Areca Nut Components Research Articles

Oral lesions in patients consuming different types of smokeless tobacco. A literature review.

Introduction: Smokeless tobacco (ST) is widely consumed around the world, with products such as gutkha and paan masala being particularly common. These products typically contain areca nut, which is known for its carcinogenic properties and negative effects on oral health. In addition, tobacco is a key ingredient in ST and has been linked to a range of general and dental health issues, particularly among younger populations. Objetive: The purpose of this study is to demonstrate the harmful effect of ST in the oral health. Method: A bibliographic search was conducted based on a sistematic review of articles published between January 2009 and March 2023, aided by PubMed, Scielo, and Google Scholar search engines. Results: Some studies have shown that ST and areca nut components can be particularly dangerous for people with cardiovascular disease and diabetes. Additionally, tobacco has a significant impact on periodontal and dental health, with pathological lesions that may lead to malignant disorders. Peer pressure and stress are common reasons for ST addiction. As such, promoting the intention to quit this habit should be repeatedly emphasized to avoid the negative consequences of these products. Conclusion: the consumption of tobacco has taken a huge toll around the world with its frequency and health consequences. The widespread of ST products such as gutkha and paan masala has affected all age groups. These are responsible for many repercussions that are harmful to health.

Bioactive Components of Areca Nut: An Overview of Their Positive Impacts Targeting Different Organs.

Areca catechu L. is a widely cultivated tropical crop in Southeast Asia, and its fruit, areca nut, has been consumed as a traditional Chinese medicinal material for more than 10,000 years, although it has recently attracted widespread attention due to potential hazards. Areca nut holds a significant position in traditional medicine in many areas and ranks first among the four southern medicines in China. Numerous bioactive compounds have been identified in areca nuts, including alkaloids, polyphenols, polysaccharides, and fatty acids, which exhibit diverse bioactive functions, such as anti-bacterial, deworming, anti-viral, anti-oxidant, anti-inflammatory, and anti-tumor effects. Furthermore, they also display beneficial impacts targeting the nervous, digestive, and endocrine systems. This review summarizes the pharmacological functions and underlying mechanisms of the bioactive ingredients in areca nut. This helps to ascertain the beneficial components of areca nut, discover its medicinal potential, and guide the utilization of the areca nut.

Development and application of mass spectrometric molecular networking for analyzing the ingredients of areca nut.

Areca nut (Areca catechu L.) is commonly consumed as a chewing food in the Asian region. However, the investigations into the components of areca nut are limited. In this study, we have developed an approach that combines mass spectrometry with feature-based molecular network to explore the chemical characteristics of the areca nut. In comparison to the conventional method, this technique demonstrates a superior capability in annotating unknown compounds present in areca nut. We annotated a total of 52 compounds, including one potential previously unreported alkaloid, one carbohydrate, and one phenol and confirmed the presence of 7 of them by comparing with commercial standards. The validated method was used to evaluate chemical features of areca nut at different growth stages, annotating 25 compounds as potential biomarkers for distinguishing areca nut growth stages. Therefore, this approach offers a rapid and accurate method for the component analysis of areca nut.

Chemical Composition of Areca Nut and Its Adverse Effects on Human Health.

Areca nut (AN) is one of the addictive substances consumed widely in the world. The composition of AN is very complex, and each component has variable properties. This study aimsto review the composition of AN and its adverse effects on humans. For this review, the literature search was performed by an electronic search of the Pubmed/Medline, Scopus, and Google Scholar databases using proper MESH headings and retrieved the articles published from 1997 to 2022. The eligibility criteria included human studies, a form of AN, the composition of AN, harmful effects of AN, the effect of AN on the oral cavity,the effect of AN on vital organs, and articles published in English. Data were extracted regarding the composition of AN, forms of AN, and harmful effects of AN on the oral cavity and on other systems. A total of 449 articles were identified from various databases, and 36 studies were selected that met the inclusion criteria. The active components of AN, which produce harmful effects, are primarily alkaloids, polyphenols, tannins, and certain trace elements. AN is consumed in different forms, and based on the form, its composition also varies. AN is known to cause deleterious effects on the oral cavity as well as various body organs. The most dangerous and widely reported impacts of AN on the oral cavity are the development of oral submucous fibrosis, a premalignant condition, and oral malignancy.However, during the chewing process, excretory products of AN are released and circulate in the body of "chronic habitual" and affect the other body parts. Thus, AN consumption may contribute to cardiac, liver, endocrinal, metabolic, respiratory, and reproductive system disorders also. AN composition is complex, and its consumption is harmful to human health. In regard to controlling the issue of the harmful effects of this habit, preventive measures should be established.

The Controversial Roles of Areca Nut: Medicine or Toxin?

Areca nut (AN) is used for traditional herbal medicine and social activities in several countries. It was used as early as about A.D. 25-220 as a remedy. Traditionally, AN was applied for several medicinal functions. However, it was also reported to have toxicological effects. In this review article, we updated recent trends of research in addition to acquire new knowledge about AN. First, the history of AN usage from ancient years was described. Then, the chemical components of AN and their biological functions was compared; arecoline is an especially important compound in AN. AN extract has different effects caused by different components. Thus, the dual effects of AN with pharmacological and toxicological effects were summarized. Finally, we described perspectives, trends and challenges of AN. It will provide the insight of removing or modifying the toxic compounds of AN extractions for enhancing their pharmacological activity to treat several diseases in future applications.

Betel quid may stimulate oral submucous fibrosis by inducing increased mitochondrial reactive oxygen species generation via copper overload

Betel quid (BQ), a smokeless chewing tobacco that includes the areca (betel) nut among its ingredients, increases the risk of oral submucous fibrosis (OSMF) and oral cancer, a disease classified as an potentially malignant oral disorder. A wound from the coarse areca nut fibers, as well as the catechin, copper, and alkaloids (e.g., arecoline, arecaidine, guvacoline, and guvacine) in BQ, may stimulate reactive oxygen species (ROS) production, causing myofibroblast differentiation and contraction, fibroblast proliferation, collagen cross-linking, and collagen phagocytosis inhibition, which could result in OSMF and oral cancer. Mitochondria, the primary source of ROS in OSMF, play a crucial role in cellular metabolism. Copper is required for mitochondrial bioenergetics, dynamics, and mitophagy, affecting metabolic reprogramming and cell fate. Given that copper levels are elevated in patients with OSMF, we hypothesize that increased mitochondrial ROS production induced by copper overload and areca nut components may regulate fibrosis-related gene expression and myofibroblast differentiation. Therefore, targeting this fundamental pathomechanism is an appropriate strategy for developing new therapeutic options for OSMF.

Evaluation of cytotoxicity of areca nut and its commercial products on normal human gingival fibroblast and oral squamous cell carcinoma cell lines

Consumption of areca nut products is the most common cause of oral cancers, particularly in South Asian countries. This study evaluates the cytotoxic and necrotizing effects of areca nut and its formulations on normal human gingival fibroblasts (HGF-1) and oral squamous cell carcinoma (OSCC, CAL-27) cell lines. Identification of various carcinogens and adulterants using LC-HR-ESI-MS/MS analysis was performed in the extracts of areca nut and its products. Apart from alkaloids and flavonoids, a major adulterant, saccharin was found in all the samples of chalia (one of the most common chewing products of areca nut) in the ranges between 1.697–7.170 mg/g of the sample. Cytotoxic studies showed that most of the areca nut products were found cytotoxic to HGF-1 cells while being relatively non-cytotoxic against CAL-27 cells, rather they promote the growth of cancer cells. Our findings revealed that the components of areca nut and its products were injurious to HGF-1 cells and caused necrosis, which may attenuate HGF-1 protection toward oral epithelial cells. Moreover, the non-cytotoxic effect of these products on cancer cell lines suggests further predisposal of the habitual chewers for developing oral carcinomas. This study will give a better understanding of the hazardous effects of areca nut products.

Molecular Pathogenesis of Oral Submucous Fibrosis: A Critical Appraisal

Oral submucous fibrosis (OSMF) is a chronic, potentially malignant condition of the oral mucosa and the habitual chewing of areca nut is believed to be the most potent etiological factor. The role of reactive oxygen species (ROS), epithelial-mesenchymal transition (EMT) and various cytokines and growth factors has been established in recent studies. The components of areca nut particularly, arecoline, flavonoids and copper have been found to affect fibroblasts, endothelial and epithelial cells through various biological pathways which are either down-regulated or up-regulated during different stages of the disease. However, the underlying molecular pathogenesis of OSMF is still partially understood.

Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

Role of Areca Nut Induced TGF-β and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis.

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-β signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-β in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-β in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-β followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-β treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-β together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-β induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut +TGF-β treated hGF cells. In concordance, areca nut along with TGF-β enhanced fibroblast activation as demonstrated by potentiation of αSMA, γSMA and collagen gel contraction by hGF cells. Furthermore, TGF-β secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

Cytotoxicity and transformation of C3H10T1/2 cells induced by areca nut components.

Betel quid (BQ) chewing is popular in Taiwan and many other countries. There are about 200-600 million BQ chewers in the world. BQ chewing is one major risk factor of oral cancer and oral submucous fibrosis (OSF). While areca nut (AN), a main component of BQ, exhibits genotoxicity, its transformation capacity and its role in the initiation and promotion stages of carcinogenesis are not fully clear. Mouse C3H10T1/2 cells were exposed to AN extract (ANE) for 24 hours. Cytotoxicity was evaluated by colony forming efficiency. For the transformation assay, C3H10T1/2 cells were exposed to ANE for 24 hours and then incubated in medium with/without 12-O-tetradecanolylphorbol-13-acetate (TPA; a tumor promoter) for 42 days. Cells were stained with Giemsa and type II and type III transformed foci were counted for analysis of the transformation capacity of ANE. ANE exhibited cytotoxicity to C3H10T/12 cells at concentrations higher than 320μg/mL as shown by a decrease in colony numbers. ANE (80-640μg/mL) alone mildly stimulated the transformed foci formation (p>0.05). In the presence of TPA, ANE (80-640μg/mL) markedly stimulated the transformed foci formation. The percentage of dishes with foci increased from 0% in controls to 20% in ANE (80μg/mL and 320μg/mL)-treated groups and further increased to 65-94% in ANE plus TPA groups. These results indicate that ANE is a weak complete carcinogen. ANE is an effective tumor initiator and can induce malignant transformation of C3H10T1/2 cells in the presence of a tumor promoter. ANE may be involved in multistep chemical carcinogenesis by its malignant transformation capacity.

Areca nut components affect COX-2, cyclin B1/cdc25C and keratin expression, PGE2 production in keratinocyte is related to reactive oxygen species, CYP1A1, Src, EGFR and Ras signaling.

AimsChewing of betel quid (BQ) increases the risk of oral cancer and oral submucous fibrosis (OSF), possibly by BQ-induced toxicity and induction of inflammatory response in oral mucosa.MethodsPrimary gingival keratinocytes (GK cells) were exposed to areca nut (AN) components with/without inhibitors. Cytotoxicity was measured by 3-(4,5-dimethyl- thiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. mRNA and protein expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. PGE2/PGF2α production was measured by enzyme-linked immunosorbent assays.ResultsAreca nut extract (ANE) stimulated PGE2/PGF2α production, and upregulated the expression of cyclooxygenase-2 (COX-2), cytochrome P450 1A1 (CYP1A1) and hemeoxygenase-1 (HO-1), but inhibited expression of keratin 5/14, cyclinB1 and cdc25C in GK cells. ANE also activated epidermal growth factor receptor (EGFR), Src and Ras signaling pathways. ANE-induced COX-2, keratin 5, keratin 14 and cdc25C expression as well as PGE2 production were differentially regulated by α–naphthoflavone (a CYP 1A1/1A2 inhibitor), PD153035 (EGFR inhibitor), pp2 (Src inhibitor), and manumycin A (a Ras inhibitor). ANE-induced PGE2 production was suppressed by piper betle leaf (PBL) extract and hydroxychavicol (two major BQ components), dicoumarol (a NAD(P)H:Quinone Oxidoreductase - NQO1 inhibitor) and curcumin. ANE-induced cytotoxicity was inhibited by catalase and enhanced by dicoumarol, suggesting that AN components may contribute to the pathogenesis of OSF and oral cancer via induction of aberrant differentiation, cytotoxicity, COX-2 expression, and PGE2/PGF2αproduction.ConclusionsCYP4501A1, reactive oxygen species (ROS), EGFR, Src and Ras signaling pathways could all play a role in ANE-induced pathogenesis of oral cancer. Addition of PBL into BQ and curcumin consumption could inhibit the ANE-induced inflammatory response.

Areca nut and its role in oral submucous fibrosis.

Areca nut, commonly called as betel nut or supari, is a fruit of areca catechu palm tree, which is native of South Asia and Pacific Islands. The seed or endosperm is consumed fresh, boiled or after sun drying or curing. Chewing areca nut is thought to have central nervous system stimulating effect and along with this it is known to have salivary stimulating and digestive properties. According to the traditional Ayurvedic medicine, chewing areca nut and betel leaf is a good remedy against halitosis. It is also used for its deworming property. Along with these beneficial effects of areca nut one of its most harmful effects on the human body in general and oral cavity in particular is the development of potentially malignant disorder called Oral Submucous Fibrosis. The present paper discusses in detail the effects of the components of areca nut on pathogenesis of Oral Submucous Fibrosis. Key words:Areca nut, oral submucous fibrosis, potentially malignant disorder, supari.

Long‐term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity

We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/β-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.

Toxicity of areca nut ingredients: Activation of CHK1/CHK2, induction of cell cycle arrest, and regulation of MMP‐9 and TIMPs production in SAS epithelial cells

There are 600 million betel quid chewers around the world. betel quid chewing is a major risk factor of oral cancer. Why betel quid components induce oral cancer is not clear. Cytotoxicity of areca nut extract and arecoline (an areca nut alkaloid) to SAS oral epithelial cell line was evaluated by trypan blue dye exclusion and MTT assays. Cell cycle distribution and apoptosis was analyzed by propidium iodide staining flow cytometry. Chk1 and chk2 activation was analyzed by Pathscan phospho-enzyme-linked immunosorbent assay. Metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase (TIMPs) production was measured by enzyme-linked immunosorbent assay. Areca nut extract (800 μg/mL) and arecoline (>0.4 mmol/L) caused cell death, apoptosis, and cell cycle arrest of SAS cells. Areca nut extract and arecoline stimulated Chk1 and Chk2 phosphorylation in SAS cells. Areca nut extract stimulated cellular MMP-9 but suppressed TIMP-1 and TIMP-2 production. Areca nut components activate Chk1/Chk2, alter cell cycle regulation/apoptosis, MMP-9, and TIMPs production, contributing to the pathogenesis of oral carcinogenesis.

Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells

Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases.

P138. Activation of Chk1/Chk2, and the induction of cell cycle arrest, IL-6 and metalloproteinase-9 production of SAS epithelial cells by areca nut ingredients

Introduction: There are about 600 million betel quid (BQ) chewers around the world and 3 million BQ chewers in Taiwan. A number of epidemiological studies indicate that BQ chewing is one of the major risk factors of oral leukoplakia, oral submucous fibrosis (OSF) and oral cancer. Areca nut (AN) is the main component of BQ. AN components exhibit cytotoxicity and genotoxicity, that may potentially lead activation of ATM/ATR-Chk1/Chk2 signaling pathways to regulate cell cycle progression. The objective of this study was to clarify the mechanisms of AN-induced oral mucosal inflammatory response and the roles of Chk1 and Chk2. Methods: Cytotoxicity of AN extract (ANE) and arecoline to SAS epithelial cells was determined by trypan blue dye exclusion technique or MTT assay. Cell cycle distribution and apoptosis were analyzed by propidium iodide (PI) staining flow cytometry. Chk1 and Chk2 activation was analyzed by Pathscan p-chk1 and p-Chk2 ELISA or western blotting. Cellular interleukin-6 and metalloproteinase-9 (MMP-9) production was measured by enzyme-linked immunosorbant assay (ELISA). Results: ANE (200–800 g/ml) and arecoline (0.1–0.4 mM), the major ingredient of AN, induced the morphological changes of SAS tongue cancer epithelial cells. Cells became round with intracellular vacuoles formation. ANE (800 g/ml) and arecoline ( 0.4 mM) caused cytotoxicity of SAS cells, and ANE further induced cellular apoptosis and G2/M cell cycle arrest. Interestingly, exposure to ANE (800 and 1200 g/ml) and arecoline (0.8 mM) for 24 h markedly stimulated the Chk1 and Chk2 phosphorylation in SAS cells. ANE also stimulated IL-6 and MMP-9 production in SAS cells and primary gingival keratinocytes (GK). However, arecoline lacked of this stimulatory effect. Pretreatment and then co-incubation by PD407824 (a Chk1 inhibitor) or 2-(4-(4-chlorophenoxy) phenyl-1H-benzimidazole-5carboxamide (a Chk2 inhibitor) showed little preventive effect on the ANEinduced IL-6 and MMP-9 production. Discussion: These results indicate that AN components may contribute to the pathogenesis of oral cancer via induction of tissue inflammation and injury. This can be associated with the activation of Chk1/Chk2, stimulation of IL-6 and MMP-9 production, cell cycle deregulation and apoptosis.

P121. Stimulation of cyclooxygenase-2 expression and prostanoids production in oral keratinocytes by areca nut components is associated with Src and Ras activation

P121. Stimulation of cyclooxygenase-2 expression and prostanoids production in oral keratinocytes by areca nut components is associated with Src and Ras activation

Autophagy induction by the 30–100 kDa fraction of areca nut in both normal and malignant cells through reactive oxygen species

Areca nut (AN) is an addictive carcinogen used by about 200-600 million people worldwide. Some AN components are shown to induce apoptosis; however, we previously demonstrated that AN extract (ANE) and the 30-100kDa fraction of ANE (ANE 30-100K) induced autophagy-like responses, such as swollen cell morphology, empty cytoplasm, acidic vesicles, and LC3-II accumulation, in an oral cancer cell line, OECM-1. To further assess the responses of other cell types to ANE 30-100K, we used both normal and malignant cells as the targets of ANE 30-100K and found that normal oral fibroblasts (CMT415), peripheral blood lymphocytes (PBLs), Jurkat leukemia T cells, and esophageal carcinoma cells (CE81T/VGH) exhibited similar responses after ANE 30-100K challenge. ANE 30-100K drastically increased acidic vesicle-containing PBLs isolated from two independent donors (from 0.1% to 92.1% and 2.9% to 64.2%). Furthermore, both ANE- and ANE 30-100K-induced LC3-II accumulation in CMT415 and CE81T/VGH was further increased in the presence of the lysosomal protease inhibitors (pepstatin A, E64d, and leupeptin). On the other hand, ANE 30-100K also increased the level of intracellular reactive oxygen species (ROS), and the ROS scavengers, N-acetylcysteine (NAC) and Tiron, inhibited ANE 30-100K-induced cell death and LC3-II accumulation. Collectively, these results suggest the existence of an autophagy-inducing AN ingredient (AIAI) in ANE 30-100K, which renders ANE as an autophagic flux inducer through ROS in both normal and malignant cells.