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Molecular pathways of oral submucous fibrosis and its progression to malignancy
Abstract
Objective:
The review aims to comprehend various factors engaged in the alteration of molecular events resulting in Oral submucous fibrosis (OSMF) and its malignant transformation.
Design:
Literature pertinent to pathways involved in OSMF were explored in databases such as PubMed, Scopus and Google Scholar. The relevant literature was reviewed and critically appraised in this narrative review.
Results:
Areca nut components influence myriad of cellular molecules such as cytokines, growth factors, myofibroblasts, non-coding RNAs and alter their expression. These aberrantly expressed molecules drive the progression of OSMF from localized inflammation to fibrosis of buccal mucosa. The oral tissue suffers from oxidative stress, hypoxia, autophagy, aberration of cell cycle and DNA damage. Apoptosis of epithelial layer results in its atrophy facilitating deeper penetration of areca nut elements. With the advance of disease, epithelial-mesenchymal transition eventuates and promotes dysplasia. The jeopardized expression of various cellular molecules, suppressed apoptosis, along with increased genetic alterations and neovascularization favors the malignant transformation.
Conclusion:
OSMF is a progressive disorder with complex mechanism of pathogenesis initiated by inflammation of oral mucosa. Continuous habit of areca nut chewing and the resulting insult to the tissues prevents healing process and is destined to debilitating disease which affects the quality of life with a higher probability of progression to malignancy.
... The biological functions of AN components and their pharmacological and toxicological effects are described in detail as below. AN provides popular compounds with several studies focusin eral articles describing the effects caused by areca nut (AN) previou some of the review articles focused on specific aspects, such as car addiction [11], AN cessation [12], genetic and epigenetic instability [ sis progression [14], oral cancer [15] and liver disease [4]. We here these reports and evaluate the controversial roles of AN. ...
... There are several articles describing the effects caused by areca nut (AN) previously [6][7][8][9]. In addition, some of the review articles focused on specific aspects, such as carcinogenic effects [10], addiction [11], AN cessation [12], genetic and epigenetic instability [13], submucous fibrosis progression [14], oral cancer [15] and liver disease [4]. We here review the results of these reports and evaluate the controversial roles of AN. ...
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.
... One of the essential constituents of the arecanut fruit is the presence of an alkaloid arecoline (AER) which is hydrolyzed to arecaidine on reacting with lime. Both AER and arecaidine are carcinogenic and mutagenic, contributing to the malignant transformation of OSF to Oral Squamous Cell Carcinoma (OSCC) [1,4,5]. OSFs worldwide malignant transformation rate is estimated to be 1.2% to 23% and is classified as a potentially malignant disorder [6,7]. ...
Oral Submucous Fibrosis is a potentially malignant disorder caused by habitual areca nut chewing, which contributes to the dispersion of active alkaloids into subepithelial tissues, stimulating excessive extracellular matrix deposition. Various treatment modalities are available; however, their efficacy in inhibiting fibrosis progression remains limited. Sulforaphane (SFN), an isothiocyanate found abundantly in cruciferous plants, is known to have effective antifibrotic properties. Objective: The present study investigated the antifibrotic effect of SFN via phosphatidylinositol 3 kinase (PI3K), Serine/Threonine Kinase 1 (AKT-1), mammalian target of rapamycin (mTOR) pathway in arecoline (AER) induced fibrosis in human gingival fibroblasts [HGFs]. Material and Methods: MTT assay determined the half-maximal inhibitory concentration of AER and SFN at 24h in the HGF cell line. Expression levels of transforming growth factor B1 (TGFB1), collagen type 1 alpha 2 (COL1A2), hydroxyproline (HYP), PI3, AKT, mTOR, and nuclear factor erythroid 2–related factor 2 (NRF2) were assessed post-AER and SFN treatment using qPCR and western blot analysis. Results: The findings of the study revealed that AER elicited a stimulatory effect, upregulating TGFB1, COL1A2, HYP, PI3K, AKT, and mTOR and downregulating NRF2 expression. Conversely, SFN treatment significantly upregulated NRF2, inhibiting TGFB1 mediated PI3/ AKT/mTOR pathway. Conclusion: These observations suggest that SFN can be used as a promising synergistic antifibrotic agent to combat fibrogenesis via the non-Smad pathway.
... OSMF has several clinical and histological features [3]. Although it is multifactorial, the primary factor responsible for bringing about these changes is considered to be the continuous use of areca or betel nut [4]. Gutka, a commercially available areca nut product, is associated with the early presentation and progression of OSMF. ...
Background: Oral Submucous Fibrosis (OSMF) is a well-established oral potentially malignant disorder (OPMD) affecting people of Pakistan, India, Bangladesh and Sri Lanka because these nations have a long-standing history of chewing areca nut. Objective: The primary objective of the current study was to assess the clinical features of OSMF in patients presented to. Clinicopathological parameters of OSMF like age, gender, habits and its frequency and duration, clinical signs and symptoms and stages were recorded. Result: There were a total of 60 patients of which 35 were males and 25 were females. The mean age of study subjects was calculated to be 36.46±11.96 years. More than 50% of the participants were using a combination of various tobacco products. Most of the patients in our study were of stage III followed by stages II, I and IV. 70% of patients had buccal fibrous bands bilaterally, 36.66% of patients had tongue restriction and 60% of patients complained of burning sensation in the mouth. Conclusion: OSMF becomes a disease of the young generation with a slight male predilection. The majority of individuals suffered from a severe and advanced form of the disease. The development of diagnostic aids is necessary to stop the disease in its early stages.
... Long-term non-healing of oral mucosal tissue damage can lead to debilitating diseases, affect the quality of life, and has a higher likelihood of developing into malignancy. Once it develops into precancerous lesions or cancerous transformation, it can even lead to hearing loss (7,8). From the pathological mechanism, OSF is featured by extreme fibrosis of the submucosa, and angiogenesis contributes to oral mucosal fibrosis (9). ...
... Another possibility is that collagen accumulation in the submucosa might lead to tissue hypoxia, which is a cancerinducing factor [7]. Chronic irritation due to areca nut usage upregulates pro-inflammatory cytokines and reduces anti-fibrotic IFN-gamma [8], which causes increased fibrosis and juxtaepithelial inflammatory reaction, eventually resulting in epithelial atrophy. Thus, multiple molecular events are happening in both epithelium and connective tissue, leading to the malignant transformation of OSMF, which is why there are numerous studies [9][10][11] on site-specific biomarkers in OSMF. Expression of P53 [12][13][14], Ki-67 [15], Bcl2 [16], and Bax [17] in the basal and parabasal layers of epithelium are reportedly high in OSMF and reported as early events of the malignant transformation of OSMF. ...
The objective of the study was to compare the expression of immunohistochemical (IHC) markers of oral submucous fibrosis (OSMF) (non-transformed group) to those of oral squamous cell carcinoma (OSCC) transformed from OSMF (transformed group). The search for comparative cross-sectional studies was carried out in PubMed and Scopus abiding to the PICO criteria, where expression of IHC markers in OSMF were compared with that of OSCC transformed from OSMF. The cellular distribution, number of positive cases, staining intensity, and mean immunoreactive score (IRS) of each IHC marker were evaluated in both groups. A total of 14 studies were included in the systematic review, in which immunoexpression of 15 epithelial and 4 connective tissue biomarkers were evaluated. Expression of β1-integrin, OCT-3, CD1a, CD207, survivin, Dickkopf-1, COX-2, hTERT, CTGF, MDM2, Ki-67, and α-SMA were increased during transformation of OSMF to OSCC. Conversely, expression of PTEN and lysyl oxidase decreased during transformation of OSMF to OSCC. Expression of a group of epithelial markers, such as COX2, hTERT, CTGF, survivin, MDM2, and p53, was 38 times lower in the non-transformed group cases compared to transformed group cases (95% CI: 58% to 10%; p = 0.01; and I2 = 90%). Meta-analysis of all markers involved in cell metabolism/apoptosis, which included β1-integrin along with the above markers also suggested 42 times lower expression in the non-transformed group as compared to the transformed group (95% CI: 58% to 10%; p = 0.01; and I2 = 90%). Sub-group analyses on cytoplasmic and nuclear epithelial markers were inconclusive. Meta-analysis of connective tissue markers was also inconclusive. No publication bias was found. Instead of delving into numerous markers without a strong basis for their use, it is advisable to further study the markers identified in this study to explore their clinical utility.
... The activation of myofibroblasts primarily contributes to this, as activated myofibroblasts are responsible for abnormal ECM deposition. 8 The expression of a-smooth muscle actin (a-SMA), a marker of differentiated myofibroblasts, 9 in fact, has a positive association with OSF. 10 Another key factor in the pathogenesis of OSF is the development of epithelial-to-mesenchymal transition (EMT) among epithelial cells, in which these cells might have the potential to become myofibroblasts. 11,12 EMT is defined as a process of the transformation of epithelial cells into migratory mesenchymal cells. ...
Background
Nature provides us with a diversity of plants and fruits. These organic plants and the products they produce might have some therapeutic potential that can be applied to the treatment of specific bacterial and pathological disorders of the body. Yet, some of these ingredients may have harmful effects on tissue and health if they are abused. In Hindu culture, areca nuts (ANs) are widely used for food, Ayurvedic medicine, and social and religious purposes. Nonetheless, frequent use may cause some changes to the oral environment.
Aim
The purpose of this review is to provide an overview of how AN addiction affects the oral environment and dental health.
Materials and methods
Electronic research of the published English literature was performed in PubMed/Medline, Science Direct, Web of Science, Embase, Google Scholar, and Scopus databases, from 2001 to 2023 using mesh keywords such as (areca nut or betel nut) and (oral tissues or oral cavity or oral environment). A manual search of all the related journals was also performed. We also checked the reference lists of the relevant articles.
Conclusions
Although the AN plays a positive function in dental and digestive health, excessive ingestion can have negative effects on the environment and oral tissues. The globe is now aware of using this sweetener with caution because of the increased prevalence and incidence of numerous oral illnesses induced by this habit for the last few years, especially in school-age youngsters. It has been discovered that this behavior is linked to even the precancerous diseases and lesions that lead to mouth cancer. Hence, it is essential to persuade them to stop chewing ANs to avoid further difficulties.
A patient had burning and pain in the mouth, reduced oral aperture, white-tan plaques on the oral mucosa, and thickened buccal mucosae bilaterally; biopsy of the lower labial mucosa showed subepithelial fibrosis. She had no history of cigarette smoking or use of chewing tobacco but had current and past history of chewing areca nuts. What is the diagnosis and what would you do next?
Background
Oral submucous fibrosis (OSMF) is a potentially malignant disorder. Although areca nut chewing is an established risk factor, its low prevalence among nut chewers indicates additional factors likely facilitates pathogenesis. We recently demonstrated high fluoride levels in smokeless tobacco products and hypothesized a potential pathological role of fluoride in OSMF. Further exploring this novel role, this study compared fluoride levels in tissue, serum, and saliva samples from OSMF patients and healthy controls.
Methods
The ethically approved study included 25 clinically confirmed OSMF patients and 25 healthy matched controls. OSMF cases underwent buccal mucosal incisional biopsy, while controls had buccal mucosa tissue sampling during third molar removal. Fasting venous blood and unstimulated saliva were collected. Fluoride levels were analysed using ion chromatography and expressed as median (IQR).
Results
OSMF cases showed significantly higher fluoride concentrations compared with controls in tissue biopsies (30.1 vs. 0 mg/kg, p < 0.0001), serum (0.4 vs. 0 mg/L, p = 0.005) and saliva (1.3 vs. 0 mg/L, p < 0.0001). Majority (68%) of controls had undetectable fluoride levels across all samples. Tissue fluoride weakly correlated with OSMF severity ( r = −0.158, p = 0.334).
Conclusion
The preliminary findings demonstrated increased tissue fluoride levels in OSMF patients compared with healthy controls. Along with a previous study showing high fluoride content in smokeless tobacco products, these findings provided early evidence suggesting fluoride could play a contributory role in OSMF pathogenesis. Further large‐scale investigation is warranted to definitively establish whether the association between fluoride exposure and OSMF is indicative of causation.
Background: Areca Nut (AN) is the fourth most commonly abused drug after nicotine, ethanol, and caffeine, due to its psychoactive properties provided by bioactive substances. Although previous studies have demonstrated AN’s anxiolytic-like activity and potential benefits in ameliorating symptoms of depression and schizophrenia, there remains limited awareness regarding its association with brief psychotic disorder.
Case Presentation: This case report presents the clinical profile of a 30-year-old male patient with a history of betel nut chewing for the past 2 years, who exhibited sudden onset delusions, hallucinations, and disorganized speech and behavior upon increasing the dosage of betel nut consumption. The patient displayed a positive response to antipsychotic treatment, and symptoms resolved upon discontinuation of betel nut consumption. However, one month after discharge, the patient experienced a recurrence of auditory hallucinations upon resuming betel nut chewing. Through counseling and support, the importance of abstaining from betel nut use and maintaining medication compliance was emphasized, resulting in no recurrence of psychotic symptoms during the six-month follow-up.
Conclusion: This case report highlights the potential role of betel nut in triggering brief psychotic disorder, especially when the chewing dosage is abruptly increased. It underscores the importance of considering betel nut as a potential precipitant of acute psychiatric disorders in clinical settings.
Betel quid (BQ) is a package of mixed constituents that is chewed by more than 600 million people worldwide, particularly in Asia. The formulation of BQ depends on a variety of factors but typically includes areca nut, betel leaf, and slaked lime and may or may not contain tobacco. BQ chewing is strongly associated with the development of potentially malignant and malignant diseases of the mouth such as oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively. We have shown recently that the constituents of BQ vary geographically and that the capacity to induce disease reflects the distinct chemical composition of the BQ. In this review, we examined the diverse chemical constituents of BQ and their putative role in oral carcinogenesis. Four major areca alkaloids—arecoline, arecaidine, guvacoline and guvacine—together with the polyphenols, were identified as being potentially involved in oral carcinogenesis. Further, we propose that fibroblast senescence, which is induced by certain BQ components, may be a key driver of tumour progression in OSMF and OSCC. Our study emphasizes that the characterization of the detrimental or protective effects of specific BQ ingredients may facilitate the development of targeted BQ formulations to prevent and/or treat potentially malignant oral disorders and oral cancer in BQ users.
The edible endosperm of Areca catechu is recognized as a potent carcinogenic agent either consumed alone or in combination with tobacco. Habitual chewing of areca nut leads to orally potential malignant disorders which are highly effective in malignant transformation and thereby lead to oral carcinogenesis. Human buccal epithelial KB carcinoma cells were used as an experimental cell system to inspect the mechanistic act of aqueous extract of areca nut on biochemical status and their implications on transcriptional activation of cancer signaling cascade that could possibly trigger numerous oncogenic players and finally decides the cells fate. Extract treated cells showed reduced viability with altered balance between oxidants and antioxidants which lead to redox status and which is known to distort various biological processes within the cell system. Results of RT-PCR demonstrated decreased expression of BCl2, cell cycle regulators along with Activator Protein −1 (AP-1) components. While Bax, p16 and p21 mRNAs showed increased expression in extract treated KB cells. Likewise, the translational levels of proliferation cell nuclear antigen (PCNA), tumor suppressor p53, retinoblastoma (Rb) and cyclin dependent kinase 4 (CDK4) were decreased along with AP-1 subunits (c-Jun/c-Fos) with increased protein levels of p21 in extract treated KB cells. Further, the downstream activation and regulation of AP-1 transcription factors could be through stress activated c-Jun – N terminal Kinase (JNK) Mitogen Activated Protein Kinases (MAPKs) which downregulated both Jun and Fos mRNA transcripts in areca nut extract exposed KB cells. Thus, outcome of the study provides insights into mechanistic path of pathogenesis of areca related disorders. Further, it could aid in designing new therapeutic modalities that specific targets these oncogenic players and help in disease management.
Background:
To explore the effectiveness of adenovirus-enhanced green fluorescent protein-vascular endothelial growth factor165 (AD-EGFP-VEGF165) transfection on fibroblasts from mice, and we assessed whether VEGF165 restores the angiogenesis of oral submucous fibrosis (OSF) in mice.
Methods:
AD-EGFP-VEGF165 and AD-EGFP were transfected into fibroblasts from mouse buccal tissues in vitro. The expression of VEGF before and after transfection was detected by RT-qPCR and ELISA in each group of fibroblasts. Fifteen OSF mice (pre-experimental construction) were randomly divided into 3 groups, and equal amounts of AD-EGFP-VEGF165 virus, AD-EGFP virus, and saline were injected into the buccal submucosal tissue of OSF mice. The expression of VEGF and local tissue angiogenesis were observed and measured in each group of animals.
Results:
The Ad-EGFP-VEGF165-transfected fibroblasts increased human and mouse VEGF expression compared to the Ad-EGFP group and control group (P<0.05). The buccal submucosal tissue of mice was injected with Ad-EGFP-VEGF165 after the 6th day, and the expression of VEGF was effectively expressed in AD-EGFP-VEGF165 group (P<0.05), while no positive expression observed in other groups. and the number of microvessels in the AD-EGFP-VEGF165 group increased significantly compared to the other groups (P<0.05).
Conclusions:
Ad-EGFP-VEGF165 can be successfully transfected into fibroblasts from mice, and restored the angiogenesis of OSF in mice.
Background:
Oral submucous fibrosis (OSMF) is a chronic, potentially malignant condition of the oral mucosa, predominantly seen in people of Asian descent. The reported malignant transformation rate of OSMF is 7%-13%. In the context of the understanding progression of OSMF, the study of prime molecular expressions is essential. Various markers have received more attention, one of them is E-cadherin. Various factors which promote epithelial-mesenchymal transition (EMT) and inhibit E-Cadherin include Snail1, Snail2, Twist and EF1/ZEB1. The intended study was undertaken to evaluate the possible role of E-cadherin and its regulatory markers Twist1 and Snail1 in OSMF.
Aims and objectives:
To evaluate the expression of E-cadherin, Twist1 and Snail1 in OSMFTo evaluate their possible association with malignant transformation of OSMF.
Materials and methods:
Histologically proven 20 cases of each OSMF with and without dysplasia were taken as the study group, 20 normal mucosa as control group and were subjected for immunohistochemical (IHC) expression with E-cadherin, Twist1 and snail1.
Results:
Immunohistochemical expression of all the three markers showed statistically significant expression of all the three markers. Intensity and percentage of staining between the groups were statistically significant for E-cadherin between normal oral mucosa (NOM) and OSMF with dysplasia (OSMFD), no significance was found between NOM and OSMF, whereas Snail1 and Twsit1 were statistically significant between NOM and OSMF and also between NOM and OSMFD. However, no significance was found for all the three markers when compared between the groups OSMF and OSMFD.
Conclusion:
The increased expression of Snail1 and Twist1 with concomitant loss of E -cadherin in OSMF suggests the role of the EMT.
Oral submucous fibrosis (OSF) is known as a potentially malignant disorder, which may result from chemical irritation due to areca nuts (such as arecoline). Emerging evidence suggests that fibrogenesis and carcinogenesis are regulated by the interaction of long noncoding RNAs (lncRNAs) and microRNAs. Among these regulators, profibrotic lncRNA H19 has been found to be overexpressed in several fibrosis diseases. Here, we examined the expression of H19 in OSF specimens and its functional role in fibrotic buccal mucosal fibroblasts (fBMFs). Our results indicate that the aberrantly overexpressed H19 contributed to higher myofibroblast activities, such as collagen gel contractility and migration ability. We also demonstrated that H19 interacted with miR-29b, which suppressed the direct binding of miR-29b to the 3′-untranslated region of type I collagen (COL1A1). We showed that ectopic expression of miR-29b ameliorated various myofibroblast phenotypes and the expression of α-smooth muscle actin (α-SMA), COL1A1, and fibronectin (FN1) in fBMFs. In OSF tissues, we found that the expression of miR-29b was downregulated and there was a negative correlation between miR-29b and these fibrosis markers. Lastly, we demonstrate that arecoline stimulated the upregulation of H19 through the transforming growth factor (TGF)-β pathway. Altogether, this study suggests that increased TGF-β secretion following areca nut chewing may induce the upregulation of H19, which serves as a natural sponge for miR-29b and impedes its antifibrotic effects.
Annexin IV (ANXA4) is highly expressed in ovarian clear cell carcinoma (OCCC); however, its underlying molecular mechanism in OCCC remains unknown. The present study aimed to identify the molecule that ANXA4 may act on and to determine its underlying molecular mechanism. Immunohistochemistry, co‑immunoprecipitation and western blotting were performed to detect the expression and interaction of ANXA4, and its associated proteins. Furthermore, MTT assay, flow cytometry, western blotting and gene expression profile enrichment analysis were performed to identify the potential role and molecular mechanism of ANXA4 in OCCC. The results demonstrated that ANXA4 and nuclear factor‑κ‑light‑chain‑enhancer of activated B cells (NF‑κB) p50 nuclear expression levels were significantly higher in OCCC tissues compared with other subtypes of ovarian cancer, such as serous and mucinous. In addition, a significantly positive correlation was observed between ANXA4 and NF‑κB p50 expression in OCCC; however, the expression levels of mutant p53 and ANXA4 were negatively correlated in a linear manner. These results suggest that ANXA4 and NF‑κB p50 may be potential independent risk factors for poor prognosis. ANXA4 and NF‑κB p50 were demonstrated to interact and their expression was co‑localized. The cBioPortal database was used to construct a protein‑protein interaction network between ANXA4, NF‑κB p50 and p53, and functional pathway analysis indicated that the genes were predominantly enriched in the cell cycle and during apoptosis. Transfection of the ANXA4 gene increased the expression of NF‑κB p50, as well as its downstream targets, Cyclin D1 and B‑cell lymphoma‑2 (Bcl‑2). Furthermore, transfection of the ANXA4 gene increased proliferation and decreased apoptosis of OCCC cells. Treatment with the NF‑κB inhibitor, BAY 11‑7082, decreased Cyclin D1 and Bcl‑2 expression levels. Collectively, the results of the present study suggest that wild p53 activates ANXA4 transcription, promotes its expression and enhances NF‑κB p50 and ANXA4 interaction. This in turn activates the NF‑κB signaling pathway, promotes cell cycle progression and inhibits apoptosis, thus contributing to the malignant progression of OCCC. Thus, ANXA4 and NF‑κB p50 may be used as prognostic biomarkers, and may be molecular therapeutic targets in OCCC.
Betel quid (BQ) chewing increased the risk of oral cancer and oral submucous fibrosis (OSMF), an oral premalignant disorder (OPMD) with malignant transformation potential. BQ components such as areca nut (AN), trauma by coarse AN fiber, catechin, copper, alkaloids, stimulated reactive oxygen species (ROS), inflammation and cytotoxicity are suggested to be the contributing factors. They may induce tissue inflammation, proliferation of fibroblasts and collagen deposition, myofibroblast differentiation and contraction, collagen cross-links and inhibit collagen phagocytosis, finally leading to the development of OSMF and oral cancer. These events are mediated by BQ components-induced changes of extracellular matrix (ECM) turnover via regulation of TGF-β1, plasminogen activator inhibitor-1 (PAI-1), cystatin, lysyl oxidase (LOX) and tissue inhibitors of metalloproteinases (TIMPs) and metalloproteinases (MMPs). Genetic susceptibility is also involved in these disease processes. Further understanding the molecular mechanisms of BQ-induced OSMF and oral cancer can be helpful for future disease prevention and treatment.
Oral submucosal fibrosis (OSF) is a premalignant disorder of the oral cavity, and areca nut chewing is known to be a major etiological factor that could induce epithelial to mesenchymal transition (EMT) and activate buccal mucosal fibroblasts (BMFs). However, this detailed mechanism is not fully understood. In this study, we showed that the upregulation of Snail in OSF samples and fibrotic BMFs (fBMFs) may result from constant irritation by arecoline, a major alkaloid of the areca nut. The elevation of Snail triggered myofibroblast transdifferentiation and was crucial to the persistent activation of fBMFs. Meanwhile, Snail increased the expression of numerous fibrosis factors (e.g., α-SMA and collagen I) as well as IL-6. Results from bioinformatics software and a luciferase-based reporter assay revealed that IL-6 was a direct target of Snail. Moreover, IL-6 in BMFs was found to further increase the expression of Snail and mediate Snail-induced myofibroblast activation. These findings suggested that there was a positive loop between Snail and IL-6 to regulate the areca nut-associated myofibroblast transdifferentiation, which implied that the blockage of Snail may serve as a favorable therapeutic strategy for OSF treatment.
Background: Pathology involving the oral epithelium may alter the level of salivary concentration of LDH. Thus its estimation can be used as a non invasive screening tool for the early detection of OPMDs and also to predict its malignant transformation especially in high risk population.
Aims and Objectives: To evaluate the salivary and serum levels of lactate dehydrogenase (LDH) in patients having of oral submucous fibrosis (OSMF) and leukoplakia and compare it with healthy individuals.
Materials and Methods: A total of 120 subjects were selected and divided into three groups comprising clinically diagnosed cases of OSMF and leukoplakia and healthy subjects as controls. Unstimulated whole saliva and blood samples were collected under aseptic conditions for biochemical estimation of LDH by Semiautomatic Analyzer using LDH kit utilizing enzymatic UV-Kinetic method. The values obtained were statistically analyzed using the SPSS software version 20.0. P-value < 0.05 was considered significant.
Results: The mean salivary LDH level in Group I (OSMF) was 631.67 + 7.67, Group II (Leukoplakia) was 492.28 + 16.17 and Group III (Healthy Control) was 140.62 + 8.87. There was a statistically significant difference between the Serum and salivary LDH levels among the various groups of study population. A positive correlation between salivary LDH and serum LDH level was seen and the regression equation for OSMF and leukoplakia was computed.
Conclusion: A significant difference was found between mean salivary LDH Levels and serum LDH levels in patients with leukoplakia, OSMF and health controls. A positive correlation was also established between salivary and serum LDH levels in patients with OSMF and leukoplakia patients making saliva a potent non invasive tool for early prediction and detection of PMOD and its malignant transformation.
Context:
Hypoxia-inducible factor (HIF)-2α is overexpressed in primary and metastatic human cancers, whose expression is correlated with tumor angiogenesis and patient mortality. HIF plays a role in the progression of fibrosis in oral submucous fibrosis (OSF).
Aim and objective:
The aim is to study and compare the expression of HIF-2α in OSF (a), oral squamous cell carcinoma (OSCC) with areca nut usage (b), OSCC without areca nut usage (c) and normal mucosa (d) by immunohistochemistry.
Subjects and methods:
Immunohistochemical detection of HIF-2α was done on 51 samples, which included 11 cases (a), 15 cases (b), 15 cases (c) and the expression was compared with that of (d).
Statistical analysis:
Data were analyzed using the SPSS™ software (ver. 21.0). Chi-square test and kappa analysis were performed to compare the intensity of staining between the groups and for inter-observer agreement, respectively. Value of P ≤ 0.05 was considered statistically significant. The mean labeling index between the groups was studied by the Kruskal-Wallis test.
Results:
All the cases of (d), (a), (b) and (c) showed HIF-2α expression (P = 0.329). About 13% cases of (c) showed intense expression (P = 0.406) and 27% of (a) cases showed expression only in the connective tissue (P = 0.023). The number of positively stained nuclei in both (b and c) cases reduced as the tumor progression was from well to poorly differentiated.
Conclusion:
Areca nut initiates fibrosis and subsequent hypoxia in OSF which triggers HIF-2α expression in the epithelium. HIF-2α could be a surrogate marker for cancer initiation and progression.
Background:
Oral submucous fibrosis (OSF) is a precancerous condition predominantly seen in people of Asian descent. About 7%-12% of OSF patients develop oral squamous cell carcinoma. Morphological features of OSF, especially fibrosis, suggest a possibility of hypoxic environment in diseased tissues. Neovascularization and increased glycolysis, represent adaptations to a hypoxic microenvironment that are correlated with tumor invasion and metastasis. The adaptation of cells to hypoxia appears to be mediated through hypoxia-inducible factor-1α (HIF-1α). HIF-1α is said to be associated with malignant transformation of epithelium in other sites.
Aim:
The aim of this study was to investigate the relationship between the expression of HIF-1α in OSMF and its role in malignant transformation.
Materials and methods:
A retrospective study which included 20 histopathologically diagnosed cases of OSF was conducted. A qualitative evaluation of HIF-1α was performed. Statistical analysis was carried out using the IBM Statistical Package for Social Sciences 20.0 version (IBM Corporation, Armonk, NY, USA).
Results:
Results showed an increased expression of HIF-1α in OSF.
Conclusion:
HIF-1α appears to play a role in malignant transformation of OSF.
Oral submucous fibrosis (OSF) has been recognized as a precancerous disorder in the oral cavity. Great effort has been made to inhibit the malignant progression of OSF over the past decades, but the cure of this fibrosis disease has not been discovered. In the present study, we found that a long noncoding RNA, LINC00312, was upregulated in OSF tissues, and positively associated with several fibrosis factors, such as α-SMA, type I collagen, and fibronectin. As such, we sought to investigate the role of LINC00312 in OSF progression and identify its interacting factor that mediated oral fibrogenesis. Our results showed that the inhibition of LINC00312 downregulated the myofibroblast activities, including collagen gel contractility, transwell migration, and wound healing, as well as the gene expression of myofibroblast markers. We verified that YBX1 was a downstream factor of LINC00312 and revealed that the downregulation of YBX1 repressed the gene expression of α-SMA and p-Smad2 along with the reduced myofibroblast phenotypes. Most importantly, we demonstrated that the LINC00312-induced myofibroblast activities were reverted by the knockdown of YBX1, suggesting that the LINC00312-mediated myofibroblast transdifferentiation was through YBX1. Collectively, our findings revealed that the LINC00312/ YBX1 axis may serve as a target for the development of therapies against OSF.
Background:
Oral squamous cell carcinoma (OSCC) is a life-threatening disease. It could be preceded by oral potentially malignant disorders (OPMDs). It was confirmed that chronic inflammation can promote carcinogenesis. Cytokines play a crucial role in this process. The aim of the study was to evaluate interleukin-1alpha (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) in tissue specimens and saliva of patients with OSCC and OPMDs.
Methods:
Cytokines were evaluated in 60 tissue specimens of pathological lesions (OSCCs or OPMDs) and in 7 controls (normal oral mucosa, NOM) by immunohistochemistry and in saliva of 45 patients with OSCC or OPMDs and 9 controls (healthy volunteers) by enzyme-linked immunosorbent assays.
Results:
Immunohistochemical analysis revealed significantly higher expression of IL-8 in OSCC specimens and TNF-α in OSCCs and OPMDs with dysplasia as compared to NOM. Moreover, expression of TNF-α was significantly higher in oral leukoplakia and oral lichen planus without dysplasia, whereas expression of IL-8 only in oral leukoplakia without dysplasia in comparison with NOM. Salivary concentrations of all evaluated cytokines were significantly higher in patients with OSCC than in controls. Moreover, levels of IL-8 were significantly higher in saliva of patients with OPMDs with dysplasia as compared to controls and in OSCC patients as compared to patients with dysplastic lesions. There was also significant increase in salivary concentrations of IL-6, IL-8 and TNF-α in patients with OSCC as compared to patients with OPMDs without dysplasia.
Conclusion:
The study confirmed that proinflammatory, NF-kappaB dependent cytokines are involved in pathogenesis of OPMDs and OSCC. The most important biomarker of malignant transformation process within oral mucosa among all assessed cytokines seems to be IL-8. Further studies on a larger sample size are needed to corroborate these results.
The molecular mechanism of oral submucous fibrosis (OSF) is yet to be fully elucidated. The identification of reliable signature genes to screen patients with a high risk of OSF and to provide oral cancer surveillance is therefore required. The present study produced a filtering criterion based on network characteristics and principal component analysis, and identified the genes that were involved in OSF prognosis. Two gene expression datasets were analyzed using meta-analysis, the results of which revealed 1,176 biologically significant genes. A co-expression network was subsequently constructed and weighted gene modules were detected. The pathway and functional enrichment analyses of the present study allowed for the identification of modules 1 and 2, and their respective genes, SPARC (osteonectin), cwcv and kazal like domain proteoglycan 1 (SPOCK1) and kruppel like factor 6 (KLF6), which were involved in the occurrence of OSF. The results revealed that both genes had a prominent role in epithelial to mesenchymal transition during OSF progression. The genes identified in the present study require further exploration and validation within clinical settings to determine their roles in OSF.
Objective
To observe the expression patterns of salivary mRNA 21 in different stages and grades of OSMF and also in habitual arecanut chewers without OSMF.
Subjects and methods
The study consisted of a total of 185 samples, where 61 patients had chewing habits (chewing gutkha and other forms of arecanut) and had OSMF (Group 1). Sixty-one patients had chewing habits but did not have OSMF (Group 2), and 63 were normal healthy patients (control group) without any chewing habits (Group 3). Unstimulated saliva samples were collected from patients following the standard operating procedures. miRNA 21 was isolated and purified from saliva samples using the miRNeasy Mini Kit, Qiagen. The primers for miRNA relative quantification analysis were designed using the Primer Express software of Applied Biosystems. Quantification of all the samples was carried out using SYBR chemistry in an Applied Biosystems Real-Time PCR.
Results
There was no statistically significant difference between the demographic characteristics of patients. There was a statistically significant difference between the expressions of miRNA 21 amongst the three groups noted in Kruskal Wallis test. (<0.001*) A post hoc test was done to confirm the statistical difference between patients within all three groups. There was no statistically significant difference noted between the OSMF group and patients with chewing habits group (G1 vs. G2 p: 0.10), but there was a significant difference when compared with normal patients. (G1 vs G3 p: <0.001*) and (G2 vs G3 <0.001*)
Conclusion
This study concludes that miRNA 21 is overexpressed in OSMF and chewing habit patients. But the expression levels were not significantly associated with the severity of the disease process. A long term and large scale study are required to assess its application as a diagnostic profibrotic marker in OSMF.
Long non-coding RNA hypoxia-inducible factor 1α-antisense RNA 1 (HIF1A-AS1) has been known to participate in various types of malignancies, but its role in the development of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we first observed the aberrant upregulation of HIF1A-AS1 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) isolated from OSF specimens. Next, we demonstrated that administration of arecoline, a natural alkaloid that is found in areca nut, induced the elevation of HIF1A-AS1 in BMFs. This finding showed that the habit of areca nut chewing may lead to an increase of HIF1A-AS1 in oral mucosa. Moreover, we found that knockdown of HIF1A-AS1 hindered the arecoline-stimulated migration capacity in BMFs, suggesting HIF1A-AS1 was critical to the transdifferentiation of BMFs into myofibroblasts. Altogether, our results demonstrated that overexpression of HIF1A-AS1 in OSF tissues may result from the use of areca nut and lead to activation of BMFs, which contribute to the progression of OSF.
Oral Submucous fibrosis (OSMF) has traditionally been described as "a chronic, insidious, scarring disease of the oral cavity, often with involvement of the pharynx and the upper esophagus". Millions of individuals are affected, especially in South and South East Asian countries. The main risk factor is areca nut chewing. Due to its high morbidity and high malignant transformation rate, constant efforts have been made to develop effective management. Despite this, there have been no significant improvements in prognosis for decades. This expert opinion paper updates the literature and provides a critique of diagnostic and therapeutic pitfalls common in developing countries and of deficiencies in management. An inter-professional model is proposed to avoid these pitfalls and to reduce these deficiencies.
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.
Background:
There are 200-600 million betel quid (BQ) chewers in the world. BQ increases oral cancer risk. Matrix metalloproteinase-9 (MMP-9) is responsible for matrix degradation, cancer invasion and metastasis. Whether areca nut extract (ANE), a BQ component, stimulates MMP-9 secretion, and the related signaling pathways awaits investigation.
Results:
ANE (but not arecoline) stimulated MMP-9 production of gingival keratinocytes and SAS cancer epithelial cells. ANE stimulated TGF-β1, p-Smad2, and p-TAK1 protein expression. ANE-induced MMP-9 production/expression in SAS cells can be attenuated by SB431542 (ALK5/Smad2 inhibitor), 5Z-7-Oxozeaenol (TAK1 inhibitor), catalase, PD153035 (EGFR tyrosine kinase inhibitor), AG490 (JAK inhibitor), U0126 (MEK/ERK inhibitor), LY294002 (PI3K/Akt inhibitor), betel leaf (PBL) extract, and hydroxychavicol (HC, a PBL component), and melatonin, but not by aspirin.
Conclusions:
AN components contribute to oral carcinogenesis by stimulating MMP-9 secretion, thus enhancing tumor invasion/metastasis. These events are related to reactive oxygen species, TGF-β1, Smad2-dependent and -independent signaling, but not COX. These signaling molecules can be biomarkers of BQ carcinogenesis. PBL, HC and melatonin and other targeting therapy can be used for oral cancer treatment.
Methods:
ANE-induced MMP-9 expression/secretion of oral epithelial cells and related TGF-β1, Smad-dependent and -independent signaling were studied by MTT assay, RT-PCR, western blotting, immunofluorescent staining, and ELISA.
Oral submucous fibrosis (OSMF) is a potentially malignant disorder, characterized by progressive fibrosis of the lamina propria and underlying connective tissue. It has high chances of malignant transformation. It is caused by betel nut which is very common habit among Indians. Thus, regular monitoring of histopathological changes is mandatory by physicians, private practitioners, and oral pathologists. Therefore, histopathological changes should be understood by everyone who is into health care. This article is a preliminary report on three-dimensional (3D) images and 3D-animation video of histopathological aspect of OSMF designed by author herself, for better understanding of histopathological aspect, which has never been reported so far. Additional hypothesis on epithelial atrophy have also been proposed.
Background:
Identification and comparison of gene expression of Lysyl oxidase (LOX) in oral submucous fibrosis and controls and to determine its role in Pathogenesis of Oral submucous fibrosis.
Material and methods:
Of total sample size (n=127), the whole blood sample were collected from case and control group in citrate vial. It is centrifused and stored at -800C. We collected and isolated RNA from blood of case group (n=127) and age and sex matched control group (n=127) recruited on the basis of inclusion criteria. The cDNA was prepared for 127 samples which were processed for gene expression of Lysyl oxidase (LOX) in relation to housekeeping genes (Beta actin and 18srRNA) and its role in pathogenesis of Oral submucous fibrosis.
Results:
In relative expression (Normalized ratio),relatively 11 cases shown down-regulation of lysyl oxidase gene while 27 cases shows up-regulation of lysyl oxidase gene while in 89 cases there were no regulation i.e expression of lysyl oxidase gene in case group was of same degree of control. In non-relative expression results (Non-normalized ratio), the 38 cases shown down regulation of LOX gene while in 53 cases, it was up-regulated however in remaining 36 cases there was neither up-regulation nor down-regulation of Lysyl oxidase gene i.e the expression of LOX gene is null.
Conclusions:
In oral submucous fibrosis, the expression of Lysyl oxidase gene is mixed type i.e either it will down regulate/upregulate or there will be no expression at all comparatively. However in majority of cases the upregulation of lysyl oxidase is relatively more common than down-regulation or non expression of Lysyl oxidase gene. Key words:Oral submucous fibrosis, lysyl oxidase, betel nut, premalignant disorders.
Oral Submucous Fibrosis (OSMF) is a chronic progressive scarring oral disease predominantly affecting people of South Asian origin. It is characterized by juxtaepithelial inflammatory cell infiltration followed by fibrosis in the lamina propria and submucosa of the oral mucosa. The pathogenesis of the disease is not well established and a number of mechanisms have been proposed regarding the pathogenesis. A renewed interest has been shown in myofibrobasts which have been implicated to play a significant role in the pathogenesis of OSMF. The myofibroblast were initially identified by means of electron microscopy in granulation tissue of healing wounds as a modulated fibroblast exhibiting features of smooth muscle cells, with prominent bundles of microfilaments, dense bodies scattered in between, and gap junctions. The presence of myofibroblasts has successively been described in practically all fibrotic situations characterized by tissue retraction and remodeling. This review paper is an attempt to identify all the studies involving myofibroblasts and explaining the pathogenesis in a simplified manner.
Background: Recently, the concept of field cancerization has questioned the accuracy of biopsy site selection clinically. Oral submucous fibrosis (OSMF) has a global malignant transformation rate of 7.6% despite having less dysplastic changes clinically or histologically. Hence, this study was undertaken to evaluate the expression of vimentin, epithelial-cadherin (E-Cad) and collagen IV in OSMF, using immunohistochemistry and polymerase chain reaction (PCR). Materials and Methods: One hundred and eighty- five patients with OSMF (61), with habits and no OSMF (61) and patients without habit and OSMF (63) were subjected to biopsy for sample collection. The samples were analyzed immunohistochemically for vimentin, E-Cad and collagen IV. The PCR values for vimentin and E-Cad were also done. Results: Vimentin expression was increased in OSMF patients, whereas E-Cad expression was decreased in OSMF patients. Conclusion: Epithelial–mesenchymal transition signatures are definitely positive in OSMF cases.
Keywords: Collagen-IV, epithelial-cadherin, epithelial–mesenchymal transition markers, immunohistochemistry, oral submucous fibrosis, vimentin
Background:
The objective of this histopathological study was to identify the expression of tumor suppressor gene p53 and to detect the correlation between p53 expression and the degree of dysplasia in oral submucous fibrosis (OSMF).
Methods:
A sample size of 30 OSMF patients irrespective of sex was taken up for the study. The tissue samples obtained were subjected to immunohistochemical method to detect p53 protein. The technique used was indirect super sensitive Polymer-HRP IHC detection system. The p53 positive samples were evaluated on a 4-point scale, which ranged from 3+ to negative.
Results:
Out of 30 cases 3(10%) cases were negative for p53 expression and 13(43.3%) showed + expression, and 14(46.6%) showed ++ expression. On application of Mantel-Haenszel Chi-Square test a statistically significant P <=0.05 i.e. (P=0.012) was obtained and there was Linear-by-Linear association between p53 expressions and dysplasia that showed the point probability of 0.006.
Conclusion:
Immunohistochemistry is a powerful tool to identify distinct patterns of gene expression in premalignant disorders and also Oral Squamous Cell Carcinomas (OSCC) from different populations. In the present study, a significant number of samples of OSMF were positive for p53 protein.
Oral submucous fibrosis (OSF) as one of the premalignant disorders endures a series of histopathological stages to invasive oral squamous cell carcinoma (OSCC) eventually. However, the role of long non-coding RNA (lncRNA) expression in OSF malignant progression still remains poorly understood. Through RNA-sequencing normal mucous, OSF and OSCC tissues, we found 687 lncRNA transcripts significantly and differentially expressed during OSF progression, including 231 upregulated lncRNAs and 456 downregulated lncRNAs, indicating that lncRNAs are involved in the regulation of different stages of OSF development. Further functional enrichment analysis showed these differentially expressed lncRNAs participated in inflammation signaling, Wnt signaling, angiogenesis, CCKR signaling, integrin signaling, PDGF signaling, p53 signaling, and EGF receptor (EGFR) signaling pathways, which contribute to inflammatory and fibroelastic pathogenetic changes of OSF and further malignant progression. Five novel lncRNAs were differentially expressed during OSF progression with varied expression levels, indicating the importance of these lncRNAs in OSF malignant development. Moreover, some lncRNAs have been previously identified to be associated with OSCC pathogenesis, including HCG22, RP11-397A16.1, LINC00271, CTD-3179P9.1, and ZNF667-AS1 . Thus, our study firstly comprehensively elucidated lncRNAs expression profile of malignant procession from OSF premalignant lesion to OSCC, which will enlighten our understanding of the importance of lncRNA involved in OSF malignant development.
Oral Submucous Fibrosis (OSMF) is an insidious, chronic, complex, crippling, debilitating, irreversible, progressive, scarring, potentially malignant and collagen metabolic disorder, induced by a known carcinogen areca nut; wherein the oral mucosa, and occasionally the pharynx and esophagus is subjected to various pathological changes with significant clinical manifestations at different stages of progression, leading to functional morbidity; and with a risk of malignant transformation in the overlying epithelium. Although the condition is mainly diagnosed based on classic clinical manifestations, the commonly used existing definition for oral submucous fibrosis is primarily based on histological features. The authors have conducted extensive clinical research studies on OSMF and intends to propose a new clinical definition as 'a debilitating, progressive, irreversible collagen metabolic disorder induced by chronic chewing of areca nut and its commercial preparations; affecting the oral mucosa and occasionally the pharynx and esophagus; leading to mucosal stiffness and functional morbidity; and has a potential risk of malignant transformation.' Thus, a new clinical definition is put forward so as to assist the academicians, researchers and clinicians in terming and grouping this disease according to its clinical and biological behaviour for its subsequent management.
Oral submucous fibrosis (OSF) is characterized by abnormal collagen deposition. It is a precancerous disorder and transforms into a malignant tumor in 1.5–15% of all cases. Symptoms include submucous fibrosis, ulceration, xerostomia, a burning sensation, and restricted mouth opening. All of these greatly interfere with patient quality of life. The present review introduces OSF from a molecular perspective and summarizes what is known about its underlying mechanisms, diagnostic biomarkers, and therapeutic interventions. In addition to the aggressive treatment of OSF, its prevention is also important. Future research should, therefore, focus on improving the oral health literacy of the patients susceptible to OSF.
Objective:
The coarse fibres of areca nut and the continuous friction from occluding teeth are major causes of mechanical stress to the oral mucosa in conditions like oral submucous fibrosis and frictional keratosis. The continuous micro trauma provided in areca nut chewers, creates an environment where the keratinocytes exhibit alteration. Loricrin, is expressed abundantly in keratinizing epithelium in response to mechanical stress. Their expression or absence could play a role in malignant transformation. This study attempts to assess the potential of Loricrin as an early diagnostic marker in patients with chewing habit.
Methods:
73 archival samples of formalin fixed, paraffin embedded tissue specimens histopathologically confirmed, were segregated as normal mucosa 11, hyperkeratotic 32 and oral submucous fibrosis 30 and stained with antibodies to Loricrin and graded as negative, mild, moderate and intense based on the staining intensity. Pearson's chi square test was done for statistical analysis.
Results:
Loricrin expression was observed in all groups with staining in the stratum granulosum showing a significant association to habits (P = 0.000).
Conclusion:
This prominent staining indicates a compensatory cytoskeletal rearrangement of surface epithelium during cell division in early oral submucous fibrosis showing potential as an early marker of the condition.
Aim:
To conduct a systematic review evaluating the cases of oral submucous fibrosis in pediatric patients.
Material and method:
Systematic review was conducted using PRISMA guidelines. The article focused on oral submucous fibrosis in pediatric patients were included. A total of five manuscripts were included in our systematic review. The prevalence of OSMF in pediatric patients, gender distribution, causes, and clinical presentation were reviewed.
Results:
On systematically reviewing, a total of 10 cases of OSMF in pediatric patients were found. The youngest patient reported to be diagnosed with OSMF was of 2.5 years of age. Female preponderance was noticed. All the patients had the habit of areca nut chewing which subsequently led to fibrosis.
Conclusion:
Such a rapid increase in the rate of OSMF among pediatric population is a potential danger to the society. The habit of areca nut chewing is the major cause for this dreadful condition. Lack of health consciousness and low level of education are the major factors for initiation of this habit among children. Therefore it is imperative for the parents and school as well as government authorities to take serious actions.
Background/purpose:
Oral submucous fibrosis (OSF) is a precancerous condition of oral cancer with a complex etiology. Our previous work has demonstrated that non-coding RNA miR-1246 contributes to the cancer stemness of oral cancer. In the current study, we sought to investigate the effect of the inhibition of miR-1246 on the oral fibrogenesis.
Methods:
The expression levels of miR-1246 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined by qRT-PCR. Collagen gel contraction and migration assays were conducted to evaluate the myofibroblast activities. The relationship between miR-1246 and type I collagen was assessed and the protein expression of type I collagen was determined by Western blot.
Results:
MiR-1246 expression was upregulated in both OSF specimen and fBMFs compared to the normal counterparts. Inhibition of miR-1246 successfully suppressed the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, the expression of miR-1246 was positively correlated with type I collagen and the expression of type I collagen was abrogated by repression of miR-1246.
Conclusion:
MiR-1246 is not only critical to the maintenance of oral stemness but also important to the activation of myofibroblasts. Our results showed that miR-1246 is positively associated with the type I collagen, which may be a downstream effector of miR-1246 and responsible for the fibrosis effect on fBMFs.
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.
Areca nut chewing habits are associated with several oral manifestations like leukoplakia, submucous fibrosis and oral squamous cell carcinoma. Although numerous evidence on areca toxicity is known but the mechanistic pathway of disease causation is to be studied. Aqueous areca nut extract treated A549 cells showed reduced cell viability by 48 h with IC50 value of 0.50%. The toxic nature of areca nut induced the production of reactive oxygen species with decreased anti-oxidant glutathione S transferase levels lead to altered redox homeostasis. PCR studies showed decreased mRNA levels of Jun and Fos AP-1 subunits on extract treatment by 48 h. The protein levels of PCNA, CDK4, RB, p53, c-Jun and c-Fos were found to be downregulated with upregulated CDK inhibitor p21 on extract treatment as compared to control. Results of FACS analysis further confirm G1/S phase cell cycle arrest on areca nut extract exposure. The regulation of downstream AP-1 subunits by MAPKs was studied by using specific inhibitors of ERK, JNK and p38 along with areca nut extract. Results showed the redox activation of MAP kinases down regulated the mRNA levels of AP-1 subunits in aqueous areca nut extract treated cells. Hence the present study aids in elucidating the role of MAP kinases in regulating the AP-1 subunits and their implications on target genes that are involved regulation of various cellular processes. Further, it would help in understanding the mechanistic aspects of the diseased state which may facilitate in designing of new therapeutic modalities that could help in cancer management.
Background
Oral submucous fibrosis (OSF) is a progressive scarring disease and has been considered as a premalignant condition of the oral cavity. However, the detailed molecular mechanisms underlying the pathogenesis of OSF are still unclear.
Method
Here, we examined the expression of a novel long non‐coding RNA LINC00974 in OSF and investigated its function role in myofibroblast transdifferentiation. Phenotypic analyses, including collagen gel contraction, migration, invasion and wound healing assays, were used to assess the myofibroblast activities following overexpression or inhibition of LINC00974.
Results
We found that the expression of LINC00974 in OSF tissues or myofibroblasts was aberrantly upregulated, and there was a positive correlation between LINC00974 and myofibroblast markers. Our results showed that inhibition of LINC00974 suppressed the myofibroblast activities, while overexpression of LINC00974 increased the activation. We demonstrated that the expression levels of α‐SMA, α‐1 type I collagen, fibronectin were downregulated in the LINC00974‐inhibited myofibroblasts. Additionally, the TGF‐β secretion and phosphorylated Smad2 expression were also repressed in the LINC00974‐inhibited myofibroblasts. We further demonstrated that silence of LINC00974 prevented the arecoline‐induced myofibroblast activation, and LINC00974‐increased myofibroblast activities were via TGF‐β pathway.
Conclusion
Altogether, these findings suggested that arecoline‐increased myofibroblast transdifferentiation was via LINC00974‐mediated activation of TGF‐β signaling.
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Epithelial–mesenchymal transition (EMT) has been implicated in fibrogenesis and carcinogenesis; however, the exact role of EMT‐inducer Slug in the progression of precancerous oral submucous fibrosis (OSF) has not been investigated. In the current study, we showed that the expression of Slug was upregulated in OSF tissues and associated with various myofibroblast markers. After silence of Slug in fibrotic buccal mucosal fibroblasts (fBMFs), the elevated myofibroblast activities and fibrosis markers were all downregulated. Our data revealed that arecoline, an areca nut alkaloid, increased the expression of Slug in normal BMFs, and inhibition of Slug successfully prevented the arecoline‐induced myofibroblast activation. Additionally, overexpression of Slug in BMFs stimulated the activities of myofibroblasts, indicating that upregulation of Slug by arecoline contributes to the myofibroblast transdifferentiation. Most importantly, Slug was able to bind to the E‐box of type I collagen, leading to increased expression of type I collagen. Altogether, this study demonstrated the abnormal elevation of Slug in OSF and its significance in arecoline‐induced fibrogenesis. Moreover, downregulation of Slug could be a potential target for OSF remedy via suppression of myofibroblast activities and type I collagen.
Background/purpose:
MicroRNA-200c (miR-200c) recently emerged as an important regulator of tumorigenesis and cancer metastasis, however, its role in regulating oral submucous fibrosis (OSF) remains unknown. In this study, we investigated the functional role of miR-200c in myofibroblastic differentiation activity and identified its potential target.
Methods:
qRT-PCR was applied to assess the expression of miR-200c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs). Arecoline, a major areca nut alkaloid, was utilized to explore whether the expression of miR-200c would alter following stimulation. Collagen gel contraction, migration and invasion capabilities were examined in arecoline-stimulated BMFs as wells as in fBMFs. Luciferase reporter assay was conducted to show the relationship between miR-200c and ZEB1.
Results:
Our results showed that the expression of miR-200c was downregulated in OSF specimen and fBMFs. Arecoline treatment dose-dependently reduced the relative expression of miR-200c in normal BMFs. Overexpression of miR-200c impeded the arecoline-induced collagen gel contraction, migration, invasion and wound healing capacities. Moreover, ectopic expression of miR-200c in fBMFs successfully reduced the increased collagen gel contractility and invasion abilities. Our results demonstrated that ZEB1 was a direct target of miR-200c, and overexpression of miR-200c inhibited the expression of ZEB1 and α-SMA.
Conclusion:
These findings suggest that downregulation of miR-200c in OSF may be involved in the pathogenesis of areca nut-associated OSF through regulation of ZEB1.
Oral submucous fibrosis (OSF) is a progressive scarring disease. MicroRNA‐200b (miR‐200b) has been reported as a tumour suppressor, but its role in the precancerous OSF remains unknown. In this study, we investigated the impact of miR‐200b on myofibroblastic differentiation activity. Arecoline is a major areca nut alkaloid and has been employed to induce the elevated myofibroblast activity in human buccal mucosal fibroblasts (BMFs). Treatment of arecoline in BMFs dose‐dependently reduced gene expression of miR‐200b, which corresponded with the decreased expression of miR‐200b in fBMFs. The arecoline‐induced myofibroblast activities were abolished by overexpression of miR‐200b in BMFs, and the same results were observed in fBMFs. In addition, α‐SMA was inhibited by an increase in miR‐200b. We further demonstrated that miR‐200b‐mediated decrease in ZEB2 led to down‐regulation of α‐SMA, vimentin. Loss of miR‐200b resulted in enhanced collagen contraction and migration capabilities, and knockdown of ZEB2 reversed these phenomena. Lastly, we showed the expression of miR‐200b was significantly less and ZEB2 was markedly higher in OSF tissues. These results suggested that down‐regulation of miR‐200b may contribute to the pathogenesis of areca quid‐associated OSF through the regulation of ZEB2 and myofibroblast hallmarks.
Background
Reduced E-cadherin expression and increased VEGF expression is known to be involved in tissue growth and transformation of Oral Potentially Malignant Disorders (OPMDs) and has been correlated with their differing histological grades in numerous studies.
Aim
To evaluate Immunohistochemical (IHC) expression of both E-cadherin and VEGF in predicting the malignant transformation potential of common OPMDs.
Materials and Methods
Ten cases each of Normal Oral mucosa (NOM), Mild Oral Epithelial Dysplasia (OED), Moderate OED, Severe OED, Oral Submucous Fibrosis, (OSMF) and Oral Squamous Cell Carcinoma (OSCC) were stained and evaluated for the expression of Ecadherin and VEGF. Quick score (QS) for expression intensity in all epithelial layers was calculated for both markers and results statistically analysed using Kruskal –Wallis ANOVA and Mann-Whitney “U” test.
Results
E-cadherin expression was continuous and membranous in all the layers of NOM and reduced with progressing grades of OED to OSCC. In OSMF, expression was intermediate between moderate and severe OED. VEGF expression increased as the disease progressed from normal to increasing grades of OED to malignancy. In OSMF, expression was similar to that in mild OED. VEGF, E-cadherin expression for basal and parabasilar cells showed a strong statistically significant negative correlation in NOM. A very strong statistically significant positive correlation with perfect monotonic relation was noted in superficial cells in severe OED group and OSCC group.
Conclusion
E-Cadherin and VEGF could be used as combination markers to predict the potential risk for malignant transformation in OEDs.
The exact process of the malignant conversion of oral submucous fibrosis (OSF) to oral cancer is not fully understood. This study aimed to detect and analyze E-cadherin expression, p63 expression, and number of mitotic figures, all correlated to cancer development, in ApoTome images of oral tissues to determine the oncogenic potentiality of OSF. ApoTome images of the study groups (6 normal, 16 OSF with dysplasia, and 10 OSF without dysplasia) were recorded. Cytoplasmic and membranous E-cadherin expression, breakages of the cell membrane, and p63 expression were detected in MATLAB 2016b. The number of mitotic figures detected by MATLAB was correlated with the number of chromosomes detected by ImageJ. A Mann–Whitney U test was done to determine a significant difference between the study groups for cytoplasmic and membranous E-cadherin distribution points. Statistical significant differences were found for cytoplasmic E-cadherin distribution between normal and OSF (with dysplasia) (p = 0.0278). There was an increase in mitotic figures, p63 expression, and cytoplasmic E-cadherin expression and a decrease in membranous E-cadherin expression from normal to diseased condition. Hence, automated detection and quantification of E-cadherin, p63, and mitotic figures in ApoTome images of oral biopsies can help in determining the oncogenic potentiality of OSF.
Background
Oral submucous fibrosis (OSMF) is a premalignant condition mainly caused by areca nut chewing and is characterized by progressive fibrosis of submucosal tissues and epithelial atrophy. Activation of transforming growth factor beta (TGF-β) signaling is considered main causative event for increased collagen production and fibrosis. In this study, molecular pathogenesis of OSMF was investigated based on the expression of the TGF-β genes in OSMF tissues compared to normal controls.
Methods
A total of 33 OSMF and 10 normal tissues were collected from patients and their clinic-epidemiological data was recorded. The expression of TGF-β isoform genes- TGF β1, TGF β2, TGF β3 and its receptor TGF βR1, TGF βR2 was studied by real time polymerase chain reaction (PCR). Comparison of the expression of these genes among normal controls and OSMF patients was done. The PCR results were confirmed by histopathological and immunohistochemical staining.
Results
The histological changes included atrophic epithelium, loss of rete ridges, presence of inflammatory cells and dense collagen bundles in connective tissue. PCR showed statistically significant upregulation of TGF-β isoforms in OSMF as compared to normal tissues. Of the three isoforms, maximum fold change was observed in TGF-β1. Similarly, both TGF-βR1 and TGF-βR2 were found to be elevated in OSMF tissues compared to normal. The semi-quantitative analysis by immunohistochemical staining revealed statistically significant difference between normal and OSMF tissues.
Conclusion
TGF-β signaling plays a major role in the molecular pathogenesis of OSMF as shown by increased mRNA expression of all the three TGF-β isotypes and their receptors.
Oral submucous fibrosis (OSF) is a serious, potentially malignant oral disorder. It is histopathologically characterized by chronic inflammation and atrophic epithelium accompanied by the accumulation of collagen fibers in the lamina propria. The molecular mechanisms leading to atrophic epithelium remain poorly understood. Therefore, the present study investigated the role of autophagy and apoptosis in atrophic epithelium in OSF. The expression of Caspase-3 and autophagy-related proteins (LC3 and P62) in OSF epithelial tissues was quantified by immunohistochemistry. The analysis demonstrated that, compared with normal oral mucosal tissues, autophagy and apoptosis increased with the progression of OSF. Flow cytometry and Western blotting showed that arecoline induces apoptosis in human oral keratinocytes (HOKs) in a time-dependent manner in vitro. Arecoline-induced autophagy was confirmed by transmission electron microscopy and Western blotting. When chloroquine was used as an inhibitor of autophagy, the apoptosis rate and Caspase-3 expression decreased compared with the use of arecoline alone. Thus, autophagy and apoptosis may be involved in atrophic epithelium in OSF, and arecoline-induced autophagy promotes apoptosis in HOKs.
Aim:
To evaluate the salivary lactate dehydrogenase (LDH) levels in clinico-pathologically confirmed oral submucous fibrosis (OSMF), oral cancer and clinically diagnosed tobacco pouch keratosis patients.
Materials and methods:
A prospective, comparative study was carried out in a tertiary healthcare centre located in Loni from October 2013 to January 2014. A total of 120 patients were separated into 4 groups depending upon the clinical diagnosis as follows. Group I: healthy control (with no addictions and diseases). Group II: oral cancer. Group III: oral submucous fibrosis. Group IV: habitual tobacco chewers (tobacco addiction without any disease). Substantiation was done using biopsy. The samples were inspected for salivary LDH levels by the technique in line with the recommendations of the International Federation of Clinical Chemistry with the help of Erba Chem semi auto analyser.
Results:
The mean salivary LDH levels in the control, oral cancer OSMF and habitual tobacco chewer group were 86.12 ± 7.05 IU/L, 592.09 ± 28.57 IU/L, 350.43 ± 5.90 IU/L and 125.19 ± 13.42 IU/L, respectively. Out of 4 groups, LDH activity was increased in saliva of patients with tobacco pouch keratosis, OSMF, and oral cancer consistently. Notable difference was found in the mean salivary levels of the above groups. Results were subjected to appropriate statistical analysis: one-way ANOVA, Student's unpaired t test for group-wise comparison followed by post hoc Tukey's test.
Conclusion:
We observed congruous higher levels of salivary LDH in oral precancer and cancer, and hence it could be a future marker.
Aim:
The aim of the present study was to correlate the immunoexpression of alpha-smooth muscle actin (α-SMA) for myofibroblasts with the serum levels of transforming growth factor-β1 (TGF-β1) in oral submucous fibrosis (OSMF).
Methods:
A total of 100 cases of histopathologically confirmed OSMF were assessed for α-SMA expression. Clinical data, such as age, sex, mouth opening, and habit history, were obtained for each case. Serum TGF-β1 levels were recorded in 73 patients with the help of enzymelinked immunosorbent assay technique.
Results:
The staining index of α-SMA increased concomitantly with higher myofibroblast count in the increasing histopathological grades of OSMF (P ≤ .05). Serum TGF-β1 levels were highest in the intermediate grades of OSMF. Clinical parameters, such as mouth opening, cheek flexibility, and tongue protrusion, showed a direct correlation with increasing clinical grades of OSMF.
Conclusions:
The progressive increase in myofibroblasts from early to advanced stages suggests their potential use as markers for evaluating the severity of OSMF. Additionally, as myofibroblasts are responsible for producing a variety of factors that are involved in the fibrotic processes; they could be the key link in the pathogenesis of OSMF. Interruption of their development, recruitment, or activation could provide a unique therapeutic target for future treatment options in patients with OSMF.
Background:
Oral submucous fibrosis (OSF) is a potentially malignant lesion characterized by epithelial-mesenchymal transition (EMT). Bone morphogenetic protein 4 (BMP4) promotes EMT in fibrotic diseases, but the underlying mechanisms and its potential role in OSF are unclear. This study investigates whether BMP4 plays a role in the pathogenesis of OSF and explores the underlying mechanisms.
Methods:
The expression of BMP4 and the EMT proteins E-cadherin and vimentin was investigated in OSF specimens by immunohistochemical staining. Pearson's correlation analysis was conducted to explore the correlation between BMP4 and the EMT markers. Western blotting and RT-PCR assays were used to analyze the effect of arecoline (a known EMT-promoting pathogenic factor in OSF) on BMP4 and identify the transcription factor involved. Confocal microscopy was used to observe the intracellular sublocalization of the identified transcription factor, Yes-associated protein 1 (YAP1). Finally, siRNA silencing of BMP4 was used to determine its effect on YAP1 activation and arecoline-induced EMT.
Results:
BMP4 is overexpressed in OSF and plays a role in EMT, as its expression correlates with the expression of E-cadherin and vimentin. Arecoline induces BMP4 expression via the activation of YAP1 (through its nuclear translocation). Furthermore, the YAP1/BMP4 mechanism is the main molecular event in arecoline-induced EMT, as knockdown of BMP4 expression affects expression of the EMT markers and inhibits extracellular matrix accumulation.
Conclusions:
Arecoline induces EMT in OSF via the YAP1/BMP4 pathway. Thus, BMP4 could be considered as a potential therapeutic target for the treatment of OSF. This article is protected by copyright. All rights reserved.
Aim:
The aim of this study was to investigate the expression of Ki67, CD105 and α-smooth muscle actin (α-SMA) expression in oral submucous fibrosis (OSF) and oral squamous cell carcinoma in the background of OSF (OSCC-SMF).
Methods:
The study was carried out on paraffin-embedded tissues of 30 normal oral mucosa (NOM), 50 OSF cases and 105 OSCC-SMF. The immunohistochemistry was carried out to evaluate the expression of Ki67, CD105 and α-SMA antigen.
Results:
Ki67 labelling index (LI), CD105 and α-SMA expression showed increasing trend from NOM, low-risk epithelial dysplasia (LRED), high-risk epithelial dysplasia (HRED), well-differentiated squamous cell carcinoma (WDSCC), moderately differentiated squamous cell carcinoma to poorly differentiated squamous cell carcinoma. However, there was no significant difference of α-SMA expression between HRED and WDSCC. In OSCC-SMF, Ki67 LI, CD105 and α-SMA were significantly higher in advanced clinical TNM stage, metastasis and less than 3 years patient survival as compared with early clinical TNM stage, non-metastasis and 3 years or more patient survival.
Conclusion:
Ki67 LI, α-SMA and CD105 expression alone or together correspond with the disease progression model of SMF. Hence, expression of these markers can be used as a predictive marker of clinical outcome of OSCC-SMF.
Objectives:
To detect the expression of protein light chain 3 (LC3) and p62-SQSTM1 (p62) in the lamina propria of oral submucous fibrosis (OSF) and to determine the association of autophagy with OSF. To investigate the role of autophagy in angiogenesis of human umbilical vein endothelial cells (HUVECs) and to assess whether this effect was induced by arecoline.
Methods:
LC3 and p62 expression was detected in OSF tissue through immunohistochemistry (IHC). Transmission electron microscopy (TEM) and Western blot were used to investigate the expression of autophagy in HUVECs. The role of autophagy in angiogenesis in HUVECs was investigated using the Matrigel assay.
Results:
1: LC3 expression was upregulated in OSF samples. In contrast, p62 was downregulated in early and intermediate stages but upregulated in advanced stages of OSF. 2: HUVECs treated with arecoline exhibited increased autophagosomes, LC3 expression and reduced p62 expression, when co-treated with chloroquine (CQ), which is a specific autophagy inhibitor, revealed the opposite trend. 3: Autophagy inhibited angiogenesis in HUVECs.
Conclusions:
Our findings suggest that arecoline induces autophagy in HUVECs. The high level of autophagy could reduce cell viability and inhibit angiogenesis in HUVECs, potentially promoting the development of OSF.
Background:
Oral cancer is one of the highly prevalent cancers worldwide being. According to data of GLOBOCAN 2018, the estimated incidence, mortality and 5-year survival rates due to lip, oral cavity and salivary gland cancer in world is (2.0%), (0.5%) and (0.3%) respectively. (Bray, Ferlay and Soerjomataram, 2018). Endothelin-1 (ET-1) is a 21-amino acid peptide; its receptors have been implicated in the growth and progression of both primary and metastatic neoplasms throughout the human body. Studies have shown that ET-1 is expressed in tissue, serum and other body fluids.
Aim:
To estimate the levels of salivary endothelin-1 in Oral potentially malignant disorders (oral leukoplakia and submucous fibrosis) and oral squamous cell carcinoma.
Materials and methods:
The study population included 60 subjects and were divided into 4 groups. All patients included in the study are clinically and histopathological diagnosed cases of oral leukoplakia, submucous fibrosis and oral cancer and assessed for salivary ET-1 levels using human ELISA kit. Significant differences between the groups were determined using one-way analysis of variance, LSD and Post HOC, unpaired t test, biserial and spearson's correlation.
Results:
The mean levels of salivary Endothelin-1 level in study groups were: 82.78 ± 5.9 pg/mL (OSCC), 65.02 ± 1.8 pg/mL (SMF), 57.76 ± 4.1 pg/mL (LEUKOPLAKIA), 29.72 ± 14.1 pg/mL (CONTROLS). The mean Salivary ET-1 levels among these four groups was compared and the difference was statistically significant (P < 0.001). We also found a significant difference in the means of ET-1 levels among the clinical and histopathological staging of the study groups.
Conclusion:
Our results demonstrate potential utility of salivary analysis for ET-1 levels to monitor patients at risk for OSCC. Although provides the basis for a larger prospective study to determine the critical levels of salivary ET-1 necessary to diagnose and monitor OPMD's and its potential to undergo malignant transformation.
Objectives
Fibrosis diseases are one of the leading causes of suffering and death. However, no systematic investigation has been carried out on fibrosis‐related genes.
Materials and Methods
By querying PubMed using keywords “fibrosis” and “gene” or “protein”, we identified fibrosis‐related genes in the last decade. Bioinformatics analysis was performed by MAS 3.0 software. Key miRNA was selected to assesse its relationship with oral submucous fibrosis (OSF) and fibroblast functions.
Results
1310 genes related to fibrosis were identified. TGF‐β1, CTGF, MMP9, HSP47 and S1P were found to be associated with mainly fibrotic organs. 244 cellular components terms, 595 molecular function terms, 1816 cellular component terms, 136 KEGG pathway annotations were predicted. MiR‐199‐5p was selected as the key miRNA, which has higher level in OSF. Upregulated miR‐199‐5p was significantly related to OSF duration and OSF histological grade (P = 0.028 and 0.012, respectively). Overexpressive miR‐199‐5p reduced proliferation and induced apoptosis in buccal fibroblasts. Additionally, expression levels of collagen I (COL I) and III (COL III) were promoted by overexpressive miR‐199‐5p in buccal fibroblasts.
Conclusion
These results indicate that fibrosis‐related genes are related to a series of complex mechanisms. The characteristics of miR‐199‐5p may supply important clues for developing therapeutic strategy for OSF.
This article is protected by copyright. All rights reserved.
Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1–7) (Ang‐(1–7)) protects against fibrosis by counteracting angiotensin II (Ang‐II) via the Mas receptor. However, the effects of Ang‐(1–7) on OSF remain unknown. NOD‐like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang‐(1–7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang‐II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang‐(1–7) improved arecoline‐induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin‐converting enzyme (ACE)/Ang‐II/AT1R axis and NLRP3 inflammasome/interleukin‐1β axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N‐acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA‐ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase‐1 blocker VX‐765. Ang‐(1–7) shifted the balance of RAS toward the ACE2/Ang‐(1–7)/Mas axis, inhibited arecoline‐induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang‐(1–7) attenuates arecoline‐induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts. In this study, we deeply revealed that NLRP3 inflammasome triggered by arecoline‐induced ROS promotes OSF. Ang‐(1–7) attenuates migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome.
Background:
IARC has classified the betel nut as a human environmental carcinogen. Previous studies have found that arecoline (AR) is the major alkaloid present in the saliva of betel quid chewers. Saliva contains a large content of AR which has been further shown to cause mutation of oral mucosa cells, resulting in oral cancer. Whereas, to date, there are only few studies reported the hepatotoxicity associated with arecoline and betel nut chewing. Therefore, the main purpose of this study was to determine the toxic effects of AR and its oxidative metabolite, arecoline N-oxide (ARNO), in normal liver cell lines.
Methods:
The cytotoxic, genotoxic, and mutagenic effects were detected by crystal violet staining, alkaline comet assay, and Salmonella mutagenicity test, respectively. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2-DCFDA assay.
Results:
Our results demonstrated that ARNO exerted higher cytotoxicity, DNA damage, and mutagenicity than its parent compound arecoline in liver cells. Antioxidants, such as N-acetylcysteine, Trolox, and penicillamine, strongly protected liver cells from ARNO-induced DNA damage and ROS production. Furthermore, co-treatment with Mito-TEMPO also effectively blocked ARNO-induced ROS production in liver cells. Besides antioxidants, co-treatment with 1-aminobenzotriazole and methimazole nearly completely suppressed ARNO-induced ROS production in liver cells.
Conclusions:
Our data suggest that arecoline ingested from the habit of chewing betel quid can be primarily oxidized to ARNO, thereby enhancing its toxicity through increased ROS production. Considering the excellent protective effects of both mitochondria-targeted antioxidant and CYP450 inhibitor on ARNO-induced ROS production in liver cells, mitochondria CYP450-mediated metabolism of ARNO may be a key mechanism. Collectively, our results provide novel cellular evidence for the positive connection between habitual betel quid chewing and the risk for liver damage.
Background and objective:
c-Jun an activator protein-1 transcription factor component is activated by a variety of extracellular stimuli. Overexpression of c-Jun has been implicated in the pathogenesis of several types of cancer including oral cancer. The aim of this study was to correlate the expression of c-Jun in the normal buccal mucosa (NM), oral submucous fibrosis (OSMF), severe epithelial dysplasia (ED), and well-differentiated squamous cell carcinoma (WDSCC).
Subjects and methods:
Qualitative and quantitative expression of c-Jun was evaluated in a total of 60 histopathologically diagnosed cases, 15 each of NM, OSMF, ED, and WDSCC. The percentage of positive cells (Nuclear labeling index [nLI]) was considered for quantitative assessment and grading of staining for qualitative assessment.
Results:
The average nLI of c-Jun expression in NM, OSMF, ED, and WDSCC was 35.02%, 35.61%, 89.09%, and 83.31%, respectively. A statistical significant difference was found in c-Jun expression quantitatively between NM and ED (P = 0.000), NM and WDSCC (P = 0.000), OSMF and ED (P = 0.000), OSMF and WDSCC (P = 0.000), and ED and WDSCC (P = 0.021). Qualitatively, statistical significant difference was seen in an intense c-Jun expression between OSMF and ED (P = 0.000), OSMF and WDSCC (P = 0.032), and ED and WDSCC (P = 0.011).
Conclusion:
The overexpression of c-Jun in ED and WDSCC reveals its role in early carcinogenesis as evidenced in this study. Therefore, c-Jun might act in different mechanisms and pathways which lead to a malignant transformation in oral lesions.