Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas

Oral Diseases (2012) 18 , 713–719 Objectives: Heat shock protein (HSP) 27 is a low‐molecular‐weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. Methods: Forty‐eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin‐3 gallate (EGCG), glutathione precursor N‐acetyl‐l ‐cysteine (NAC), cyclooxygenase‐2 inhibitor NS‐398, HSP inhibitor quercetin, extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose‐ and time‐dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline‐induced HSP27 expression (P < 0.05). Conclusions: Heat shock protein 27 expression is significantly elevated in areca quid chewing‐associated OSCCs. Arecoline‐induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

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

Objectives

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.

Materials

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.

Results

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).

Conclusions

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.

Clinical relevance

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.

     

The Upregulation of Transglutaminase‐2 by Cyclosporin A in Human Gingival Fibroblasts Is Augmented by Oxidative Stress

Background: Transglutaminase‐2 (TGM‐2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM‐2 via ROS, and this regulation may have a role in the pathogenesis of CsA‐induced gingival overgrowth. Methods: Cytotoxicity, 2′,7′‐dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal‐regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase inhibitor LY294002, glutathione precursor N‐acetyl‐L‐cysteine (NAC), curcumin, epigallocatechin‐3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM‐2 protein induced by CsA was found in HGFs in a dose‐ and time‐dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM‐2 expression induced by CsA (P <0.05). Conclusions: These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM‐2 expression in HGFs. In addition, TGM‐2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.     

Impact of interleukin-8 gene polymorphisms and environmental factors on oral cancer susceptibility in Taiwan

Oral Diseases (2012) 18 , 307–314 Objectives: Interleukin‐8 (IL‐8), which is an angiogenic chemokine with a high expression level in tumor tissues, plays important roles in developing many human malignancies including oral squamous cell carcinoma (OSCC). This study was designed to examine the association of IL‐8 gene polymorphisms with the susceptibility and clinicopathological characteristics of OSCC. Methods: A total of 270 patients with OSCC and 350 healthy control subjects were recruited. Four single nucleotide polymorphisms (SNPs) of IL‐8 genes were analyzed using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) genotyping analysis. Results: Results showed that four IL‐8 SNPs (?251 T/A, +781 C/T, +1633 C/T, and +2767 A/T) were not associated with oral cancer susceptibility as well as clinicopathological parameters. But among 345 smokers, IL‐8 polymorphisms carriers with betel quid chewing were found to have a 17.41‐ to 23.14‐fold risk to have oral cancer compared to IL‐8 wild‐type carriers without betel quid chewing. Among 262 betel quid chewers, IL‐8 polymorphisms carriers with smoking have a 10.54‐ to 20.44‐fold risk to have oral cancer compared to those who carried wild type without smoking. Conclusions: Our results suggest that the combination of IL‐8 gene polymorphisms and environmental carcinogens might be highly related to the risk of oral cancer.     

Salivary arecoline levels during areca nut chewing in human volunteers

J Oral Pathol Med (2010) 39 : 465–469 Background: Arecoline stimulates cultured cells above 0.1 μg/ml and is cytotoxic above 10 μg/ml. Although this alkaloid seems important for areca nut induced oral carcinogenesis, little is known of the levels achieved during chewing. Materials and methods: Saliva was collected in 3‐ to 5‐min intervals over 50 min in 32 habitual chewers: before, for 25 min during, and for 20 min after chewing areca nut (0.5 g) without any other additives. Salivary arecoline was quantitated by HPLC‐MS. Controls comprised six subjects who denied areca nut use, and who were given rubber‐base material to chew during experiments instead. Results: Arecoline was detected before chewing in 22 subjects, exceeding the 0.1 μg/ml threshold in 20 cases. Salivary arecoline exceeded either the 0.1 or 10 μg/ml thresholds in all participants during chewing (P < 0.001). Maximum concentrations ranged from 5.66 to 97.39 μg/ml. All subjects reached 0.1 μg/ml salivary arecoline in at least 85% of time points studied (P < 0.0001), whereas 10 μg/ml was reached in 11 participants in at least 30% of the time points (P < 0.003). Arecoline concentrations varied greatly over time between individuals, and levels were much lower when peak concentrations were reached before 3 min, than in cases where arecoline peaked later (P < 0.02). No salivary arecoline was found in control saliva. Conclusions: Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre‐malignancy and malignancy.     

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.     

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Epidemiological survey of oral submucous fibrosis and leukoplakia in aborigines of Taiwan

A population-based survey was designed to investigate the prevalence of areca/betel quid chewing, oral submucous fibrosis and leukoplakia in a typical aboriginal community of southern Taiwan. Three hundred and twelve people 20 years of age or older were collected in the study. The prevalence of chewing areca/betel quid was 69.5%, with an average of 17.3 portions a day for an average 24.4 years. More women (78.7%) than men (60.6%) chewed areca/betel quid. The prevalences of oral submucous fibrosis and leukoplakia were 17.6% and 24.4%, respectively. It was found that the odds ratio for chewing areca/betel quid and having at least one of the above oral mucosal lesions was 8.21. Any additional smoking or drinking habits were not significant for having oral mucosal lesions. Although the areca/betel quid in Taiwan does not contain any tobacco, a significant association was still identified between areca/betel quid chewing and oral mucosal lesions.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

Association of HPV infections with second primary tumors in early-staged oral cavity cancer

Oral Diseases (2012) 18 , 809–815 Objective: The infection of human papilloma virus (HPV) has been reported in head and neck cancer; however, the clinical significance of HPV infection on the pathogenesis of oral cavity squamous cell carcinoma (OSCC) is still uncertain. Materials and Methods: The study recruited 103 patients with pathological early‐stage OSCC between March 1997 and December 2003 from Chang Gung Memorial Hospital, Taiwan. Tumor specimens were HPV‐genotyped by the EasychipVR HPV Blot method. Clinical association study was performed by using chi‐square, Kaplan–Meier, and logrank tests. Results: Thirty‐one patients (30.1%) were positive for HPV infection. The most frequent HPV types were types 16 (16 patients, 51.6%) and 18 (seven patients, 22.6%). HPV infection was not associated with tumor aggressiveness (pathological tumor stage or differentiation status), risk exposure (alcohol, cigarette, or areca quid chewing habit), or the treatment outcome (disease‐free survival or overall survival). However, infection with HPV‐18 was associated with the occurrence of a second primary cancers (P = 0.033), indicating the infection of HPV in OSCC enhances the susceptibility of developing secondary malignancy. Conclusions: There are 30% of the patients with OSCC infected with HPV, with most high‐risk types. HPV‐18 infection may enhance the susceptibility of second primary tumors. Large scale of validation study will be needed to confirm this result.     

The effects of chewing areca/betel quid with and without cigarette smoking on oral submucous fibrosis and oral mucosal lesions

OBJECTIVES: The purpose of this study was to investigate the risk of areca/betel quid chewing with or without cigarette smoking on oral submucous fibrosis (OSF) and other oral mucosal lesions. METHODS: A stratified case-control study was designed. There were in total 102 patients with oral mucosal lesions or OSF (confirmed pathologically) in the case group. OSF (n = 62) and oral mucosal lesions (n = 62) in 102 subjects were separately analyzed for men and women investigating their risks. RESULTS: For OSF, people with both smoking and chewing habits had a statistically significant odds ratio (OR) 8.68 (95% CI = 1.87, 40.23). For the group of people with chewing habit only and without any lifetime cigarette smoking habit, the OR was 4.51 (95% CI = 1.20, 16.94). For other oral mucosal lesions, people with mixed habits and chewing only had also significant risks (OR = 8.37 and 3.95, respectively). For both OSF and other oral lesions, the ORs of mixed habits and chewing only were both higher in women than in men. Conclusions: The areca/betel quid used in Taiwan does not contain any tobacco product. The only way of areca/betel quid could synergize with any tobacco product is through cigarette smoking. A statistically significant association with oral mucosal lesions and OSF was still found in the group of areca/betel quid chewing only.     

Oral precancerous disorders associated with areca quid chewing, smoking, and alcohol drinking in southern Taiwan

OBJECTIVE: To investigate the prevalence and the associated risk factors of oral precancerous disorders in southern Taiwan. METHODS: We conducted a cross-sectional community survey interviewing 1075 adult subjects, 15 years of age and over, gathered from randomly selected 591 households, and spanning five villages in southern Taiwan. The study protocol included a visual oral soft tissue examination and a questionnaire-based interview. The chi-square test was used to test the differences in prevalence of oral precancerous lesions and conditions by different "life styles" relating to current risk habits of current areca quid chewing, smoking, and alcohol drinking. To control for possible confounding, a logistic regression model was used to estimate the Odds Ratios (OR) for leukoplakia and oral submucous fibrosis (OSF). RESULTS: 136 precancerous lesions and conditions were detected among 1075 subjects (12.7%). The analysis of the spectrum of oral precancerous disorders detected, leukoplakia (n = 80), OSF (n = 17) and verrucous lesions (n = 9), demonstrated an association with gender (P < 0.001). There were statistically significant associations among leukoplakia (P < 0.01), OSF (P < 0.0001), and verrucous lesions (P < 0.0001) and the life style of current areca quid chewing, smoking, and alcohol drinking. The synergistic effect of smoking and areca quid chewing habit on leukoplakia and OSF was demonstrated. CONCLUSION: This study reinforces the association of current areca quid chewing without tobacco, cigarette smoking, and alcohol drinking to leukoplakia, OSF, and verrucous lesions in Taiwan.     

Molecular and cellular cues of diet‐associated oral carcinogenesis—with an emphasis on areca‐nut‐induced oral cancer development

In modern times, potent dietary carcinogens are key contributors for neoplastic development. For oral squamous cell carcinoma (OSCC), one of the leading cancer types in developing countries, main oncogenic inducers/enhancers, including areca nut chewing, tobacco smoking, and alcohol consumption, were shown to promote cancer initiation/progression. Over decades, studies from different laboratories have identified underlying cellular and molecular mechanisms for carcinogen‐induced OSCC. In this review, we will give an overview of where we are in understanding potential oral carcinogenic factors stimulated OSCC tumorigenesis, especially those associated with areca nut chewing in Asians, aiming to provide future scope of possible interception.     

Elevated expression of cyclooxygenase (COX)-2 in oral squamous cell carcinoma – evidence for COX-2 induction by areca quid ingredients in oral keratinocytes

BACKGROUND: Elevated expression of cyclooxygenase (COX)-2 has been demonstrated in several human cancers. Whether COX-2 is up-regulated in areca quid (AQ) related oral squamous cell carcinoma (OSCC) is unknown and the potential of AQ ingredients to induce COX-2 expression has not been studied. METHODS: COX-2 expression was analyzed by immunohistochemistry and RT-PCR in oral tissues. The COX-2 mRNA and protein induction potential of AQ ingredients were analyzed by real-time RT-PCR and Western blotting in normal human oral keratinocyte (NHOK). RESULTS: COX-2 protein expression was significantly higher (P < 0.01) in OSCC (n = 27) as compared to their adjacent non-cancerous matched tissue (NCMT). COX-2 protein was nearly undetectable in control normal oral mucosa. The level of COX-2 mRNA was markedly elevated in 63% (12/19) of OSCC compared to NCMT. Hydroxychavicol induced COX-2 mRNA and protein expression in NHOK. CONCLUSIONS: COX-2 protein as well as mRNA expression were significantly enhanced in OSCC as compared to NCMT. Hydroxychavicol, a unique ingredient in AQ, induced COX-2 expression in NHOK, which highlighted early involvement of COX-2 in AQ-associated oral oncogenesis.     

Polymorphisms in the apoptosis‐associated genes FAS and FASL and risk of oral cancer and malignant potential of oral premalignant lesions in a Taiwanese population

J Oral Pathol Med (2010) 39: 155–161 Background: Our aim was to measure the relationship of FAS (?1377G>A and ?670A>G), FASL (?844C>T) gene variants and risk of oral cancer. Methods: Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis was used to determine the FAS and FASL polymorphisms in 294 oral squamous cell carcinoma (OSCC), 53 oral submucous fibrosis (OSF), and 84 oral leukoplakia (OL) patients, as well as in 333 healthy controls. A standardized questionnaire was applied to collect demographic data, and potential confounding factors. JMP statistical software was used to analyze the association. Results: FAS and FASL polymorphisms were not correlated with OSCC development or the malignant potential of OL by simple and multivariate logistic regression. However, a two‐ to fourfold difference in the risks of betel quid chewing, alcohol consumption, and smoking on OSCC development were observed between participants with different FAS polymorphisms. FAS polymorphisms were significantly correlated with the malignant potential of OSF. Multivariate logistic regression analysis indicated that FAS A_?1377‐G_?670 vs. G_?1377‐A_?670 haplotype (OR = 2.26, 95% CI = 1.16–4.41) was correlated with the malignant potential of OSF. Conclusions: We suggest that FAS and FASL polymorphisms are not significantly correlated with OSCC development or malignant potential of OL. The impact of substance usage on OSCC development could be differentiated by FAS polymorphisms. FAS A_?1377‐G_?670 haplotype may play a role in the malignant potential of OSF.