Areca Quid Chewing Research Articles

Downregulation of PAX1 in OSCC Enhances Stemness and Immunosuppression via IFIT1 and PD‐L1 Pathways

ABSTRACTObjectiveOur study investigated how arecoline‐induced extracellular vesicle (EV) secretion suppresses PAX1 protein production through DNA hypermethylation and examined whether PAX1 downregulation enhances cancer stemness and immunosuppression in the tumor microenvironment.Materials and MethodsEVs were isolated from SAS/TW2.6 cancer cell lines using ultracentrifugation and identified using transmission electron microscopy. PAX1 DNA methylation was tested in an ISO17025‐certified lab, with and without EV pretreatment. Stemness and epithelial–mesenchymal transition markers were assessed by western blotting and 3D culture. PAX1, IFIT1, and PD‐L1 co‐expression were examined through immunofluorescence. Flow cytometry detected various T cells.ResultsArecoline‐induced EVs enhanced PAX1 methylation, suppressing its tumor‐suppressive function. Reduced PAX1 mRNA in OSCCs was linked to larger tumors, nodal metastasis, late‐stage disease, areca quid chewing, and poor survival. Downregulated PAX1 protein negatively correlated with IFIT1 and PD‐L1 expression. Reduced PAX1 promoted stemness via the IFIT1 pathway, increasing PD‐L1 secretion and aiding immune evasion. PD‐L1 expression correlated with Treg and CD8+ T cell levels in OSCC tissues, and the CD4+/CD8+ T cell ratio was lower in OSCC patients than in controls.ConclusionArecoline‐induced EV production, which influences PAX1/IFIT1/PD‐L1 function, may serve as a reliable biomarker for targeted therapy in OSCC patients.

Abstract LB379: HLA-DQB1*05:02: An independent genetic marker for oral cavity squamous cell carcinoma susceptibility

Abstract Environmental exposure to carcinogens causes mucosal damage in the upper aerodigestive tract, which can lead to cancers. The genetic basis of head and neck cancer is controversial. To identify susceptibility genes in head and neck cancers, we enrolled patients with early-onset disease in Taiwan. This case-control study included 54 young male patients with oral squamous cell carcinoma (OSCC) who were treated between March 1996 and December 2016, as well as 2,400 healthy controls. A single-nucleotide polymorphism (SNP) array was used to determine genetic loci that increase susceptibility to OSCC. Sequencing-based typing (TBG Biotechnology Corp., Taipei, Taiwan) was used to determine the HLA-DQB1 genotype in another cohort of 147 OSCC patients. We analyzed the allele frequencies of 664,994 autosomal SNPs in the 54 OSCC cases. In a genome-wide association analysis, four SNPs within loci on chromosomes 6, 7, 9, and 12 were significantly different between OSCC patients and controls (corrected P < 1.0 × 10−6). HLA-DQB1 was closest to rs28451423 on chromosome 6. HLA-DQB1*05:02 in OSCC (18.5%) was significantly different from normal population (7.0%) (P = 0.009). The influence of disease onset was independently significant after adjusting cigarette smoking, alcohol drinking and areca-quid chewing (P = 0.015, OR: 3.922, 95% confidence interval: 1.311 - 11.734). Furthermore, HLA-DQB1*05:02 was associated with early-onset OSCC (P = 0.004). HLA-DQB1*05:02 is associated with OSCC independent of environmental exposures. HLA-DQB1*05:02 is also related with early onset of OSCC. It provides evidence of a genetic basis for OSCC. Citation Format: Shiang-Fu Huang, Huei-Tzu Chien, Chi-Kuan Young, Yun-Shien Lee, Chun-Ta Liao, Kai-Lun Cho, Ching-Han Chen. HLA-DQB1*05:02: An independent genetic marker for oral cavity squamous cell carcinoma susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB379.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP β-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) β-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP β-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP β-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

Analysis of erythrocytes in oral submucous fibrosis - A morphometric study.

Oral submucous fibrosis (OSMF) is a chronic progressive scarring oral disease caused due to areca quid chewing. The constituents of areca nut can enter into the circulation by swallowing the liquid mixture of areca quid which evokes oxidative stress on RBC membrane integrity. To study the morphometric characteristics of erythrocytes under light microscopy and to assess the role of areca quid constituents on the morphology of circulating erythrocytes. Peripheral blood smears prepared from blood samples of 50 patients selected according to Lai's classification. Group I: Normal individuals (10 cases); Group II: Mouth opening > 35 mm (10 cases); Group III: Mouth opening 30-35 mm (10 cases); Group IV: Mouth opening 20-30 mm (10 cases) and Group V: Mouth opening < 20 mm (10 cases). The slides were stained with Leishman's stain and assessed by light microscopy. A total of 100 randomly selected RBCs from 5 different fields in each smear were selected and the RBC circumference was measured and tabulated. Data was analyzed using GraphPad Prism 5.03 software. Tukey's multiple comparison test showed statistically significant difference (p < 0.05) between groups I and IV; I and V; II and IV; II and V; III and IV; III and V. These results suggest the possibility of cytotoxic effect of areca quid constituents on circulating erythrocytes in advanced cases of OSMF, which might result in microcytic anaemia.

Safrole-induced expression of proinflammatory responses is associated with phosphorylation of mitogen-activated protein kinase family and the nuclear factor-κB/inhibitor of κB pathway in macrophages.

Objective:Safrole, also called shikimol and Sassafras, is the carcinogenic and phenylpropanoid compound extracted from Sassafras tree and anise, betel, and camphor. Moreover, a high concentration of safrole can be occur in the saliva because of betel nut or areca quid chewing which a common habit observed in Southern and Southeastern Asia. Notably, macrophages are crucial phagocytic cells of the immune system. Nonetheless, to date, no evidence has been reported regarding safrole-induced proinflammatory response and the corresponding mechanism in macrophages.Materials and Methods:In the present study, the cytokines expression, NO generation, protein phosphorylation, and expression were assessed by enzyme-linked immunosorbent assay, Griess reagent, and Western blot assay, respectively.Results:In this study, we determined that safrole induces the generation of nitric oxide and proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and IL-6 in RAW264.7 macrophages in a concentration-dependent manner. Furthermore, inhibitor of κB (IκB) degradation was caused by safrole in a concentration-dependent manner. In addition, the phosphorylation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) family, including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase, was induced by safrole began to increase at 10 μM and attained a plateau at 100 μM.Conclusion:These results indicated that safrole induces the expression of proinflammatory responses in macrophages through the NF-κB/IκB pathway and its upstream factor, MAPK family phosphorylation.

Increased salivary AKR1B10 level: Association with progression and poor prognosis of oral squamous cell carcinoma.

Aldo-keto reductase family 1 member B10 (AKR1B10) expression in oral squamous cell carcinoma (OSCC) tissue specimens is correlated with the progression and prognosis of OSCC. Saliva samples were obtained from 35 normal controls and 86 patients with OSCC before cancer surgery. The AKR1B10 levels were determined using enzyme-linked immunosorbent assay (ELISA). The mean salivary AKR1B10 levels were significantly higher in the patients with OSCC than in the normal controls (P < .001). Higher salivary AKR1B10 levels were significantly associated with larger tumor size, more advanced clinical stage, and areca quid chewing habit. Patients with OSCC with a higher salivary AKR1B10 level (>646 pg/mL) had a significantly poorer survival than those with a lower (≤646 pg/mL) salivary AKR1B10 level (P = .026). The salivary AKR1B10 level may be a promising biomarker for screening high-risk patients with OSCC and monitoring the progression of OSCC.

Expression of AKR1B10 as an independent marker for poor prognosis in human oral squamous cell carcinoma.

Aldo-keto reductase family 1 member B10 (AKR1B10) is implicated in xenobiotic detoxification and has disparate functions in tumorigenesis that are dependent on the cell types. The purpose of this study was to investigate the clinicopathological significance of AKR1B10 as a prognostic marker for oral squamous cell carcinomas (OSCCs). AKR1B10 protein expression was analyzed by immunohistochemistry in 77 patients with OSCC. The AKR1B10 labeling score for OSCCs (1.16 ± 1.14) was significantly higher than that for normal oral mucosa (0.10 ± 0.23; p < .0001). High expression of AKR1B10 significantly correlated with large tumor size (p = .041), advanced TNM classification (p = .037), and patient's areca quid chewing habit (p = .025). Multivariate analysis revealed that high AKR1B10 labeling score >1.16 (hazard ratio, 3.647; p = .001) significantly correlated with mortality. AKR1B10 overexpression is an independent poor prognostic biomarker for OSCC. AKR1B10 inhibitors may be promising in clinical trials against OSCC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1327-1332, 2017.

Characterization of a Novel Dermal Fibrosis Model Induced by Areca Nut Extract that Mimics Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is an oral potentially malignant disorder and areca quid chewing is the main etiological factor. However, the molecular mechanism underlying OSF remains unclear, partly due to the lack of an appropriate animal model. The present study aimed to establish and characterize an animal model of areca nut extract (ANE)-induced skin fibrosis that mimics OSF. Mice were divided into 4 groups: the control group; the bleomycin group; and the ANE10 and ANE20 groups, which received 10mg/ml and 20mg/ml subcutaneous (SC) injection of ANE, respectively. Skin fibrosis was evaluated by histological analyses. Additionally, the expression levels of the fibrotic marker genes were determined by immunohistochemical staining and immunoblotting. ANE administration significantly increased dermal thickness and collagen deposition compared with the control group. Moreover, ANE induced the expression of the fibrotic marker genes alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in the skin lesions. The SC injection of ANE successfully induced skin fibrosis, exhibiting characteristics similar to those of OSF. This model may facilitate future studies of the mechanism underlying OSF.

Hypermethylated ZNF582 and PAX1 are effective biomarkers for detection of oral dysplasia and oral cancer

ObjectivesThis study investigated whether the methylation of ZNF582, PAX1, SOX1, NKX6.1, and PTPRR genes in oral scrapings could be used to detect oral dysplasia and oral cancer and to predict oral cancer recurrence. Materials and methodsOral scrapings were collected from 65 normal oral mucosa subjects, 107 oral precancer patients, and 95 oral squamous cell carcinoma patients. Methylation levels of the five genes were quantified by real-time methylation-specific PCR after bisulfite conversion. ResultsAmong the five tested genes, methylated ZNF582 (ZNF582m) and PAX1 (PAX1m) were found to be appropriate biomarkers for oral dysplasia and oral cancers. ZNF582m could detect mild dysplasia or worse oral lesions with the sensitivity and specificity being 0.85 and 0.87, respectively. PAX1m performed better in identifying moderate dysplasia or worse oral lesions with the sensitivity and specificity being 0.72 and 0.86, respectively. Moreover, the methylation levels and positive rates for ZNF582m and PAX1m were increased when disease severity increased. Thus, they may be applicable as a triage tool for patients with abnormal visual oral examinations. After cancer excision, both ZNF582m and PAX1m levels decreased. However, their levels increased again at the subsequently recurrent sites in some patients approximately 3–4 months before cancer recurrence. Finally, areca-quid chewing alone and in combination with cigarette smoking or alcohol drinking were found to be correlated with ZNF582 and PAX1 hypermethylation. ConclusionWe conclude that hypermethylated ZNF582 and PAX1 are effective biomarkers for the detection of oral dysplasia and oral cancer and for the prediction of oral cancer recurrence.

Oral submucous fibrosis: a review article on etiopathogenesis.

Areca quid chewing related oral mucosal lesions are potential hazard to a large population worldwide. Commercially freeze dried products such as pan masala, guthka and mawa have high concentration of areca nut per chew and appear to cause OSMF more rapidly than by self prepared conventional betel quid that contain smaller amounts of areca nut. The basic constituent of areca nut is either raw or dried or boiled or baked. Diverse agents including lime, tobacco, catechu, cloves, saffron and leaf of piper betel leaves may form a part of formulation. Many of the undesirable aspects of areca nut have been attributed to arecoline. These chemical appear to interfere with the molecular processes of deposition and or degradation of extracellular matrix molecules such as collagen, causing imbalance in the normal process. The most likely events that take place with regards to the above imbalance may be reduced phagocytosis of collagen by fibroblasts, up or down regulation of copper dependent enzyme lysyl oxidase, matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases . It has been postulated that areca nut may also induce the development of the disease by increased levels of cytokines in the lamina propria. Current evidence implicates collagen related genes in susceptibility and pathogenesis of OSMF. The individual mechanisms operating at various stages of the disease--initial, intermediate and advanced--need further study in order to propose appropriate therapeutic interventions.

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

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

Abstract 3868: Clinical significance of MRE11A/ATM/CHEK1 DNA damage response genes in oral cavity squamous cell carcinomas

Abstract Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers in Taiwan and worldwide. Except cigarette smoking and alcohol drinking, areca quid (AQ) chewing is also the major risk factor of OSCC in Southeast Asia including Taiwan. Despite better methods of diagnosis and new treatment procedures, there has not been a significant improvement in the five year survival rate for OSCC in the past 30 years. To explore some clues for clinical management of OSCC, we carried out whole genome loss of heterozygosity (LOH) and copy number alterations (CNAs) analysis and found high frequency of minimal deleted region (MDR) in11q13.4-q25 and significant CNA region in 11q22.3-q24.3. In this region, several DNA damage response (DDR) genes are located including MRE11A, ATM, H2AFX and CHEK1. The DDR pathway represents a complex network of multiple signaling pathways involving cell cycle checkpoints, DNA repairs, transcriptional programs, and apoptosis, through which cells maintain genomic integrity following various endogenous or environmental stress. To investigate the clinical significance of DDR pathway in OSCC, CNAs and LOH of ATM, MRE11A, and CHEK1 gene were analyzed in the present study. A total number of 324 OSCCs were first examined for CNAs using TaqMan CNV analysis and the copy number neutral cases were then examined for LOH using TaqMan SNP analysis and denaturing high performance liquid chromatography (DHPLC). Preliminary results indicated that only 8% (26/324) of OSCCs did not have any alterations in these 3 genes. The frequency of loss of MRE11A, ATM and CHEK1 gene was 7.10% (23/324), 15.74% (51/324) and 13.89% (45/324), respectively. Loss of MRE11A, ATM, and CHEK1 was associated with tumor differentiation and lymph node metastasis. The frequency of LOH in MRE11A, ATM, and CHEK1 gene was 37.84% (42/111), 50% (61/122) and 58.28% (95/163), respectively and LOH of MRE11A was associated with tumor differentiation. Combined the loss and LOH as the alteration event, we found that alterations of MRE11A, ATM, and CHEK1 gene were associated with tumor differentiation. Alterations of MRE11A and ATM were associated with early lymph node metastasis, while alterations of CHEK1 were associated with late lymph node metastasis. Alterations of MRE11A, ATM, and CHEK1 gene were associated with poor disease-free survival (DFS) and overall survival (OS) and gene loss had a stronger effect than LOH. Furthermore, alterations of MRE11A/ATM/CHEK1 as a whole were strongly associated with poor DFS and OS. These results confirmed our previous MDR findings and indicated that MRE11A/ATM/CHEK1 pathway might play a crucial role in the development and progression of OSCCs. Citation Format: Huei-Tzu Chien, Shiang-Fu Huang, I-How Chen, Chun-Ta Liao, Hung-Ming Wang, Ling-Ling Hsieh. Clinical significance of MRE11A/ATM/CHEK1 DNA damage response genes in oral cavity squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2015-3868

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

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

High frequencies of vitamin B12 and folic acid deficiencies and gastric parietal cell antibody positivity in oral submucous fibrosis patients.

Oral submucous fibrosis (OSF) is a chronic progressive scaring oral disease associated with areca quid chewing. This study evaluated whether OSF patients had anemia, hematinic deficiencies, and serum gastric parietal cell antibody (GPCA) positivity. The blood hemoglobin (Hb), iron, vitamin B12, and folic acid concentrations, mean corpuscular volume, and serum GPCA in 68 male OSF patients were measured and compared with the corresponding data in 136 age-matched male healthy control individuals. We found that five (7.4%), 14 (20.6%), 34 (50.0%), 28 (41.2%), and nine (13.2%) of the 68 male OSF patients had Hb (< 13g/dL), iron (≤ 70μg/dL), vitamin B12 (≤ 450pg/mL), and folic acid (≤ 6ng/mL) deficiencies, and serum GPCA positivity, respectively. Furthermore, OSF patients had a significantly higher frequency of Hb (p=0.006), vitamin B12 (p<0.001), or folic acid (p<0.001) deficiency and of serum GPCA positivity (p=0.011) than healthy control participants. Of the five OSF patients with anemia, two had thalassemia trait, one had iron deficiency anemia, and two had macrocytic anemia (mean corpuscular volume≥100fL). In addition, of the nine OSF patients with serum GPCA positivity, six had vitamin B12 deficiency, five had folic acid deficiency, and two had iron deficiency. However, none of the nine GPCA-positive OSF patients had pernicious anemia based on the strict World Health Organization definition. We conclude that there are high frequencies of vitamin B12 and folic acid deficiencies and of serum GPCA positivity in our male OSF patients.

Knockdown of S100A4 impairs arecoline-induced invasiveness of oral squamous cell carcinomas

Metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. The physiological function of S100A4 in the pathogenesis of areca quid chewing-associated OSCC has not been uncovered. OSCC tissues from areca quid chewers were analyzed by immunohistochemistry for S100A4 expression. The functions of S100A4 in invasiveness of arecoline-treated oral epithelial (OE) cells were determined by loss function approaches. Expression of S100A4 was positively correlated with clinical grading and lymph node metastasis of OSCC. Upregulated S100A4 is correlated with poor survival outcome of OSCC patients. Arecoline led to dose-dependent elevation of S100A4 expression in oral epithelial (OE) cells. Down-regulation of S100A4 significantly reversed arecoline-induced oncogenecity in OE cells. The additions of pharmacological agents LY294002, SP600125, and CAY10585 were found to inhibit arecoline-induced S100A4 expression in OE cells. Arecoline-induced S100A4 expression was down-regulated by LY294002, SP600125, or CAY10585 treatment. Targeting S100A4 might offer a new strategy for the treatment of OSCC patients with metastasis.

ZEB1 as an indicator of tumor recurrence for areca quid chewing-associated oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is the sixth most prevalent malignancy worldwide and the third most common cancer in developing nation. Most OSCC patients relapse within months after receiving treatment. Therefore, searching the biomarkers of recurrence is urgently required to improve OSCC patient survival. We set out to explore whether expression of ZEB1 could be triggered in oral epithelial cells (SG and FaDu) by arecoline in vitro. Control and ZEB1-knockdown arecoline-stimulated SG and FaDu were subjected to migration/invasiveness/anchorage-independent growth assay. Primary and recurrent OSCC tissues from areca quid chewers were analyzed using real-time RT-PCR analysis for ZEB1 expression. Arecoline led to dose-dependent elevation of ZEB1 expression in SG and FaDu cells. Downregulation of ZEB1 by lentiviral infection significantly reversed arecoline-induced oncogenicity including migration ability, cell invasiveness, and anchorage-independent growth in SG and FaDu cells. Clinically, the level of ZEB1 expression was higher in recurrent OSCC tumor samples but lower in primary lesions. Targeting ZEB1 might offer a new strategy for the treatment of OSCC patients. ZEB1 can serve as a progression and relapse marker in OSCC patients.

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

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

Abstract 4146: Polymorphisms of GSTT1, APE1 and MUTYh gene and the risk of oral squamous cell carcinoma

Abstract In Taiwan, oral cancer (including sub-sites in the oral cavity, oropharynx and hypopharynx) is the fourth most common cancer in men. According to the cancer registry report from the Ministry of Health and Welfare of the Executive Yuan, the annual age-adjusted incidence for oral cancer in males has shown a 1.55-fold increase in the past decade. Cigarette smoking, areca quid (AQ) chewing and alcohol drinking have been unveiled as the major risk factors. Previous studies have implicated that oxidative stress generated from cigarette smoking, AQ chewing and alcohol drinking contributed to oral cancer development. It is well known that GSTM1 and GSTT1 are involved in the detoxification of oxidative free radicals and nearly all oxidative DNA lesions are repaired via the DNA base excision repair (BER) pathway. To investigate the role of GSTM1, GSTT1 and BER genes (including OGG1, APE1, MUTYh, WRN and XRCC1) involved in OSCC risk in Taiwan, a comprehensive study on evaluation of polymorphisms of above genes was carried out in 1139 OSCC male cases and 1470 general males. A significant effect of AQ chewing or AQ chewing combined with cigarette smoking or alcohol drinking on the risk of OSCC was observed. GSTT1 null genotype was significantly associated with an increased risk of OSCC (odds ratio (OR), 2.10; 95% confidence interval (CI), 1.64-2.68, p = 0.000000003). After adjustment for cigarette smoking, AQ chewing and alcohol drinking, GSTT1 null-type variant was still significantly associated with an increased risk of OSCC (OR= 2.00; 95% CI, 1.42-2.81, p = 0.00007), while GSTM1 was not associated with OSCC risk. Polymorphisms of OGG1 Ser326Cys, APE1 Asp148Glu, APE1 T-656G, MUTYh Gln324His, WRN Cys1367Arg, and XRCC1 Arg399Gln were not associated with OSCC risk in general. However, stratification analysis indicated that polymorphisms of APE1 Asp148Glu and MUTYh Gln324His were associated with OSCC risk in individuals with cigarette smoking combined with AQ chewing. Furthermore, those individuals with either APE1 148 Asp/Asp or MUTYh 324 His/His genotype had an increased OSCC risks when compared to those with APE1 148 Glu and MUTYh 324 Gln allele (OR = 1.48; 95% CI, 1.13-1.95, p = 0.005). In conclusion, our results demonstrated that polymorphisms of genes involved in detoxification and BER of oxidative stress could influence the OSCC risk especially for those have history of cigarette smoking and AQ chewing. Citation Format: Chih-Hsiung Lai, Li-Ling Chiu, Huei-Tzu Chien, Saou-Hsing Liou, Shiang-Fu Huang, I-How Chen, Chun-Ta Liao, Ling-Ling Hsieh. Polymorphisms of GSTT1, APE1 and MUTYh gene and the risk of oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4146. doi:10.1158/1538-7445.AM2014-4146

Quinone Methide Bioactivation Pathway: Contribution to Toxicity and/or Cytoprotection?

The formation of quinone methides (QMs) from either direct 2-electron oxidation of 2- or 4-alkylphenols, isomerization of o-quinones, or elimination of a good leaving group could explain the cytotoxic/cytoprotective effects of several drugs, natural products, as well as endogenous compounds. For example, the antiretroviral drug nevirapine and the antidiabetic agent troglitazone both induce idiosyncratic hepatotoxicity through mechanisms involving quinone methide formation. The anesthetic phencyclidine induces psychological side effects potentially through quinone methide mediated covalent modification of crucial macromolecules in the brain. Selective estrogen receptor modulators (SERMs) such as tamoxifen, toremifene, and raloxifene are metabolized to quinone methides which could potentially contribute to endometrial carcinogenic properties and/or induce detoxification enzymes and enhance the chemopreventive effects of these SERMs. Endogenous estrogens and/or estrogens present in estrogen replacement formulations are also metabolized to catechols and further oxidized to o-quinones which can isomerize to quinone methides. Both estrogen quinoids could cause DNA damage which could enhance hormone dependent cancer risk. Natural products such as the food and flavor agent eugenol can be directly oxidized to a quinone methide which may explain the toxic effects of this natural compound. Oral toxicities associated with chewing areca quid could be the result of exposure to hydroxychavicol through initial oxidation to an o-quinone which isomerizes to a p-quinone methide. Similar o-quinone to p-quinone methide isomerization reactions have been reported for the ubiquitous flavonoid quercetin which needs to be taken into consideration when evaluating risk-benefit assessments of these natural products. The resulting reaction of these quinone methides with proteins, DNA, and/or resulting modulation of gene expression may explain the toxic and/or beneficial effects of the parent compounds.

Arecoline‐induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1

Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial–mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid–associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs.