Chewing-associated Oral Squamous Cell Carcinomas Research Articles

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.

Correction: Elevated Snail Expression Mediates Tumor Progression in Areca Quid Chewing-Associated Oral Squamous Cell Carcinoma via Reactive Oxygen Species

Correction: Elevated Snail Expression Mediates Tumor Progression in Areca Quid Chewing-Associated Oral Squamous Cell Carcinoma via Reactive Oxygen Species

Elevated Snail Expression Mediates Tumor Progression in Areca Quid Chewing-Associated Oral Squamous Cell Carcinoma via Reactive Oxygen Species

BackgroundSnail is an important transcription factor implicated in several tumor progression and can be induced by reactive oxygen species (ROS). Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). Therefore, we hypothesize that the major areca nut alkaloid arecoline may induce Snail via ROS and involve in the pathogenesis of areca quid chewing-associated OSCC.Methodology/Principal FindingThirty-six OSCC and ten normal oral epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. Cytotoxicity, 2′, 7′-dichlorofluorescein diacetate assay, and western blot were used to investigate the effects of arecoline in human oral keratinocytes (HOKs) and oral epithelial cell line OECM-1 cells. In addition, antioxidants N-acetyl-L-cysteine (NAC), curcumin, and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. Initially, Snail expression was significantly higher in OSCC specimens (p<0.05). Elevated Snail expression was associated with lymph node metastasis (p = 0.031) and poor differentiation (p = 0.017). Arecoline enhanced the generation of intracellular ROS at the concentration higher than 40 µg/ml (p<0.05). Arecoline was also found to induced Snail expression in a dose- and time-dependent manner (p<0.05). Treatment with NAC, curcumin, and EGCG markedly inhibited arecoline induced Snail expression (p<0.05).Conclusion/Significance:Our results suggest that Snail overexpression in areca quid chewing-associated OSCC is associated with tumors differentiation and lymph node metastasis. Arecoline-upregulated Snail expression may be mediated by ROS generation. In addition, arecoline induced Snail expression was downregulated by NAC, curcumin, and EGCG.

β-catenin expression in areca quid chewing-associated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

Nuclear localization of β-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare β-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce β-catenin expression. A total of 40 areca quid chewing-associated OSCCs 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, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl-l-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. β-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for β-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate β-catenin expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycin-A, SB203580, and LY294002 markedly inhibited the arecoline-induced β-catenin expression (p < 0.05). β-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of β-catenin expression is correlated with the tumor size and clinical stage. In addition, β-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002.

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

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. 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. 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). 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.

The upregulation of metallothionein-1 expression in areca quid chewing-associated oral squamous cell carcinomas

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, inducible, intracellular proteins that bind heavy metals with high affinity. MT-1 is known as a stress-inducible protein and functions as an antioxidant enzyme. Areca quid chewing is a major risk factor in the development and further progression of oral squamous cell carcinoma (OSCC). The aim of this study was to compare MT-1 expression in normal human oral epithelium and OSCC and further explore the potential mechanism that may lead to induce MT-1 expression. Thirty four OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. The oral epithelial cell line GMN cells were challenged with arecoline, a major areca nut alkaloid, by reverse-transcriptase polymerase chain reaction. Furthermore, tobacco smoke carcinogen benzo[a]pyrene (BaP) and glutathione (GSH) precursor N-acetyl-l-cysteine (NAC) were added to find the possible regulatory mechanisms. The results from immunohistochemistry demonstrated that MT-1 expression was significantly higher in OSCC specimens (p<0.05). No significant difference in MT-1 expression was observed with respect to age, sex, T category, and stage (p>0.05). The high MT-1 expression was associated with lymph node metastasis (p=0.012). In addition, arecoline was found to elevate MT-1 mRNA in a dose-dependent manner (p<0.05). Furthermore, the addition of BaP enhanced the arecoline-induced MT-1 expression (p<0.05). The addition of NAC markedly inhibited the arecoline-induced MT-1 expression (p<0.05). These results lead to the conclusion that MT-1 expression is significantly upregulated in areca quid chewing associated-OSCC. The expression profile suggests MT-1 could be used clinically as a marker for tumors possessing the potential for lymph node metastasis. The compounds of tobacco products may act synergistically in the pathogenesis of OSCC in areca quid chewers. The regulation of MT-1 expression induced by arecoline is critically dependent on the intracellular GSH concentration.

P53 and MDM2 co-expression in tobacco and betel chewing-associated oral squamous cell carcinomas.

Oral cancers of tobacco and betel chewers represents a unique in-vivo model to understand the genotoxic effect of tobacco and betel carcinogens on oncogenes and tumor suppressor genes. Coordinated interactions of p53 and MDM2 play an important role in regulation of critical growth control gene following exposure to DNA damaging agents. The purpose of this study is to determine if the tumor suppressor function of p53 is inactivated by mutation or other alternative mechanisms in carcinogen-induced oral squamous cell carcinoma (SCC), and to investigate the clinicopathological significance of p53 and MDM2 expression. The p53 mutation in oral SCC of tobacco and betel chewers (n=40) was detected by polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and immunohistochemistry (IHC) was done to investigate p53 and MDM2 proteins overexpression. The incidence of p53 mutation was relatively low (17.5%), but there was a high prevalence of MDM2 overexpression (72.5%). In the total of 40 cases, IHC phenotype showed p53 positive immunostaining with MDM2 positive immunostaining (p53+/MDM2+) 62.5%, p53 negative immunostaining with MDM2 negative immunostaining (p53-/MDM2-) 15%, p53 positive immunostaining with MDM2 negative immunostaining (p53+/MDM2-) 12.5%, and p53 negative immunostaining with MDM2 positive immunostaining (p53-/MDM2+) 10%. A significant correlation was found between MDM2 and p53 overexpression (p=0.0289). Moreover, p53+/MDM2+ phenotype was significantly associated with poorly differentiated tumors (p= 0.0007). These results conclude that other factors than p53 mutation is likely to be the targets of tobacco/betel carcinogens and MDM2 may play an important role in tobacco/betel chewing-related oral SCCs. Overexpression of MDM2 protein may constitute an alternative mechanism for p53 inactivation.