Systematic Review of Roles of Arecoline and Arecoline N-Oxide in Oral Cancer and Strategies to Block Carcinogenesis.

Betel quid and areca nut are complex mixture carcinogens, but little is known about whether their derived single-agent arecoline or arecoline N-oxide (ANO) is carcinogenic, and the underlying mechanisms remain unclear. In this systematic review, we analyzed recent studies on the roles of arecoline and ANO in cancer and strategies to block carcinogenesis. In the oral cavity, flavin-containing monooxygenase 3 oxidizes arecoline to ANO, and both alkaloids conjugate with N-acetylcysteine to form mercapturic acid compounds, which are excreted in urine, reducing arecoline and ANO toxicity. However, detoxification may not be complete. Arecoline and ANO upregulated protein expression in oral cancer tissue from areca nut users compared to expression levels in adjacent normal tissue, suggesting a causal relationship between these compounds and oral cancer. Sublingual fibrosis, hyperplasia, and oral leukoplakia were diagnosed in mice subjected to oral mucosal smearing of ANO. ANO is more cytotoxic and genotoxic than arecoline. During carcinogenesis and metastasis, these compounds increase the expression of epithelial-mesenchymal transition (EMT) inducers such as reactive oxygen species, transforming growth factor-ß1, Notch receptor-1, and inflammatory cytokines, and they activate EMT-related proteins. Arecoline-induced epigenetic markers such as sirtuin-1 hypermethylation, low protein expression of miR-22, and miR-886-3-p accelerate oral cancer progression. Antioxidants and targeted inhibitors of the EMT inducers used reduce the risk of oral cancer development and progression. Our review findings substantiate the association of arecoline and ANO with oral cancer. Both of these single compounds are likely carcinogenic to humans, and their mechanisms and pathways of carcinogenesis are useful indicators for cancer therapy and prognosis.

Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

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.

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.

Vildagliptin, a dipeptidyl peptidase-4 inhibitor, reduces betel-nut induced carcinogenesis in female mice.

AIM: Betel-nut, a popular masticatory among Southeast Asian populations is a class I carcinogen, previously associated with dyslipidemia and aberrant lipid metabolism, and is reported to be used more frequently by females, than males. This study investigates the potential of repurposing the anti-diabetic drug, vildagliptin, a dipeptidyl peptidase-4 inhibitor, for alleviating the oncogenic condition in female Swiss Albino mice administered an aqueous extract of betel-nut (AEBN) orally (2 mg ml-1) for 24 weeks. MAIN METHODS: Tissues were investigated by histopathological, immunohistochemical and apoptosis assays. Biochemical analyses of oxidative stress markers and lipid profile were performed using different tissues and sera. The expressions of different proteins involved in lipid metabolism and oncogenic pathways were evaluated by Western blotting. KEY FINDINGS: AEBN induced carcinogenesis primarily in the liver by significantly impairing AMPK signaling, inducing oxidative stress, activating Akt/mTOR signaling, increasing Ki-67 immunoreactivity and cyclin D1 expression, and significantly diminishing apoptosis. Co-administration of AEBN with vildagliptin (10 mg kg-1 body weight) for 8 weeks reduced liver dysplasia, and significantly decreased free palmitic acid, increased free oleic acid, normalized lipid profile, decreased oxidative stress, cyclin D1 expression, Ki-67 immunoreactivity, and Bcl2 expression, and increased the ratio of apoptotic/non-apoptotic cells. Mechanistically, vildagliptin elicited these physiological and molecular alterations by restoring normal AMPK signaling and reducing the cellular expressions of FASN and HMGCR, restoring AMPK-dependent phosphorylation of p53 at Ser-15 and reducing Akt/mTOR signaling. SIGNIFICANCE: These results indicate that vildagliptin may alleviate betel-nut induced carcinogenesis in the liver of female mice.

Oral cancer: Clinicopathological features and associated risk factors in a high risk population presenting to a major tertiary care center in Pakistan.

Oral squamous cell carcinoma (OSCC) has the highest prevalence in head and neck cancers and is the first and second most common cancer in males and females of Pakistan respectively. Major risk factors include peculiar chewing habits like areca nut, betel quid, and tobacco. The majority of OSCC presents at an advanced stage with poor prognosis. On the face of such a high burden of this preventable cancer, there is a relative lack of recent robust data and its association with known risk factors from Pakistan. The aim of this study was to identify the socioeconomic factors and clinicopathological features that may contribute to the development of OSCC. A total of 186 patients diagnosed and treated at a tertiary care hospital, Karachi Pakistan were recruited. Clinicopathological and socioeconomic information was obtained on a structured questionnaire. Descriptive analysis was done for demographics and socioeconomic status (SES) while regression analysis was performed to evaluate the association between SES and chewing habits, tumor site, and tumor stage. The majority of patients were males and the mean age of OSCC patients was 47.62±12.18 years. Most of the patients belonged to low SES (68.3%) and 77.4% were habitual of chewing. Gender (male) and SES were significantly associated with chewing habits (p<0.05). Odds of developing buccal mucosa tumors in chewers (of any type of substance) and gutka users were 2 and 4 times higher than non-chewers respectively. Middle age, chewing habits, and occupation were significantly associated with late stage presentation of OSCC (p<0.05). In conclusion, male patients belonging to low SES in their forties who had chewing habits for years constituted the bulk of OSCC. Buccal mucosa was the most common site in chewers and the majority presented with late stage tumors.

Arecoline induces epithelial mesenchymal transition in HK2 cells by upregulating the ERK-mediated signaling pathway.

Arecoline, a component of betel nuts, is a known carcinogen that causes oral cancers among those who chew betel nuts. Betel nut chewing is also associated with an increased risk of chronic kidney disease (CKD), but the role of arecoline in this association is unclear. This in vitro study investigates the effects of arecoline on cultured human kidney (HK2) cells. We observed that arecoline had no effect on cell viability but increased cell migration in a dose-dependent manner. Western blot analysis showed that arecoline treatment caused a dose-dependent decrease in E-cadherin expression and dose-dependent increases in N-cadherin, vimentin, α-SMA, and collagen expression; reverse transcriptase-polymerase chain reaction analysis revealed dose-dependent increases in α-SMA and collagen mRNA. Arecoline treatment upregulated the expression of phosphorylated extracellular signal-regulated kinase through epithelial mesenchymal transition and renal fibrosis in HK2 cells. These findings demonstrate that arecoline plays a role in inducing the epithelial mesenchymal transition and fibrogenesis in renal tubule cells and suggest that arecoline promotes the progression of CKD.

Betel quid-associated cancer: Prevention strategies and targeted treatment.

Betel quid (BQ) and areca nut use are at risk of cancer. This review includes the latest evidence of carcinogenesis caused by BQ exposure, suggests possible prevention strategies. We conducted a systematic literature search in the PubMed and Web of Science databases to identify relevant articles published in the past 10 years according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria. Arecoline N-oxide, a metabolite of areca nut, is likely an initiator in carcinogenesis and is detoxified by N-acetylcysteine. Oral potentially malignant disorder and reactive oxygen species involved in carcinogenesis pathways may be treatable using antioxidants. Screening programs conducted by trained physicians are useful for identifying patients with early stages of oral cancer in high-risk groups. Anti-inflammatory medications may be used as chemopreventive agents in the disease-free stage after surgery. The association between survival and tumor somatic mutations in patients who chew BQ should be addressed in cancer studies. Current evidence on the natural course from BQ exposure to cancer occurrence and development provides information for developing primary, secondary, and tertiary prevention strategies against BQ-associated cancer at clinical or translational levels.

How Each Component of Betel Quid Is Involved in Oral Carcinogenesis: Mutual Interactions and Synergistic Effects with Other Carcinogens-a Review Article.

PURPOSE OF REVIEW: The roles of the components of betel quid in oral carcinogenesis remain unclear. The purpose of the present review is to highlight the effect of each component of betel quid and to discuss the synergistic effects of other carcinogens along with betel quid in the development of oral cancer in habitual betel quid chewers. RECENT FINDINGS: Betel quid may synergistically participate in carcinogenesis by disrupting the compositions of oral microbiota, accompanied by endotoxins secretion and reactive oxygen species (ROS) production. Microbiome dysbiosis mediated by synergistic effects of betel quid chewing, smoking, and alcohol drinking is possibly linked to oral carcinogenesis, which is firstly discussed in this report. Betel quid and other carcinogenic components, mainly contribute to downregulate the antioxidant proteins and lead to the induction of ROS. The elimination of ROS may prove most effective chemoprevention for betel quid-mediated oral carcinogenesis.

Arecoline suppresses epithelial cell viability through the Akt/mTOR signaling pathway via upregulation of PHLPP2.

Arecoline, the major active ingredient of the betel nut, is involved in the pathogenesis of oral submucous fibrosis. However, the underlying mechanism of this pathogenesis remains unclear. In this study, we found that arecoline suppresses the cell proliferation of the HaCaT epithelial cell and induces cell cycle arrest at the G1/S phase with an IC50 of 50 µg/mL. Furthermore, we found that arecoline reduces the protein level of cyclin D1, but it has no effect on its mRNA level and protein stability, implying that arecoline may modulate the translation of cyclin D1. We also observed the downregulation of the Akt/mTOR signaling pathway after treatment with arecoline, which may be related to the translation of cyclin D1. RNA-seq analysis identified that PHLPP2, the direct upstream target of Akt, is significantly upregulated after arecoline treatment. siRNA-mediated knockdown of PHLPP2 recovered the phosphorylation state of Akt, as well as attenuated the effect of arecoline on cell viability. Thus, our study revealed the crucial role of PHLPP2 in arecoline-induced cell viability suppression.

Evaluation of 13-week repeated oral dose toxicity of Areca catechu in F344/N rats.

This study investigated the potential toxicity of the Areca catechu water extract after 13-week repeated oral administration at 0, 166.7, 500, and 1500 mg/kg/day in rats. During the study period, clinical signs, mortality, body weight, food consumption, water consumption, urinalysis, estrous cycle, sperm count and motility, ophthalmoscopy, hematology, serum biochemistry, gross pathology, organ weights, and histopathology were examined. At 1500 mg/kg/day, both sexes exhibited an increase in the incidence of abnormal clinical signs, which included, decreased body weight gain and food consumption, and increased urine bilirubin, ketone bodies, specific gravity, and protein and kidney weight. An increase in liver weight and estrous cycle alterations was observed in females. Serum biochemical and histopathological investigations revealed an increase in the levels of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, and the incidence of hepatic necrosis in females. At 500 mg/kg/day, an increase in the incidence of abnormal clinical signs including diarrhea and soiled perineal region, was observed in both sexes. No treatmentrelated effects were observed at 166.7 mg/kg/day. Under the present experimental conditions, the target organs were determined to be the liver, kidney, and female reproductive system in rats. The no-observedeffect level was considered to be 166.7 mg/kg/day in rats.

Low dietary n-6/n-3 polyunsaturated fatty acid ratio prevents induced oral carcinoma in a hamster pouch model.

Dietary fatty acid patterns have been linked to the prevalence of certain cancers, however in oral carcinoma is limited. Thus, we investigated the chemopreventive effects of various dietary n-6 and n-3 fatty acids in a 9,10-dimethyl-1,2-benz[a]-anthracene (DMBA)- and betel quid extract (BQE) -induced hamster oral cancer model. Thirty 6-week-old adult male hamsters were housed and divided into normal, low, and high dietary n-6 and n-3 fatty acid groups under DMBA + BQE treatment for 16 weeks. The right buccal pouch of all hamsters were evaluated by tumor number, volume, burden and selected inflammatory parameters. The results indicate that the low dietary n-6/n-3 fatty acid group exhibited a significantly lower tumor number, volume, and burden than those of the other groups. Furthermore, this group had significantly lower nuclear factor-κB, proliferating cell nuclear antigen, and cyclin D1 expression in the right buccal pouch tissue. In conclusion, the lower dietary n-6/n-3 fatty acid ratio exerted chemopreventive effects in the DMBA- and BQE-induced hamster oral cancer model.

[Systematic evaluation for safety of traditional Chinese medicine Areca catechu and its preparations].

The authors systemically evaluated and analyzed the safety of Areca catechu from domestic and foreign literatures about the herbal origin, toxicity recorded in ancient/current documents, safety case reports of clinical A. catechu, experimental studies on toxicity in recent years, and differences of safety risk between edible and medicinal A. catechu. Subsequently, they proposed a preliminary summary about the clinical characteristics and potential risk factors of safety related cases of A. catechu and its preparations. According to the authors, although clinical adverse events of A. catechu were fewer and controllable, clinicians shall stillstrictly standardize its application, and rationally combine it with other herbs, while strengthening fundamental and clinical studies related to safety, so as to give better guidance to safety application of A. catechu in clinic.

Using the "target constituent removal combined with bioactivity assay" strategy to investigate the optimum arecoline content in charred areca nut.

Charred areca nut (CAN) is used to treat dyspepsia and abdominal distension in children. However, reports revealed that arecoline, the most important active constituent of CAN, possesses potential toxicities. This study was designed to investigate the optimum arecoline content in CAN, using the "target constituent removal combined with bioactivity assay" strategy. Based on PTLC method, we prepared CAN lacking all arecoline (WAC-100R) and a series of different ratios of arecoline-removed CAN samples (WAC-Rx). MTT and acute toxicity assays indicated that decreasing content by 50% decreased CAN toxicity significantly. Animal results revealed arecoline contents over 50% could guarantee the beneficial effects of CAN on gastrointestinal tract. Additionally, decreasing arecoline content in CAN by 50% decreased its pro-apoptotic effects significantly. Furthermore, decreasing 50% arecoline content in CAN down-regulated the expressions of Cleaved-Caspase-3, c-jun, c-fos, COX-2, PGE2, and IL-1α. Thus, our results revealed that CAN with 50% arecoline content (WAC-50R) has similar beneficial effects on the gastrointestinal tract to CAN, whereas its toxicity was decreased significantly. Collectively, our study suggested that the strategy of "target constituent removal combined with bioactivity assay" is a promising method to identify the optimum arecoline content in CAN, which is approximately 0.12%.

Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.

MicroRNA-17-5p regulated apoptosis-related protein expression and radiosensitivity in oral squamous cell carcinoma caused by betel nut chewing.

Betel nut chewing is associated with oral cavity cancer. Radiotherapy is one of the therapeutic approaches. Here, we used miR-17-5p antisense oligonucleotides (AS-ODNs) and human apoptosis protein array to clarify which apoptosis-related proteins are increased or decreased by miR-17-5p in betel nut chewing- oral squamous cell carcinoma OC3 cells. Furthermore, miR-17-5p AS-ODN was used to evaluate the radio-sensitization effects both in vitro and in vivo. An OC3 xenograft tumor model in severe combined immunodeficiency mice was used to determine the effect of miR-17-5p AS ODN on tumor irradiation. We simultaneously detected the relative expressions of 35 apoptosis-related proteins in irradiated OC3 cells that were treated with miR-17-5p AS-ODN or a control ODN. Several proteins, including p21, p53, TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1, were found to be up- or downregulated by miR-17-5p in OC3 cells; their expression patterns were also confirmed by Western blotting. We further clarified the role of p53 in irradiated OC3 cells, using a p53 overexpression strategy. The results revealed that the enhancement of p53 expression significantly enhanced radiation-induced G2/M arrest of the OC3 cells. In the in vivo study, treatment of miR-17-5p AS-ODN before irradiation significantly enhanced p53 expression and reduced tumor growth. These results suggest that miR-17-5p increases or decreases apoptosis-related proteins in irradiated OC3 cells; its effect on p53 protein expression contributes to the modulation of the radiosensitivity of the OC3 cells.

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.