Major Areca Nut Alkaloid Research Articles

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.

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.

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.

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.

STRO-1 confers myofibroblast transdifferentiation in fibroblasts derived from oral submucous fibrosis.

STRO-1 is a mesenchymal cell marker present on all clonogenic stromal precursors. Current evidence has indicated that the pathogenesis of fibrotic changes may be mediated by stemness properties. The aim of this study was to investigate the role of STRO-1 in areca quid chewing-associated oral submucous fibrosis (OSF). Thirty OSF specimens and ten normal buccal mucosae were examined by immunohistochemistry. The activity of STRO-1 from fibroblasts cultured from normal buccal mucosa (BMFs) and OSF (OSFFs) were measureed and the effect of arecoline, a major areca nut alkaloid, on STRO-1 in BMFs was evaluated. Compared the activities between sorted STRO-1+ cells and STRO-1- cells from OSFF were measured by collagen gel contraction, migration, invasion abilities, and the expression of α-smooth muscle actin (α-SMA) and pro-α1 (I) chain of type I collagen. Our results first showed that the expression of STRO-1 was more evident in areca quid chewing-associated OSF than normal buccal mucosa tissues (P < .05). Arecoline dose-dependently activated the level of STRO-1 in BMFs (P < .05). The relative expression of STRO-1 was significantly higher in OSFFs compared with BMFs (P < .05). In addition, the sorted STRO-1+ cells from OSFFs exhibited higher collagen gel contraction, migration, and invasion abilities as well as elevated expression of α-SMA and pro-α1 (I) chain of type I collagen than the negative subset (P < .05). These findings suggested that the stemness marker STRO-1 may be a crucial factor in the pathogenesis of areca quid chewing-associated OSF.

Arecoline activates latent transforming growth factor β1 via mitochondrial reactive oxygen species in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFβ in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFβ1 signaling and to inspect the detailed mechanisms involved. Western blot and TGFβ1 Emax® ImmunoAssay were used to measure the effect of arecoline on the TGFβ signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS). Arecoline induced latent TGFβ1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFβ-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFβ1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFβ1 activation and mitochondrial ROS in BMFs. Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFβ1 leading to the initiation of TGFβ1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF.

Regulation of hypoxia-inducible factor-1α in human buccal mucosal fibroblasts stimulated with arecoline.

Hypoxia-inducible factor (HIF)-1α is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HIF-1α expression from fibroblasts derived from human normal buccal mucosa and oral submucous fibrosis (OSF) specimens and further to explore the potential mechanisms that may lead to induce HIF-1α expression. OSF buccal mucosal fibroblasts (BMFs) demonstrated significantly higher HIF-1α mRNA expression than normal BMFs (p<0.005). Arecoline, the major areca nut alkaloid, was also found to elevate HIF-1α mRNA expression in a dose-dependent manner (p<0.05). Moreover, arecoline-induced HIF-1α expression was downregulated by mitogen-activated protein kinase inhibitor U0126, phosphatidylinositol 3-kinase inhibitor LY294002, p38 inhibitor SB203580, cyclooxygenase-2 inhibitor NS-398, and glutathione precursor N-acetyl-L-cysteine (p<0.05). Taken together, hypoxia plays an important role in the pathogenesis of areca quid chewing-associated OSF. These pharmacological agents may be further used as chemoprevention agents for OSF.

Acquisition cancer stemness, mesenchymal transdifferentiation, and chemoresistance properties by chronic exposure of oral epithelial cells to arecoline.

Oral squamous cell carcinoma (OSCC), one of the most deadliest malignancies in the world, is caused primarily by areca nut chewing in Southeast Asia. The mechanisms by which areca nut participates in OSCC tumorigenesis are not well understood. In this study, we investigated the effects of low dose long-term arecoline (10 μg/mL, 90-days), a major areca nut alkaloid, on enhancement cancer stemness of human oral epithelial (OE) cells. OE cells with chronic arecoline exposure resulted in increased ALDH1 population, CD44 positivity, stemness-related transcription factors (Oct4, Nanog, and Sox2), epithelial-mesenchymal transdifferentiation (EMT) traits, chemoresistance, migration/invasiveness/anchorage independent growth and in vivo tumor growth as compared to their untreated controls. Mechanistically, ectopic miR-145 over-expression in chronic arecoline-exposed OE (AOE) cells inhibited the cancer stemness and xenografic. In AOE cells, luciferase reporter assays further revealed that miR-145 directly targets the 3′ UTR regions of Oct4 and Sox2 and overexpression of Sox2/Oct4 effectively reversed miR-145-regulated cancer stemness-associated phenomenas. Additionally, clinical results further revealed that Sox2 and Oct4 expression was inversely correlated with miR-145 in the tissues of areca quid chewing-associated OSCC patients. This study hence attempts to provide novel insight into areca nut-induced oral carcinogenesis and new intervention for the treatment of OSCC patients, especially in areca nut users.

Arecoline Induces Neurotoxicity to PC12 Cells: Involvement in ER Stress and Disturbance of Endogenous H2S Generation.

Arecoline is a major alkaloid of areca nut and has been effect on central nervous system. Although arecoline-induced neurotoxicity has been reported, the possible underlying neurotoxic mechanisms have not yet been elucidated. Increasing evidences have shown that both excessive endoplasmic reticulum (ER) stress and disturbance of hydrogen sulfide (H2S) production are involved in the pathophysiology of numerous neurodegenerative diseases. Here, the purpose of present study was to verify whether ER stress and the disturbance of endogenous H2S generation are also involved in arecoline-caused neurotoxicity. We found that treatment of PC12 cells with arecoline induced the down-regulation of cells viability and up-regulation of apoptosis and the activity of caspase-3, indicating the neurotoxic role of arecoline to PC12 cells. In addition, arecoline also increased the expression of Bax (pro-apoptotic protein) and attenuated the expression of Bcl-2 (anti-apoptotic protein) in PC12 cells. Simultaneously, arecoline caused excessive ER stress in PC12 cells, as evidenced by the up-regulations of Glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), and Cleaved caspase-12 expressions. Notably, the level of H2S in the culture supernatant and the expressions of cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase (two major enzymes for endogenous H2S generation in PC12 cells) were also reduced by arecoline treatment. These results indicate that arecoline-caused neurotoxicity to PC12 cells is involved in ER stress and disturbance of endogenous H2S generation and suggest that the modulation of ER stress and endogenous H2S generation may be potential therapeutic approach in treatment of arecoline-caused neurotoxicity.

Arecoline-induced pro-fibrotic proteins in LLC-PK1 cells are dependent on c-Jun N-terminal kinase.

Areca nut (AN) chewing is associated with chronic kidney disease (CKD). However, the molecular mechanisms of AN-induced CKD are not known. Thus, we studied the effects of arecoline, a major alkaloid of AN, on proximal tubule (LLC-PK1) cells in terms of cytotoxicity, fibrosis, transforming growth factor-β (TGF-β) and c-Jun N-terminal kinase (JNK). We found that arecoline dose (0.1-0.5mM) and time (24-72h)-dependently induced cytotoxicity without causing cell death. Arecoline (0.25 mM) also time-dependently (24-72h) increased fibronectin and plasminogen activator inhibitor-1 (PAI1) protein expressions. Arecoline (0.25 mM) time-dependently (24-72h) increased TGF-β gene transcriptional activity and supernatant levels of active TGF-β1. Moreover, arecoline (0.25 mM) activated JNK while SP600125 (a JNK inhibitor) attenuated arecoline-induced TGF-β gene transcriptional activity. SP600125, but not SB431542 (a TGF-β receptor type I kinase inhibitor), attenuated arecoline-induced fibronectin and PAI1 protein expressions. Finally, tubulointerstitial fibrosis occurred and renal cortical expressions of fibronectin and PAI1 proteins increased in arecoline-fed mice at 24 weeks. We concluded that arecoline induced tubulointerstitial fibrosis in mice while arecoline-induced TGF-β and pro-fibrotic proteins (fibronectin, PAI1) are dependent on JNK in LLC-PK1 cells.

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

Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation. Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2', 7'-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. TGM-2 expression was significantly higher in OSF specimens than normal specimens (p<0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p<0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p<0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p<0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p<0.05). Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation. TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

Effects of arecoline on cell growth, migration, and differentiation in cementoblasts

Background/purposeStudies have supported a higher prevalence of periodontal disease among areca quid chewers than non-chewers. However, few studies have stated the effects of areca quid on periodontal tissues. The aim of this study was to investigate the inhibitory effects of arecoline, the major alkaloid of areca nut, on murine immortalized cementoblast cell line (OCCM.30). Materials and methodsCytotoxicity was judged using tetrazolium bromide reduction assay. Cell migration was evaluated by Transwell assay. In vitro mineral nodule formation was assayed by von Kossa staining. Cell differentiation was examined by alkaline phosphatase activity with substrate assay. The production of osteoprotegerin was evaluated using enzyme-linked immunosorbent assay. ResultsArecoline demonstrated cytotoxicity to cementoblasts in a dose-dependent and time-dependent manner (P < 0.05). Arecoline attenuated cell migration in a dose-dependent manner (P < 0.05). Arecoline treatment markedly suppressed cementoblast-mediated biomineralization in vitro compared to untreated cells at Day 8. Arecoline was found to inhibit alkaline phosphatase activity in a time-dependent manner (P < 0.05). In addition, arecoline decreased the secretion of osteoprotegerin in a dose-dependent manner (P < 0.05). ConclusionTaken together, these results suggest that arecoline could inhibit cell growth, migration, and differentiation in cementoblasts. Areca quid chewers might be more susceptible to the destruction of periodontium and less responsive to regenerative procedure during periodontal therapy.

Elevation of Twist expression by arecoline contributes to the pathogenesis of oral submucous fibrosis.

Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a precancerous condition of oral mucosa. In this study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF. Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented. We observed that the treatment of arecoline dose-dependently increased Twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues. This evidence suggests that upregulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity.

Elevated Lin28B expression is correlated with lymph node metastasis in oral squamous cell carcinomas.

Lymph node (LN) metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. Searching the detailed molecular mechanisms involved LN metastasis in OSCC is still an open question. Paired tissue samples from tumor (T) and adjacent non-cancerous matched tissues (NCMT) parts, as well as LN metastatic lesions in patient with OSCC tissues were subjected to quantitative real-time PCR analysis for the expression levels of Lin28B. Arecoline, a major areca nut alkaloid,was to explore whether expression of Lin28B could be changed dose dependent in oral epithelial cells. Control and Lin28B-knockdown arecoline-stimulated oral epithelial cells were subjected to migration/invasion/anchorage-independent growth assay. Compared with NCMT samples from the same OSCC patient, the expression of Lin28B was increased in all of the tumor samples. A similar upregulation of Lin28B was also observed in LN metastatic when compared with local tumors. Arecoline treatment dose dependently induced Lin28B expression in SG and FaDu cells. Lentiviral-mediated silencing Lin28B expression significantly attenuated arecoline-induced oncogenicity including proliferation, migration, invasiveness, and anchorage-independent growth in SG and FaDu cells. Lin28B may be a useful biomarker and novel molecular target for LN metastasis OSCC patients' treatment.

Betel Nut Chewing Associated With Severe Periodontitis

Betel quid chewing has been in practice among people in many Asian countries since ages. It is the most widely used psychoactive substance and an important environmental risk factor for development of oral premalignant lesions and cancer. Arecoline, the major alkaloid of areca nut, has been known to cause cytotoxicity and genotoxicity in mammalian cells in vivo and in vitro and even contributes to carcinogenicity. It also increases the risk and severity of periodontal disease. This paper presents a case of betel nut chewing associated with severe periodontitis in a 32 year old male patient.DOI: http://dx.doi.org/10.3126/hren.v12i1.11990Health Renaissance 2014;12(1):57-60

Effect of arecoline, the major alkaloid of areca nut, on the number of N-benzyl-N-methylnitrosamine (NMBA)-induced esophageal papilloma in F344 male rats.

53 Background: Arecoline is the major alkaloid of areca nut which is one major risk of developing esophageal squamous cell carcinoma (ESCC) in the Asian population. This study aims to establish one rat model to study the relationship of areca nut exposure with the development of ESCC. Methods: Six-week-old Fischer 344 male rats were injected subcutaneously with 0.5 mg/kg NMBA thrice a week for the first 5 weeks and given water without or with arecoline (500 mg/ml) for 30 weeks. Results: With co-treatment of arecoline, NMBA-injected rats had significantly more esophageal papillomas (p = 0.02) and marginally significantly more tongue papillomas (p= 0.09) per rat, compared to those with NMBA only in the end of week 30. No papilloma was found in the lung, liver and/or stomach among all experimental groups. The histological features of esophageal papilloma showed the hyperplastic changes of squamous epithelium. Conclusions: This established animal model can be ideally used to study areca nut-associated esophageal carcinogenesis.

Hypoxic regulation of plasminogen activator inhibitor-1 expression in human buccal mucosa fibroblasts stimulated with arecoline.

Oral submucous fibrosis (OSF) is regarded as a pre-cancerous condition with fibrosis in oral subepithelial connective tissue. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal buccal mucosa tissues and OSF specimens and further explore the potential mechanisms that may lead to the induction of HIF-1α expression. Twenty-five OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. The expression of HIF-1α from fibroblasts cultured from OSF and normal buccal mucosa was measured by Western blot. Arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether HIF-1α expression could affect by arecoline. In addition, the effects of arecoline on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. HIF-1α expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, epithelial cells, and inflammatory cells. Fibroblasts derived from OSF were found to exhibit higher HIF-1α protein expression than BMFs (P<0.05). Arecoline was found to upregulate HIF-1α protein in a dose-dependent manner (P<0.05). Hypoxia increased arecoline-induced PAI-1 protein expression than normoxic conditions (P<0.05). These results suggest that HIF-1α expression is significantly upregulated in OSF tissues from areca quid chewers, implying a potential role as a biomarker for local tissue hypoxia. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in oral submucosa leading to fibrosis.