation of BMFs as well as the persistent activation of fBMFs. Meanwhile, Snail increased the expression of numerous fibrosis factors (e.g. α-SMA and collagen I) as well as IL-6. Results from bioinformatics software and luciferase-based reporter assay revealed that IL-6 was a direct target of Snail. Moreover, IL-6 in BMFs was found to further increase the expression of Snail and contribute to the Snail-induced myofibroblast activation. Interpretation: Our results suggested that there was a positive loop between Snail and IL-6 to regulate the areca nut-associated myofibroblast transdifferentiation. These findings implied that blockage of Snail may serve as a favourable therapeutic strategy for OSF treatment. Funding Statement: This work was supported by grants from the Chung Shan Medical University Hospital (grant number CSH-2018-C-013); the China Medical University (grant numbers CMU108-N-10, CMU108-MF-06); and the Ministry of Science and Technology (grant number MOST108-2314-B-040-001, MOST107-2314-B-040 -023) in Taiwan. Declaration of Interests: The authors declare that there are no conflicts of interest. Ethics Approval Statement: All procedures performed in this study involving human participants were in accordance with the tenets of the Declaration of Helsinki and reviewed by the Institutional Review Committee at Chung Shan Medical University, Taichung, Taiwan.

Chemical Markers for Short- and Long-Term Areca Nut Exposure

Background: Areca nut (AN) chewing causes oral cancer. AN cessation programs are the most effective approach to reduce AN chewing induced cancers but require biomarkers to determine program compliance and success. Objectives: To explore chemical markers for short- and long-term AN exposure using non-invasively collected saliva, buccal cells (BCs), and scalp hair of chewers. Methods: Saliva was collected from a male chewer before and up to 2 days after AN chewing. Saliva was separated into supernatant and pellet (BCs) then analyzed by spectrophotometry and liquid chromatography (LC) with UV/VIS detection. Scalp hair was collected from four chewers and analyzed for areca alkaloids using direct analysis in real time-tandem mass spectrometry (DART-MSMS). Results: The red pigmented saliva after chewing showed no valuable signals when either the saliva supernatant or pellet (BCs) were analyzed by spectrophotometry. Saliva analysis by LC–UV/VIS showed diagnostically valuable signals at 488 nm up to 5 and 24 h post chewing in the supernatant and pellet, respectively. DART-MSMS analysis detected two of the four AN specific alkaloids (arecoline and arecaidine) in male but none in female hair. Conclusions/Importance: LC–UV/VIS analysis of the red pigments extracted from saliva and BCs after AN chewing showed distinct signals up to 24 h post chewing while DART-MSMS analysis in BCs and scalp hair showed selective signals of AN alkaloids for several weeks or months after AN exposure. Chemical hair treatment might prevent detection of areca alkaloids in hair. AN cessation trials and other programs now have essential tools for bioverification.

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.

MiR-200c inhibits the arecoline-associated myofibroblastic transdifferentiation in buccal mucosal fibroblasts.

MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target. qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1. Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA. These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1.

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.

Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc and miR-22, directly targeting oncostatin M.

Arecoline, the major alkaloid of areca nut, is known to induce oral carcinogenesis, however, its mechanism is still needed to elucidate. This study investigated the effects of arecoline on cell viability and cell-cycle progression of oral squamous cell carcinoma (OSCC) cells as well as a relevant cellular gene expression. The results showed that a low concentration of arecoline (0.025 μg/ml) increased OSCC cell viability, proportion of cells in G2/M phase and cell proliferation. Simultaneously, it induced IL-6, STAT3 and c-Myc expression. Interestingly, c-myc promoter activity was also induced by arecoline. MiR-22 expression in arecoline-treated OSCC cells was suppressed and comparable to an upregulated c-Myc expression. In arecoline-treated OSCC cells, oncostatin M (OSM) expression was significantly upregulated and inversely correlated with miR-22 expression. Likewise, OSM expression and its post-transcriptional activity were significantly decreased in miR-22-transfected OSCC and 293FT cells. This result demonstrated that miR-22 directly targeted OSM. Interestingly, miR-22 played an important role as a tumor suppresser on suppressing cell proliferation, migration and cell-cycle progression of OSCC cells. This result suggested the effect of arecoline to promote cell proliferation and cell-cycle progression of OSCC cells might be involved in induction of c-Myc expression and reduction of miR-22 resulting in OSM upregulation.

Perspectives on areca nut with some global implications: Symposium report

Areca nut and products made from the nut, evaluated by the International Agency for Research on Cancer as carcinogenic to humans since 2004, are being increasingly used in India and South East Asia and exported to many countries. There is thus an urgent need to identify best control policies and research gaps. On 1 February 2018, a group of experts shared their latest knowledge and perspectives on areca nut at Healis-Seksaria Institute for Public Health in Navi Mumbai, Maharashtra, India. By itself, areca nut has been implicated in oral and oesophageal cancer. Areca nut users are also at high risk of oral submucous fibrosis, a debilitating and potentially malignant condition. Users may also suffer adverse health outcomes due to smokeless tobacco, commonly mixed with areca nut. The use of areca nut commonly begins in childhood and often proceeds to use with tobacco. Gutka, a widely consumed, industrially made areca nut product containing tobacco, has been banned in all states and union territories of India, but it is still widely available. Areca nuts contain arecoline, an alkaloid that stimulates various brain receptors, promoting physical dependence. Areca nut alkaloids have also been implicated in carcinogenesis. Other chemicals, introduced during cultivation and processing of areca nuts, contribute to oral submucous fibrosis and cancer. The wide variety of products, different storage conditions and lack of regulation can lead to significant variation in the content of toxicants, carcinogens and alkaloids, making the psychological and physical dependence issues even more challenging. Regulation of product contents is needed as a part of control measures to supplement product bans.