A Critical Interpretive Synthesis of the Role of Arecoline in Oral Carcinogenesis: Is the Local Cholinergic Axis a Missing Link in Disease Pathophysiology?

Arecoline is the primary active carcinogen found in areca nut and has been implicated in the pathogenesis of oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). For this study, we conducted a stepwise review process by combining iterative scoping reviews with a post hoc search, with the aim of identifying the specific mechanisms by which arecoline initiates and promotes oral carcinogenesis. Our initial search allowed us to define the current trends and patterns in the pathophysiology of arecoline-induced OSF and OSCC, which include the induction of cell proliferation, facilitation of invasion, adhesion, and migration, increased collagen deposition and fibrosis, imbalance in immune and inflammatory mechanisms, and genotoxicity. Key molecular pathways comprise the activation of NOTCH1, MYC, PRDX2, WNT, CYR61, EGFR/Pl3K, DDR1 signaling, and cytokine upregulation. Despite providing a comprehensive overview of potential pathogenic mechanisms of OSF, the involvement of molecules functioning as areca alkaloid receptors, namely, the muscarinic and nicotinic acetylcholine receptors (AChRs), was not elucidated with this approach. Accordingly, our search strategy was refined to reflect these evidence gaps. The results of the second round of reviews with the post hoc search highlighted that arecoline binds preferentially to muscarinic AChRs, which have been implicated in cancer. Consistently, AChRs activate the signaling pathways that partially overlap with those described in the context of arecoline-induced carcinogenesis. In summary, we used a theory-driven interpretive review methodology to inform, extend, and supplement the conventional systematic literature assessment workflow. On the one hand, the results of this critical interpretive synthesis highlighted the prevailing trends and enabled the consolidation of data pertaining to the molecular mechanisms involved in arecoline-induced carcinogenesis, and, on the other, brought up knowledge gaps related to the role of the local cholinergic axis in oral carcinogenesis, thus suggesting areas for further investigation.


Introduction
Arecoline, an active carcinogenic alkaloid found in areca nut, has been recognized as an important factor in the pathogenesis of premalignant and malignant oral disorders [1], specifically, oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC).However, the precise molecular mechanisms underlying arecoline-induced OSF and OSCC development remain unclear [2,3].
OSF is characterized by progressive fibrosis and inflammation of the submucosal tissues [4].It is a potentially malignant disorder that is associated with an increased risk of OSCC, a malignant neoplasm originating from the stratified squamous epithelium of the oral mucosa [5].OSF progression to OSCC takes place in approximately 7-14% of patients [6].OSCC is one of the most frequently reported malignancies in the world, especially in Taiwan and India, accounting for approximately 90% of all oral cavity cancers [7].
Previous research has identified key biological changes underlying arecoline-induced oral carcinogenesis, including abnormal cell proliferation [8], the dysregulation of cell cycle control [9], invasion and metastasis [10], fibrotic alterations [11], altered inflammatory and immune responses [12], and epigenetic modifications [13].However, the specific signatures associated with arecoline-induced oral carcinogenesis and their roles in disease pathogenesis are widely heterogeneous.
Understanding the mechanisms that are specifically related to the effect of arecoline in the oral mucosa is particularly challenging.In humans, dissecting the arecoline-specific pathways leading to carcinogenesis is virtually impossible as areca nut contains several potentially detrimental alkaloids [14].In addition, areca nut is often consumed together with betel leaves and other ingredients in a mixture-the so-called betel quid (BQ).Different BQs have diverse chemical compositions and the interaction of different constituents confers a distinct disease-inducing capacity to the quids [15].Hence, conventional systematic review methodology may be inappropriate in tackling this question.
Critical interpretive synthesis draws on the traditions of qualitative research inquiry and systematic review methodology and can be used to synthesize both qualitative and quantitative forms of evidence [16].This methodology is explicitly oriented toward theorybuilding and proposes an iterative and dynamic approach to question formulation, searching, and the selection of materials for inclusion in reviews.For the systematic assessment of the existing literature, a scoping review was deemed to be the most suitable approach as it comprehensively examines the existing literature, mapping key concepts, evidence sources, and knowledge gaps.This approach provides an overview, identifies the research trends, and highlights areas for further investigation [17].
Our study aims to investigate the mechanisms through which arecoline promotes the progression of OSCC.Understanding the molecular pathways and diagnostic markers associated with arecoline-induced oral carcinogenesis is paramount for the early detection, prevention, and, potentially, treatment of oral cancer.

Initial Scoping Review
The first round of this review assessing the mechanisms of arecoline-associated carcinogenesis identified 196 records, with 90 eligible articles included in the qualitative synthesis (Figure 1).Of these 90 studies, there were 14 in vivo (Table 1) and 86 in vitro investigations (Supplementary Table S1), with multiple studies containing both in vitro and in vivo components.Most of the in vitro studies reported on the molecular markers involved in arecoline-induced oral carcinogenesis (Supplementary Table S1).There were 32 studies that used fibroblasts, 23 studies that used keratinocytes, and 1 that involved endothelial cells.
Exposure of epithelial cells to arecoline was found to suppress viability and promote apoptosis and atrophy in a dose-dependent manner [26,39].Arecoline inhibits epithelial cell proliferation and affects cell morphology, including cell cycle arrest in the G1/S phase and survival in a dose-dependent manner [35,40,41].
In in vitro experiments, arecoline has been found to promote EMT in oral epithelial cells through DEC1 upregulation activating FAK/AKT downstream [49], the upregulation of proteasome activator complex subunit 3 (PA28γ), and the phosphorylation of MEK-1 [53] and was found to promote the expression of EMT-related genes [27,34].Arecoline resulted in a dose-and time-dependent increase in zinc finger protein 1 (SNAI1) expression in human oral keratinocytes (HOKs) and OECM-1 [54].The long-term exposure of buccal mucosal fibroblasts (BMFs) resulted in the dose-dependent upregulation of transcription factor zinc-finger E box-binding homeobox 1 (ZEB1) and the upregulation of insulin-like growth factor receptor 1 (IGF-R1) [55,56].
In terms of cellular adhesion, arecoline has been found to upregulate αvβ6 integrin expression in oral keratinocytes, modulated by the M4 muscarinic receptor in OKF6/TERT-1 cells [57].Arecoline led to the increased attachment of U397 mononuclear cells to EAhy926 cells [58].

Immune Responses and ROS/Antioxidant Activity
One in vivo study conducted on murine models observed increased antioxidant activity in heat shock protein 27 (HSP27) when the mice were exposed to arecoline [50].

Genotoxicity and Epigenetics
The induction of DNA damage and the alteration of repair mechanisms are widely regarded as the central mechanisms responsible for arecoline-induced carcinogenesis.In vitro, arecoline stimulated an increase in O6-methyl-guanine-DNA methyltransferase (MGMT) expression in HOKs and an increase in the phosphorylation of H2AX variant histone (γH2AX) [19,24,45], as well as the induced markers of irreparable DNA doublestranded breaks in normal human oral fibroblasts and p53-binding protein 1 (53BP1) [45].Low doses of arecoline induced elevated cell proliferation and DNA repair [24]; however, long-term and high-dose exposure reduced DNA repair [69].Arecoline also resulted in the reduced expression of sirtuin 1 (SIRT1) mRNA [79].In contrast, a study found that arecoline is cytotoxic, while no genotoxicity was found in human buccal fibroblasts affecting DNA [80].

Second Round of the Scoping Review, with a Post Hoc Search
The second scoping review focusing on the role of acetylcholine receptors in arecolineinduced carcinogenesis returned 16 results (PubMed, n = 6; Web of Science, n = 10).Following deduplication and screening, only one study was eligible for inclusion [57].The post hoc manual search, including a proximity search of the studies identified in the second scoping review, offered mechanistic insights into the pathways activated by arecoline and allowed us to generate a framework that fits a model of receptor-mediated arecoline-induced oral carcinogenesis.

Arecoline-Mediated Acetylcholine Receptor Signaling in Oral Carcinogenesis
A study by Gareth Thomas' group demonstrated that arecoline upregulated keratinocyte αvβ6 expression, a process modulated through the M(4) muscarinic acetylcholine receptor [57].Arecoline-dependent αvβ6 upregulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation.In another study, long-term nicotine-derived nitrosamine ketone (NNK) and arecoline exposure resulted in an increase in cancer stem cell properties, anti-apoptotic pathways, and a resistance to cisplatin in head and neck squamous cell carcinoma (HNSCC) cells in vitro [84].The EGFR protein was pivotal in inducing tumor promotion and in impeding apoptosis in cancer cells by inducing phosphorylated AKT serine/threonine kinase 1 (pAKT) and nuclear factor kappa B (NFκB).While the authors pointed out that both NNK and arecoline exert agonist activity with the alpha-7-nicotinic acetylcholine receptor (α7-nAChR), the study did not directly investigate the role of nAChR in mediating the effects reported and, hence, was not included in the qualitative synthesis.Both studies, however, point to the possibility that arecoline promotes carcinogenesis via receptor-mediated mechanisms, an aspect that has not been captured in the available literature.The putative signaling pathways are depicted in Figure 2. Putative pathways involving the receptor-mediated signaling of arecoline.Arecoline binds preferentially to muscarinic acetylcholine receptors (mAChR) but can also serve as a partial agonist of nicotinic receptors (nAChR).Two key molecular pathways involve EGFR and integrins.mAChR activates EGFR signaling via a so-called "triple-membrane-passing" pathway, whereby metalloproteinases cleave and activate EGF-like ligands, which, in turn, bind to EGFR and trigger downstream kinase signaling, including the Ras/Raf/MEK/ERK pathway.MAPK signaling can also be activated via canonical second messenger-mediated signals, as well as via nAChR.The two receptors also work synergistically to promote survival and inhibit apoptosis via PI3K/Akt and p21, respectively.Together, these pathways promote the expression of proliferation and survival genes, as well as migration/invasion and fibrosis/senescence via integrins and TGF-beta signaling, respectively (brown arrow).See Abbreviations part for the abbreviations and acronyms.

The Effects of Arecoline in the Oral Mucosa Could Be Mediated by the Local Cholinergic Axis
Despite a substantial body of evidence demonstrating the pro-carcinogenic effects of arecoline, both in vivo and in vitro, our iterative scoping reviews failed to shed light on the receptor-mediated signaling that is probably responsible for these effects.Therefore, we undertook a post hoc search to elucidate the link between arecoline, AChRs, and oral cancer.
Previous research has convincingly demonstrated that both arecoline and guvacoline activate muscarinic acetylcholine receptors 1 and 3 (M1 and M3 mAChRs) [85], while only arecoline produces significant activation of the α4 nicotinic receptor and acts as a silent agonist of α7 nAChR [86].A molecular docking simulation and antagonist co-exposure experiments also showed that arecoline has a strong affinity to muscarinic receptors M1-M4 [87].Hence, it is likely that arecoline elicits cholinergic signals in the oral mucosa via the M2, M3, and M4 mAChR subtypes that are expressed in oral keratinocytes [88].Importantly, muscarinic receptors work synergistically with nicotinic receptors to regulate Putative pathways involving the receptor-mediated signaling of arecoline.Arecoline binds preferentially to muscarinic acetylcholine receptors (mAChR) but can also serve as a partial agonist of nicotinic receptors (nAChR).Two key molecular pathways involve EGFR and integrins.mAChR activates EGFR signaling via a so-called "triple-membrane-passing" pathway, whereby metalloproteinases cleave and activate EGF-like ligands, which, in turn, bind to EGFR and trigger downstream kinase signaling, including the Ras/Raf/MEK/ERK pathway.MAPK signaling can also be activated via canonical second messenger-mediated signals, as well as via nAChR.The two receptors also work synergistically to promote survival and inhibit apoptosis via PI3K/Akt and p21, respectively.Together, these pathways promote the expression of proliferation and survival genes, as well as migration/invasion and fibrosis/senescence via integrins and TGF-beta signaling, respectively (brown arrow).See Abbreviations part for the abbreviations and acronyms.

The Effects of Arecoline in the Oral Mucosa Could Be Mediated by the Local Cholinergic Axis
Despite a substantial body of evidence demonstrating the pro-carcinogenic effects of arecoline, both in vivo and in vitro, our iterative scoping reviews failed to shed light on the receptor-mediated signaling that is probably responsible for these effects.Therefore, we undertook a post hoc search to elucidate the link between arecoline, AChRs, and oral cancer.
Previous research has convincingly demonstrated that both arecoline and guvacoline activate muscarinic acetylcholine receptors 1 and 3 (M1 and M3 mAChRs) [85], while only arecoline produces significant activation of the α4 nicotinic receptor and acts as a silent agonist of α7 nAChR [86].A molecular docking simulation and antagonist co-exposure experiments also showed that arecoline has a strong affinity to muscarinic receptors M1-M4 [87].Hence, it is likely that arecoline elicits cholinergic signals in the oral mucosa via the M2, M3, and M4 mAChR subtypes that are expressed in oral keratinocytes [88].Importantly, muscarinic receptors work synergistically with nicotinic receptors to regulate keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities [89].Given that alpha 3, alpha 5, alpha 7, and beta 2, as well as the alpha 9 nAChR subunits, are expressed in oral keratinocytes [88], a non-neuronal cholinergic system of the oral mucosa exists that regulates key biological functions such as cell viability, proliferation, migration, adhesion, terminal differentiation, and the secretion of cytokines and growth factors [90].This keratinocyte cholinergic system has been shown to play a role in oral mucosal diseases [91] and also mediates nicotine toxicity in oral keratinocytes and in epithelial cancers [92].It is now known that the nAChRs expressed on the cell membrane and mitochondria mediate both growth-promoting and anti-apoptotic effects synergistically.Other mechanisms associated with nicotine toxicity include the genotoxic action of reactive oxygen species [93].With regard to mAChRs, accumulating evidence suggests that mAChRdependent signaling pathways can promote cell proliferation and cancer progression [94].In particular, previous experimental results indicated that M3 receptor activation may promote malignancy in epithelial cancers.In one example, M3-deficient mice displayed reduced epithelial cell proliferation and decreased tumor number and size in models of intestinal neoplasia [95,96].Similarly, M1 receptor deficiency inhibited mAChR-mediated prostate cancer invasion and metastasis in mouse models of prostate cancer [97].
In summary, there is increasing evidence that the non-neuronal cholinergic system in epithelial tissues is involved in carcinogenesis.Similar to the effects of nicotine, it is reasonable to speculate that AChR ligands, such as arecoline and other areca alkaloids, induce pro-tumorigenic effects in the oral mucosa via receptor-mediated signaling.

Discussion
In the present study, we employed a theory-driven interpretive review methodology to inform, extend, and supplement the conventional systematic review workflow.This approach is particularly useful when attempting to make sense of heterogeneous evidence in diverse contexts, in a similar manner to that employed in realistic reviews [98].The results identified cell proliferation, invasion, adhesion, and migration, increased collagen deposition and fibrosis, and altered the immune, inflammatory and epigenetic mechanisms as important events in the pathogenesis of arecoline-induced OSF and OSCC.We generated a framework whereby AChRs were putatively identified as important mediators of these pathophysiological processes.
The initial scoping review identified 90 direct evidence-based primary studies that addressed arecoline-induced carcinogenesis in oral tissues and the potential diagnostic markers associated with the process, all published between 1994 and 2023.All the primary in vivo studies involved the use of murine models challenged with arecoline, while the in vitro studies used a range of human oral cell cultures, such as HOKs or OSCC cell lines that were challenged with arecoline.The subsequent iteration of the scoping review, with a refined search string focused on AChRs, only retrieved one study.Despite this limited result, we collected substantial evidence to show that arecoline activates the local cholinergic axis via AChRs.These receptors are expressed in the oral mucosa and are known to mediate the pro-carcinogenic effects of nicotine [93].Given that arecoline binds to the same receptor family as nicotine, albeit with a preferential affinity to mAChRs, it is not unreasonable that this areca alkaloid promotes oral carcinogenesis in a similar fashion to nicotine.Hence, we propose that future research should focus on this conceptual framework.
Most of the in vivo studies examined in this review directly challenged mice with arecoline and/or its metabolites, whereas 3 studies injected arecoline-induced OSCC cell lines into their BALB/c nude mice.
These three studies demonstrated the pro-fibrotic property of arecoline in stimulating collagen deposition in the oral mucosa of mice [8,19,49].TGF-β, the key player in fibrosis, was shown to increase in response to arecoline exposure [12,21,48].In one particular study, the injection of arecoline into the buccal mucosa of mice directly induced the OSF associated with the DEC1/FAK/Akt signaling pathway [49].As AChRs are expressed in oral fibroblasts [99], it is plausible that the effect of arecoline on this cell type may involve receptor-mediated mechanisms that may be involved in the pathogenesis of OSF.
One study observed that arecoline exposure increased HSP27, an antioxidant protein, suggesting that arecoline could play a role in the cellular adaptation and survival of tumor cells in the presence of oxidative stress [50].
Arecoline was also found to promote invasion.In one study, injecting mice with arecoline-stimulated OSCC resulted in a 50% increase in mice with cervical LN metastasis [48].Activation of the invasion property in epithelial cells is associated with EMT.Several factors and signaling pathways have been implicated in promoting EMT in mouse models, including TGF-β signaling, DEC1 upregulation leading to FAK/Akt activation [49], PTk6 expression with E-cad suppression [10,27,34], and Krt17 upregulation [52].Pertinently, the role of AChRs in invasion, migration, and metastasis is well established [100,101].
Analysis of the in vitro research yielded conflicting results.Studies showed that arecoline exposure led to increased or uncontrolled cellular proliferation and tumor cell growth, via the upregulation of oncogenes such as PCNA, Ki67, MEK1, ERK, B-Raf protooncogene, serine/threonine kinase (BRAF), ZEB1, FAT atypical cadherin 1 (FAT1), NOTCH1 via increased IL-1β, CYR61, and lysine-specific demethylase 1 (LSD1), while simultaneously downregulating the expression of tumor suppressor genes such as tumor proteins 53, 21, and 27 (p53, p21, p27), DUSP4, and the more upstream activators of p53 such as the maternally expressed 3 gene (MEG3) [8,24,[31][32][33][34]36,43,53,55,56,70].Generally, arecoline increases the proportion of mitotic cells and could arrest cells at the prometaphase, resulting in misaligned chromosomes such as cyclin B1 [37].However, conflicting results were found in relation to arecoline's effects on cell cycle arrest and apoptosis.Some studies reported increased cellular proliferation and the inhibition of apoptosis in oral keratinocytes, while others found that arecoline promoted apoptosis and cell-cycle arrest in the G1/S phase and reduced cellular proliferation in SAS cancer cells and epithelial cells, as well as increasing the expression of ADHFE1 and ALDH1A2, and increased the phosphorylation of Chk1 and Chk2 [26,32,[39][40][41][42].These contrasting results are probably due to the different arecoline concentrations and cell lines used.Increased DNA damage was commonly observed in multiple studies through increased γH2AX and increased 53BP1, which are biomarkers for double-stranded DNA breaks [19,24,45,79,80,102]. Interestingly, DNA repair was often increased initially in the cells and then subsequently decreased as the exposure time and dose of arecoline increased [24,69], which was observed through the reduced phosphorylated ataxia telangiectasia-mutated (p-ATM) gene level [24,46] and increased miR-23a expression [82].
EMT has been shown to play a major role in carcinogenesis; our study highlighted the finding that arecoline directly promotes EMT in a dose-dependent manner in oral epithelial cells via specific factors and multiple signaling pathways, including TWIST overexpression leading to the loss of E-cad [27,62,79], DEC1/FAK/AKT pathway upregulation [49], PA28γ leading to the BRAF/MEK1/ERK pathway [53], and snail family SNAI1 expression [54], which downstream leads to enhanced EMT.The biomarkers for EMT, such as the increased expression of N-cadherin and vimentin, were found to be elevated as a result of arecoline exposure in multiple studies [13,27,34].Arecoline was also found to alter cellular adhesion, which is paramount in carcinogenesis, via the upregulation of αvβ6 integrin expression in HOK and the increased attachment of U397 mononuclear cells to EAhy926 cells [57,58].Arecoline was found to promote cellular migration by upregulating S100A4, Lin28B, and COX-2 expression in OSFs and OE cells [25,[59][60][61].EMT is associated with the acquisition of invasion and metastasis.Clinical markers for predicting lymph node spread and metastasis, such as Lin28B and HSP47, were increased in OSCC compared to normal epithelium in a dose-and time-dependent manner related to arecoline exposure [25,63].It is possible that these effects are mediated by the local cholinergic system as there is abundant evidence in the literature to show that AChRs control cell-cell cohesion and EMT in the keratinocytes [103][104][105].These effects are probably pleiotropic in epithelial cells as nicotine has been shown to induce proliferation, invasion, and EMT in a variety of human cancer cell lines, including breast, lung, and pancreatic cells [106].
The link between arecoline, fibrosis, and OSF was also explored.Arecoline is linked to fibrosis, a condition that causes the lining of the mouth to become thick and fibrous.Multiple in vitro studies have explored the potential underlying mechanisms and signaling pathways, including TGF-β signaling, the downregulation of the c terminus of Hsp70 interacting protein (CHIP), leading to increased myofibroblast activities [66], α-SMA and MAD proteins (SMAD) [55,57,66], miR-10b upregulation [79], TWIST expression [62,79], early growth response-1 (EGR-1) expression, leading to Wnt5a activation [41], TIMP-1 and TIMP-2 upregulation leading to inhibition of matrix metalloproteinases [42,46,47], and altered MMPs expression [42,45,47,64].These findings were suggested as showing the upregulation of transdifferentiation and the activity of myofibroblasts, contributing to increased collagen deposition and fibrosis.This possibly accounts for the onset of OSF, which is associated with a high risk of progression to cancer.Arecoline-treated BMFs resulted in the increased expression of various fibrosis markers, such as PAI-1, and elevated ECM synthesis and secretion through the upregulation of S100A4 [47, 60,61,[63][64][65].Arecoline-induced fibrosis is often associated with micro-hypoxia, which was observed through an increased CAIX level [44].
The production of ROS and inflammatory cytokines are major contributors to carcinogenesis.Arecoline has been shown to induce PGE2, IL6, IFN-y, and TNF-a production by oral keratinocytes, inducing inflammation [8,77,78].In another study, it was implicated in the adaptive immune response by increasing Th17 and decreasing T-reg T-lymphocyte pathways, linking to a potential dysregulated immune response [107].Arecoline also induced immune evasion in tumor cells in one study, where PD-L1 was upregulated in cancer cells [28].nROS production was increased in multiple studies when cells were challenged with arecoline or its metabolites [12,26,54,68,69].In another study, arecoline upregulates CYP26B1, which is thought to play a role in arecoline catalysis, potentially being associated with ROS production [41].In relation to these findings, some studies showed a concordant decrease in the antioxidant activities of cells challenged by arecoline, such as a significant reduction in catalase [12,69].Meanwhile, others found an increase in activity [64,70,[72][73][74], for instance, in increased heme oxygenase-1 enzyme (HO1); however, this could play a role in enhancing the cell survival of tumor cells against oxidative injury.Most of our studies found the elevated expression of several inflammatory cytokines such as TGF-β, but one study observed a reduction in IL-6 [8,12,19,45,48,57,[75][76][77][78].In this regard, the relationship between oxidative stress and AChRs is well-established, although the prevailing literature suggests that acetylcholine suppresses oxidative stress-mediated pathways [108][109][110].
While our iterative review process was aimed at a scoping review of original studies addressing the molecular mechanisms of arecoline-induced carcinogenesis, we note that several systematic reviews on this topic have been published.Ko et al. linked arecoline and ANO to an increase in the expression of EMT inducers, such as reactive oxygen species, TGF-β1, NOTCH1, and inflammatory cytokines, and to the activation of EMTrelated proteins [112].Our mixed-methods approach is original in that it used the current knowledge base to identify gaps in the literature and inform further theory-driven searches that allowed us to identify potentially crucial mechanisms in OSF pathophysiology.
As with all study designs, our interpretive review does not come without its limitations.Our data synthesis was derived from only two relevant databases, which means that we could potentially have missed relevant studies in other databases.No clinical studies could be included, as there were no studies that tested the oral carcinogenic effect of arecoline in isolation (i.e., not in the context of areca nut or betel quid) on live humans.It should be noted that the vast majority of the included studies were from either Taiwan or India, so the sample and researcher variations are low and some systematic biases might have been introduced.Although in vivo animal models are invaluable in studying oral carcinogenesis, the results may be neither reliable nor applicable to human tissues, and most studies had a relatively small sample size.Finally, there was heterogeneity of data, with a wide breadth of signatures being implicated in carcinogenesis and conflicting results regarding their respective mechanisms.

Study Design
The iterative review process included four phases: (1) a scoping review, including qualitative synthesis of the results; (2) refinement of the search strategy, based on selected themes and/or relevant adjacent literature; (3) a second round of scoping review complemented by (4) a post hoc manual search, leading to the generation of key concepts.

Scoping Review Methodology
The scoping reviews were conducted in accordance with PRISMA-ScR guidelines [17].The following databases were searched: PubMed and the Web of Science.
The research questions being addressed in the initial scoping were: What are the molecular mechanisms underlying arecoline-induced oral carcinogenesis?Which specific signatures are associated with arecoline-induced oral carcinogenesis and how do they contribute to disease pathogenesis?
To comprehensively screen a wide range of studies, the search strategy incorporated terms such as "cancer", "carcinoma", and "carcinogenesis".Given the interest in oral cancer, the search was confined to terms related to the "mouth", "oral", or "oral cavity" search terms.The search strategy encompassed the "diagnostic markers" associated with oral carcinogenesis and terms relevant to "pathogenesis".
There were three main concepts in our initial search strategy: 1.The condition: cancer; 2.
Location of interest: oral.
The following search string and related terms were used to find the relevant publications: (cancer OR carcinogenesis OR carcinoma) AND (mouth OR oral OR oral cavity) AND (arecoline) AND (pathogenesis OR diagnostic markers).
The second round of searches included the following string and related terms: (cancer OR carcinogenesis OR carcinoma) AND (mouth OR oral OR oral cavity) AND (arecoline) AND (acetylcholine OR AChR*).
There were no restrictions on the publication dates within the searched databases.Publications released up until April 2023 were included in this review.
Although additional keywords such as "OSCC" and "biomarkers" were included in the pilot calibration, they did not significantly alter the search results.The inclusion of the terms "areca nut" and "betel nut" did yield a notable increase in the number of articles retrieved.However, for the purpose of this study, the focus was specifically on the role of arecoline (and its metabolites) in the mechanism of carcinogenesis, excluding the broader context of betel nut.Therefore, the search string was considered comprehensive enough to encompass the various terminologies related to oral carcinogenesis and to specifically target the involvement of arecoline as the etiological factor.
During the review process, careful consideration was given to the inclusion and exclusion criteria.Despite our focus on cancer, studies reporting on OSF were included in this review, as we believe that these could be relevant to understanding the mechanisms of oral carcinogenesis.Studies that were not published in the English language, as well as reviews (systematic and meta-analyses), book chapters, and non-peer-reviewed literature, were manually excluded.We excluded those studies that did not test arecoline in isolation, those that conducted an investigation on cells derived from outside of the oral cavity, and studies with indirect evidence drawn from different treatment modalities.Duplicate articles were manually excluded by comparing the search results from PubMed and the Web of Science.

Data Screening
All reviewers evaluated the same set of 196 publications that were obtained from PubMed and the Web of Science.Through discussions, the screening process was refined for this review.The data screening process was rigorous, involving five independent reviewers who meticulously assessed the titles and abstracts of the articles while following predetermined exclusion criteria.For articles that were initially unclear in terms of eligibility, a thorough examination of the full text was conducted to determine their inclusion status.Disagreements regarding study selection and data extraction were resolved by means of consensus among the reviewers.Inter-rater agreement was measured using the kappa score, and any disagreements were resolved by an impartial third-party adjudicator.The inter-rater agreement was 89.9%.

Data Extraction and Synthesis
To ensure consistency and standardization, a pre-established data extraction sheet was utilized to extract the relevant information from the full texts of the eligible studies.
The evidence obtained from the selected studies was presented in two ways.Firstly, the data extracted from the final search were organized and presented in the form of extraction tables.These tables included details such as the study type, population/sample size, intervention and control groups, outcomes, cell culture/chemical investigations, the compounds used, and diagnostic markers, as well as the key findings and conclusions.The tables presented in this paper followed a PICO format for brevity and visual clarity.
Secondly, a narrative component was integrated into the results section, providing a comprehensive overview of the investigations and consistently reporting the outcomes across multiple studies.

Conclusions
This critical interpretive synthesis enabled the identification of prevailing trends and the consolidation of data pertaining to the molecular mechanisms involved in carcinogenesis, while also highlighting crucial gaps and inconsistencies in the literature.The iterative review process and the final synthesis allowed for possible explanations to be found for arecoline-induced carcinogenesis.
In our scoping reviews, we discussed the molecular mechanisms by which arecoline promotes OSF and OSCC and recognized a wide range of specific diagnostic markers implicated in arecoline-induced carcinogenesis.We were able to highlight common mechanisms in the pathogenesis of disease, including pro-fibrotic effects, increases in DNA damage and apoptosis, increased cellular proliferation, and EMT promotion, from both in vivo and in vitro studies.This comprehensive overview of the diagnostic and prognostic biomarkers implicated in OSF carcinogenesis can guide future research directions toward evaluating diagnostic potential and accuracy and, thus, aid earlier detection and intervention.
Nonetheless, the data extracted was heterogeneous and inconsistencies were observed, necessitating a further post hoc search.The results show that most of the mechanisms that are potentially responsible for the detrimental effects of arecoline in the oral cavity may be explained by the activation of the local cholinergic system via arecoline-AChR binding.
With the results of our review, we can present insight into the most promising molecular targets in arecoline-induced OSF and OSCC, which can inform future research into topics such as the downstream effects of AChR-mediated pathways and the development of potential preventative and therapeutic options.

Pharmaceuticals 2023 , 21 Figure 1 .
Figure 1.PRISMA flow diagram demonstrating the selection process for the initial scoping review.PRISMA: preferred reporting items for systematic reviews and meta-analysis.

Figure 1 .
Figure 1.PRISMA flow diagram demonstrating the selection process for the initial scoping review.PRISMA: preferred reporting items for systematic reviews and meta-analysis.

Figure 2 .
Figure 2.Putative pathways involving the receptor-mediated signaling of arecoline.Arecoline binds preferentially to muscarinic acetylcholine receptors (mAChR) but can also serve as a partial agonist of nicotinic receptors (nAChR).Two key molecular pathways involve EGFR and integrins.mAChR activates EGFR signaling via a so-called "triple-membrane-passing" pathway, whereby metalloproteinases cleave and activate EGF-like ligands, which, in turn, bind to EGFR and trigger downstream kinase signaling, including the Ras/Raf/MEK/ERK pathway.MAPK signaling can also be activated via canonical second messenger-mediated signals, as well as via nAChR.The two receptors also work synergistically to promote survival and inhibit apoptosis via PI3K/Akt and p21, respectively.Together, these pathways promote the expression of proliferation and survival genes, as well as migration/invasion and fibrosis/senescence via integrins and TGF-beta signaling, respectively (brown arrow).See Abbreviations part for the abbreviations and acronyms.

Figure 2 .
Figure 2.Putative pathways involving the receptor-mediated signaling of arecoline.Arecoline binds preferentially to muscarinic acetylcholine receptors (mAChR) but can also serve as a partial agonist of nicotinic receptors (nAChR).Two key molecular pathways involve EGFR and integrins.mAChR activates EGFR signaling via a so-called "triple-membrane-passing" pathway, whereby metalloproteinases cleave and activate EGF-like ligands, which, in turn, bind to EGFR and trigger downstream kinase signaling, including the Ras/Raf/MEK/ERK pathway.MAPK signaling can also be activated via canonical second messenger-mediated signals, as well as via nAChR.The two receptors also work synergistically to promote survival and inhibit apoptosis via PI3K/Akt and p21, respectively.Together, these pathways promote the expression of proliferation and survival genes, as well as migration/invasion and fibrosis/senescence via integrins and TGF-beta signaling, respectively (brown arrow).See Abbreviations part for the abbreviations and acronyms.

Table 1 .
Summary of the in vivo studies included in the scoping review *.