Global DNA hypomethylation is associated with the development and poor prognosis of tongue squamous cell carcinoma.

BACKGROUNDS: Oral cancer is the 4th leading cause of cancer death for males and the top cancer in young adult males in Taiwan. Tongue squamous cell carcinoma (TSCC) is a common oral cancer and generally associated with poor prognosis. Global DNA hypomethylation at the 5 position of cytosine (5mC) is a well-known epigenetic feature of cancer. Therefore, the purpose of this study was to investigate the relationship of the global 5mC content with the tumorigenesis and prognosis of patients with TSCC. METHODS: The levels of global 5mC were evaluated by immunohistochemistry using tissue microarray slides of 248 surgically resected TSCC and 202 corresponding tumor adjacent normal (TAN) tissues. RESULTS: We found that the level of 5mC in TSCC (P < 0.001) was significantly decreased as compared to TAN. Among TSCC tissues, decreased levels of 5mC were associated with female gender (P = 0.036). In addition, the global hypomethylation was associated with the poor disease-specific survival in TSCC patients (adjusted hazard ratio: 1.55, P = 0.043), especially for patients in older age group (> 50 years, P = 0.013), with moderate or poor cell differentiation (P = 0.044), early stage of disease (I-II, P = 0.046), small tumor size (T1-T2, P = 0.005), without lymph node involvement (P = 0.041), and ever received postoperative radiotherapy (P = 0.009). CONCLUSIONS: Global hypomethylation was an independent biomarker for the development and poor prognosis of TSCC.

Association of OCT4, SOX2, and NANOG expression with oral squamous cell carcinoma progression.

BACKGROUND: OCT4, SOX2, and NANOG are major transcription factors related to stem cell self-renewal and differentiation. The aim of this study was to examine the association of OCT4, SOX2, and NANOG expression levels with the development and prognosis of patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Expression levels of OCT4, SOX2, and NANOG were evaluated by immunohistochemistry with tissue microarray slides of 436 OSCC, 362 corresponding tumor-adjacent normal (CTAN) tissues, and 71 normal uvula epithelium tissues. The clinicopathologic and follow-up data of the OSCC patients were recorded. RESULTS: OCT4 expression was significantly higher in normal and CTAN tissues than in tumor tissue (both P < 0.001). SOX2 expression in CTAN tissue was significantly higher than that in normal (P = 0.021) and tumor tissues (P < 0.001). However, NANOG expression was significantly higher in CTAN (P = 0.014) and tumor tissues (P = 0.009) than in normal tissue. Higher OCT4 and SOX2 expressions were associated with earlier AJCC stage (P = 0.002 and P < 0.001), small tumor size (P = 0.017 and P = 0.001), and the absence of lymph node metastasis (P = 0.015 and P = 0.025). Higher levels of SOX2 expression were associated with better disease-specific survival (P = 0.002) even after adjustment for clinicopathologic factors. DISCUSSION: OCT4 and SOX2 are biomarkers of tumorigenesis and early stage OSCC. SOX2 is an independent prognostic factor for OSCC.

Association of SDF-1 and CXCR4 Polymorphisms With Susceptibility to Oral and Pharyngeal Squamous Cell Carcinoma.

BACKGROUND/AIM: Long-term exposure to betel quid (BQ)-, cigarette-, and alcohol-induced chronic inflammation is a crucial risk factor for oral and pharyngeal squamous cell carcinoma (OPSCC) progression. We analyzed the genotypes of stromal-cell-derived factor-1 (SDF-1) and CXC-chemokine receptor-4 (CXCR4) and determined the association between their polymorphisms and the risk of OPSCC. MATERIALS AND METHODS: This study consisted of 452 patients with pathologically proved OPSCC and 424 sex- and age-matched cancer-free controls. The genotypes of SDF-1 and CXCR4 were detected through the TaqMan real-time polymerase chain reaction (PCR) method. RESULTS: Our data indicated that the C allele and C/C genotypes of CXCR4 were significantly associated with OPSCC [adjusted odds ratio (AOR)=1.41, 95% confidence interval (CI):1.02-1.96, p=0.037 and AOR=1.51, 95% CI:1.05-2.17, p=0.028, respectively] and OSCC (AOR=1.41, 95%CI:1.00-2.00, p=0.049 and AOR=1.49, 95%CI:1.01-2.20, p=0.044, respectively) risk. Patients with genetic polymorphisms of the genotype combination SDF-1/CXCR4 had a higher risk of OSCC (p trend=0.033). We analyzed the effects of CXCR4 genetic variants on susceptibility to OPSCC in patients with different risk habits of BQ chewing, tobacco smoking and alcohol consumption, and revealed that C/T+T/T genotypes exerted an increased risk only in patients with one (AOR=2.68, p=0.036) or two risk habits (AOR=2.02, p=0.027) compared to patients with the C/C genotype. CONCLUSION: We concluded that CXCR4 C>T can be used as a genetic marker of susceptibility to OPSCC, particularly in OPSCC patients with one or two types of risk habits with a synergistic effect.

Roles of the genetic variation of alcohol-metabolizing enzymes on biomarkers in trauma patients with excessive alcohol intake at emergency department.

BACKGROUND: Alcohol abuse has been implicated as an important factor for accidents. We evaluated the roles of different genetic combinations of the ADH2 and ALDH2 genotypes on biomarkers in trauma patients with excessive alcohol intake at our emergency department. METHODS: Blood samples were obtained from 80 patients and 88 age-matched controls. The biomarkers, including AST, ALT, GGT, and MDA, were assayed. The polymerase chain reaction-restriction fragment length polymorphism method was used to determine the genetic polymorphisms of ADH2 and ALDH2. RESULTS: There were significant differences in the levels of AST, ALT, GGT, MDA, and AST/ALT ratios between the 2 groups. In addition, MDA values and AST/ALT ratios were significantly higher in the patients with normal activity of ADH2 than the patients with low activity of ADH2. Meanwhile, regarding ALDH2 genotypes, there were significantly higher ratios of AST/ALT in the patients with low activity of ALDH2. The highest AST/ALT ratios and MDA values were in the patients with ADH2 (*2/*2) and ALDH2 (*1/*2 and *2/*2). CONCLUSIONS: In conclusion, our results indicated that alcohol-induced liver damage or oxidative stress might be influenced by the genetic variation of ADH2 or ALDH2. Therefore, the combinations of different ADH2 and ALDH2 genotypes may be influential markers for susceptibility to alcohol-induced liver damage.

Distribution of alcohol-metabolizing enzyme genotypes in trauma patients with excessive alcohol consumption in the emergency department.

OBJECTIVES: The objective of this study was to investigate the distribution of genetic polymorphisms of alcohol-metabolizing enzymes in trauma patients with excessive alcohol consumption in the emergency department (ED). DESIGN AND METHODS: A total of 100 trauma patients and age-matched control subjects composed of 98 participants were enrolled in this study. The activities of liver enzymes and genotypes of alcohol-metabolizing enzymes, including ADH2, ALDH2, and CYP2E1, were analyzed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: There was a significant difference in the allele frequencies of ALDH2 between the two groups. For the genotypes, there were significant differences in the genotype frequencies of ADH2 and ALDH2. There was also a significantly lower frequency in patients with the ALDH2*2 phenotype than those of the controls. For the activities of liver enzymes, there were significant differences between the two groups. For ADH2 and ALDH2, there were significantly higher ORs (odds ratios) in trauma patients with normal activity than those with weak or intermediate activity but there were no significant difference in CYP2E1 genotype between two groups. To investigate the interaction of alcohol-metabolizing enzyme genotypes, we have estimated the odds ratios in two alcohol-metabolizing pathways. The ORs of the combined genotypes of ADH2 (*1/*1+*1/*2) and ALDH2 (*1/*1) and the combined genotypes of either CYP2E1 (*c1/*c1) or CYP2E1 (*c1/*c2+*c2/*c2) and ALDH2 (*1/*1) were significantly higher than that of the reference group in the major and the minor pathway, respectively. CONCLUSIONS: Genetic variation of alcohol-metabolizing enzymes, especially ALDH2, may play an important role on the occasions of alcohol problems in the emergency department.

The variant of pri-mir-26a-1 polymorphism is associated with decreased risk of betel quid-related oral premalignant lesions and oral squamous cell carcinoma.

OBJECTIVES: This case-control study evaluated the association of the single nucleotide polymorphism rs7372209 (T>C) in pri-mir-26a-1 with the risk and progression of betel quid (BQ)-related oral premalignant lesions (OPLs) and oral squamous cell carcinoma (OSCC). STUDY DESIGN: In total, 597 BQ chewers were recruited: 196 healthy controls, 241 patients with OPLs, and 160 patients with OSCC. Genotypes were determined using the TaqMan real-time assay. RESULTS: The C/T + T/T genotypes and T allele in pri-mir-26a-1 were correlated with a decreased risk of BQ-related OPLs (P = .038 and .005, respectively), oral leukoplakia (P = .01 and .001, respectively), and advanced-stage OSCC (P = .021 and .004, respectively). The effects of the C/T + T/T genotypes and T allele on the decreased risk of OPLs were potent in the older age group (both Pinteraction < .001), heavy smokers (Pinteraction ≤ .003 and .006, respectively) and alcohol drinkers (Pinteraction ≤ .004 and .001, respectively). Furthermore, among patients with OSCC, the C/T + T/T genotypes and T allele were associated with a decreased risk of advanced pathologic stage (P = .032) and lymph node involvement (P = .017). CONCLUSIONS: BQ chewers carrying the T allele or C/T + T/T genotypes in pri-mir-26a-1 may have a decreased risk of oral leukoplakia, OPLs, and advanced-stage OSCC.

Whey protein concentrate promotes the production of glutathione (GSH) by GSH reductase in the PC12 cell line after acute ethanol exposure.

Excessive ethanol consumption may increase the production of reactive oxygen species (ROS), which results in the damage of tissues, especially the neurons and glial cells in the central nervous system (CNS). The purpose of this study is to evaluate the effects of whey protein concentrate (WPC) on the glutathione (GSH) status after acute ethanol exposure in the pheochromocytoma (PC12) cell line. In this study, we assayed the cell viability, the percentage of lactate dehydrogenase released (% LDH released), the level of GSH, and the activity of GSH reductase (GRx). The results showed that with the supplement of WPC, the cell viability displayed no significant difference after acute exposure of ethanol in groups with or without ethanol treatment. The ethanol-induced cytotoxicity showed a slight decrease ,and the level of GSH showed a significant increase. The activity of GRx significantly increased when 0.1, 10mg/ml of WPC was supplied. In conclusion, these results suggest that WPC in a moderate concentration should be a precursor agent to promote the production of GSH and will enhance the antioxidant capacity in the PC12 cell line.

Aberrant DNA hypomethylation of miR-196b contributes to migration and invasion of oral cancer.

MicroRNAs (miRs) are a class of small endogenous non-coding RNAs of ~21-24 nucleotides in length. Previous studies have indicated that miR-196b has either an oncogenic or tumor-suppressive function in various types of cancer. However, the biological role of miR-196b in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, the expression levels of miR-196b were examined in oral cancer tissues and corresponding adjacent normal tissues from 69 OSCC patients using stem-loop reverse transcription-quantitative polymerase chain reaction. The results indicated that miR-196b was significantly overexpressed in OSCC tissues compared with the corresponding adjacent normal tissue samples (64 of 69, 92.7%, P<0.001). Analysis of the methylation status of the miR-196b gene indicated more frequent hypomethylation of the CpG islands located upstream of the miR-196b gene in the OSCC tissues than in the adjacent normal tissues (32 of 69, 46.3%), and the methylation status of miR-196b correlated inversely with its expression levels. Furthermore, the unmethylated status of the miR-196b promoter correlated with poor disease-specific survival in OSCC patients (P=0.035). Functional analysis revealed that ectopic miR-196b expression promoted oral cancer cell migration and invasion abilities, and that silencing of miR-196b could abrogate in vitro migration and invasion of oral cancer cells. Collectively, the present findings indicate that the epigenetic regulation of miR-196b expression plays a crucial role in modulating cell migration and invasion during OSCC progression, and thus may serve as a potential prognosis marker or therapeutic target for OSCC.

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.

Second primary tumors and myeloperoxidase expression in buccal mucosal squamous cell carcinoma.

OBJECTIVE: The present study investigated the relationship between the expression of manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase (GPx), and myeloperoxidase (MPO) in buccal mucosal squamous cell carcinoma (SCC), and the risk of second primary tumors (SPTs). STUDY DESIGN: Immunohistochemistry was performed to examine the expression of MnSOD, GPx, catalase, and MPO in tissue microarray slides of 173 male patients with buccal mucosal SCC who had undergone potentially curative resections. RESULTS: Forty-five (26.01%) patients developed SPTs. The prevalent subsites of SPTs were buccal mucosa (48.89%), tongue (13.33%), and lip (11.11%). High expression level of MPO was correlated with an increased risk of SPTs, even after adjustment for development of clinicopathologic parameters (high vs. low expression, adjusted hazard ratio = 3.89; 95% confidence interval, 1.33-11.41; P = .013). CONCLUSIONS: SPTs are common in male buccal mucosal SCC patients. Higher MPO expression in buccal mucosal SCC is a risk factor for SPTs.

Roles of the genetic polymorphisms of alcohol-metabolizing enzymes on the immunology in high-risk drinkers.

Alcohol metabolism involves several enzymes and the individual genetic variations in the alcohol metabolism are related to the absorption, distribution, and elimination of alcohol and metabolites such as acetaldehyde. Therefore, the genetic variations of alcohol-metabolizing enzymes are responsible for the different toxicity of alcohol in several organs like liver and immunological systems. The purpose of this study was to evaluate if the life styles such as drinking and smoking and the genetic variations of alcohol-metabolizing enzymes (ADH2, ALDH2, CYP2E1, and CAT) were associated with the immunological biomarkers. In this study, 105 high-risk drinkers and 102 low-risk drinkers who were excluded from the immune-related diseases and other critical diseases were enrolled to evaluate the immunological functions. Counts of white blood cells, mononuclear cells, and lymphocyte subpopulations, and liver and immunological function tests were measured. Genotypes of alcohol-metabolizing enzymes were assayed by a real-time PCR and PCR-restriction fragment length polymorphism. Generally, the activity of aspartate aminotransferase (AST) was higher than that of alanine aminotransferase (ALT) in alcoholics; however, the activities of AST and ALT were simultaneously elevated in general hepatitis except for alcohol-induced hepatitis. Thus, the higher ratio of AST/ALT was used to be a marker for the alcohol-induced abnormal liver function. Glutamyltransferase (GGT) is produced by the liver cell microsomes and is a useful laboratory marker as an indicator of early liver cell damage. An increase in GGT concentration has been regarded as a marker of alcohol consumption or liver disease. In addition, the synergistic effects of smoking and drinking on the count of white blood cell (WBC) and mononuclear cells were found to be significant. Furthermore, there were higher OR to become high-risk drinkers in subjects with the combination of ALDH2 (*1/*1) genotype and either genotype of ADH2 or CYP2E1 than the others with other combinations of genotypes. Additionally, there were more abnormal immunological tests in the subjects with higher activity of ADH2 and lower activity of ALDH2. Our results suggested that the habits of drinking, smoking, and betel chewing, and genetic variations of alcohol metabolism were associated with the immunological biomarkers.

Effects of alcohol-induced human peripheral blood mononuclear cell (PBMC) pretreated whey protein concentrate (WPC) on oxidative damage.

Excessive alcohol consumption can induce apoptosis in a variety of tissues and influence the antioxidant status in peripheral blood mononuclear cells (PBMC). This paper investigates the effects of whey protein concentrate (WPC) pretreated in PBMC on the apoptosis and antioxidant status after the treatment of alcohol. The results show that the percentages of apoptotic cells in the alcohol-treated group were higher than those in the group without alcohol treatment. Additionally, there was higher glutathione (GSH) peroxidase (GPx) activity when the PBMC were treated with 300 mg/dL of alcohol. With regard to the activity of GSH reductase (GRx), there was higher activity in the group pretreated with WPC than in the group with the treatment of alcohol only. On the contrary, the levels of GSH were reduced after the treatment of alcohol, but there was a higher level of GSH in the group pretreated with WPC. In this study, it was found that the increased level of GSH in PBMC might not be attributed to the effect of GRx because there was still a higher level of GSH in the group with the treatment of WPC and BCNU (a GRx inhibitor) in this study. The results indicated that PBMC pretreated with WPC might ameliorate alcohol-induced effects such as imbalance of the antioxidant status.

Intake of vitamin A-rich foods and lung cancer risk in Taiwan: with special reference to garland chrysanthemum and sweet potato leaf consumption.

A case-control study was conducted to investigate the association between the consumption of local common foods that are rich in vitamin A and the risk of lung cancer in Taiwan. A total of 301 incident lung cancer cases, 602 hospital controls, and 602 neighborhood controls were recruited. The consumption of 13 food items and vitamin supplements was estimated by use of a food frequency questionnaire. The conditional logistic regression models were used to estimate the adjusted odds ratios (AOR) and 95% confidence intervals (CI) for lung cancer risk with each control group as reference by adjustment of covariates. A reduced risk for lung cancer was found to be associated with increased intakes of vitamin A, alpha-carotene, and beta-carotene from 13 food items. More servings of vegetables (AOR for the highest versus the lowest quartile = 0.67-0.70, 95% CI = 0.42-1.08, (plinear trend )= 0.04), garland chrysanthemum (AOR for the highest versus the lowest tertile = 0.58-0.74, 95% CI = 0.37-1.14, (plinear trend )<= 0.04) and sweet potato leaves (AOR for the highest versus the lowest tertile = 0.43-0.65, 95% CI = 0.28-0.96, (plinear trend )<= 0.03) were associated with the reduced risk for lung cancer. In conclusion, higher consumption of vitamin A-rich vegetables, especially garland chrysanthemum and sweet potato leaves might provide potential protection from lung cancer.