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BACKGROUND:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Human WW domain-containing oxidoreductase (WWOX) gene has been identified as a tumor suppressor gene in multiple cancers. We hypothesize that genetic variations in WWOX are associated with HCC risk. METHODOLOGY/PRINCIPAL FINDINGS:Five single-nucleotide polymorphisms (SNPs) of the WWOX gene were evaluated from 708 normal controls and 354 patients with HCC. We identified a significant association between a WWOX single nucleotide polymorphism (SNP), rs73569323, and decreased risk of HCC. After adjustment for potential confounders, patients with at least one T allele at rs11545028 of WWOX may have a significantly smaller tumor size, reduced levels of α-fetoprotein and alanine aminotransferase (ALT). Moreover, the A allele at SNP rs12918952 in WWOX conferred higher risk of vascular invasion. Additional in silico analysis also suggests that WWOX rs12918952 polymorphism tends to affect WWOX expression, which in turn contributes to tumor vascular invasion. CONCLUSIONS:In conclusion, genetic variations in WWOX may be a significant predictor of early HCC occurrence and a reliable biomarker for disease progression.

BACKGROUND:Oral squamous cell carcinoma (OSCC), which is the most common head and neck cancer, accounts for 1%-2% of all human malignancies and is characterized by poor prognosis and reduced survival rates. WNT1-inducible signaling pathway protein 1 (WISP1), a cysteine-rich protein belonging to the Cyr61, CTGF, Nov (CCN) family of matricellular proteins, has many developmental functions and may be involved in carcinogenesis. This study investigated WISP1 single-nucleotide polymorphisms (SNPs) to elucidate OSCC susceptibility and clinicopathologic characteristics. METHODOLOGY/PRINCIPAL FINDINGS:Real-time polymerase chain reaction was used to analyze 6 SNPs of WISP1 in 900 OSCC patients and 1200 cancer-free controls. The results showed that WISP1 rs2929970 polymorphism carriers with at least one G allele were susceptible to OSCC. Moreover, compared with smokers, non-smoker patients with higher frequencies of WISP1 rs2929970 (AG + GG) variants had a late stage (stages III and IV) and a large tumor size. In addition, OSCC patients who were betel quid chewers and carried WISP1 rs16893344 (CT + TT) variants had a low risk of lymph node metastasis. CONCLUSION:Our results demonstrate that a joint effect of WISP1 rs2929970 with smoking as well as WISP1 rs16893344 with betel nut chewing causally contributes to the occurrence of OSCC. WISP1 polymorphism may serve as a marker or a therapeutic target in OSCC.

Melatonin plays an important role in immunity and has been linked to autoimmune diseases. Possible associations of single-nucleotide polymorphisms (SNPs) of melatonin receptor type 1A (MTNR1A) and 1B (MTNR1B), with autoimmune thyroid disease in an ethnic Chinese (i.e., Taiwanese) population were examined.Totally, 83 Hashimoto's thyroiditis patients, 319 Graves' disease (GD), and 369 controls were recruited. Three SNPs (rs6553010, rs13140012, and rs2119882) of MTNR1A and three SNPs (rs1387153, rs10830963, and rs1562444) of MTNR1B were genotyped.There were a reduced frequency of the C allele of rs2119882 and a reduced percentage of the CC+CT genotype in the GD group compared to the control group (p = 0.039, odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.63~0.99, and p = 0.032, OR = 0.72, 95% CI = 0.53~0.97, respectively). There was a significant difference in the percentage of the AT haplotype of the combination of rs13140012 and rs2119882 between the GD and control groups (p = 0.010, OR = 1.34, 95% CI = 1.07~1.67). In addition, there were significant associations of anti-thyroid peroxidase antibody titers with rs13140012 and rs2119882, and the AATT genotype of the combination of rs13140012 and rs2119882 (p = 0.003, 0.003, and 0.004, respectively). There were no significant associations of SNPs and possible haplotypes of MTNR1B with susceptibility to GD.Genetic variants of rs2119882 of MTNR1A and the AT haplotype of the combination of rs2119882 and rs13140012 were associated with GD susceptibility in an ethnic Chinese population. The results support the involvement of the melatonin pathway in the pathogenesis of GD.

[This corrects the article DOI: 10.1371/journal.pone.0031055.].

BACKGROUND:ADAMTS14 is a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), which are proteolytic enzymes with a variety of further ancillary domain in the C-terminal region for substrate specificity and enzyme localization via extracellular matrix association. However, whether ADAMTS14 genetic variants play a role in hepatocellular carcinoma (HCC) susceptibility remains unknown. METHODOLOGY/PRINCIPAL FINDINGS:Four non-synonymous single-nucleotide polymorphisms (nsSNPs) of the ADAMTS14 gene were examined from 680 controls and 340 patients with HCC. Among 141 HCC patients with smoking behaviour, we found significant associations of the rs12774070 (CC+AA vs CC) and rs61573157 (CT+TT vs CC) variants with a clinical stage of HCC (OR: 2.500 and 2.767; 95% CI: 1.148-5.446 and 1.096-6.483; P = 0.019 and 0.026, respectively) and tumour size (OR: 2.387 and 2.659; 95% CI: 1.098-5.188 and 1.055-6.704; P = 0.026 and 0.034, respectively), but not with lymph node metastasis or other clinical statuses. Moreover, an additional integrated in silico analysis proposed that rs12774070 and rs61573157 affected essential post-translation O-glycosylation site within the 3rd thrombospondin type 1 repeat and a novel proline-rich region embedded within the C-terminal extension, respectively. CONCLUSIONS:Taken together, our results suggest an involvement of ADAMTS14 SNP rs12774070 and rs61573157 in the liver tumorigenesis and implicate the ADAMTS14 gene polymorphism as a predict factor during the progression of HCC.

BACKGROUND:Oral squamous cell carcinoma (OSCC) is the sixth and fourth most common cause of cancer death in men worldwide and in Taiwan, respectively. AURKA, which encodes a centrosome-related serine/threonine kinase, is frequently amplified and overexpressed in many human cancers, particularly advanced OSCC. We conducted a hospital-based case-control study to estimate AURKA single-nucleotide polymorphisms (SNPs) and environmental risk factors to determine OSCC susceptibility and clinicopathological characteristics. METHODOLOGY/PRINCIPAL FINDINGS:We enrolled a total of 876 OSCC patients and 1200 controls. Four SNPs of AURKA, namely rs1047972, rs2273535, rs2064863, and rs6024836, were analyzed using real-time polymerase chain reaction (PCR). Among the 1420 smokers, the AURKA polymorphism carriers with the betel nut chewing habit had a higher risk of oral cancer than AURKA wild-type (WT) carriers without the betel nut chewing habit. Patients with the AURKA rs2064863 gene had a 1.365-fold higher risk of stage III or IV OSCC (95% confidence interval [CI] 1.029-1.811) than those with the rs2064863 WT gene. Furthermore, carriers of the AURKA rs1047972/rs2273535/rs2064863 C-A-T haplotype had a 1.736-fold (95% CI 1.110-2.715) higher risk of OSCC than controls (C-T-T, the most common haplotype). Among patients with the betel quid chewing habit, carriers of other haplotypes (C-T-T, C-A-G, T-A-T, T-A-G, T-T-T, and C-T-G) had a 12.857-fold (95% CI 10.731-15.404) increased risk, and carriers of the C-A-T haplotype had the highest risk (AOR: 31.120; 95% CI 13.864-69.850) of OSCC, compared with those without the betel quid chewing who harbored other haplotypes. CONCLUSIONS:In conclusion, betel nut chewing combined with the AURKA C-A-T haplotypes lead to a high risk of OSCC. These findings reveal a novel genetic-environmental predisposition for oral tumorigenesis.

BACKGROUND:We identified rs17071138 T/C, rs3744941 C/T, and rs8089104 T/C gene polymorphisms of SERPINB5 (mammary serine protease inhibitor) that are specific to patients with oral cancer susceptibility and their clinicopathological status. METHODOLOGY/PRINCIPAL FINDINGS:In total, 1342 participants, including 601 healthy controls and 741 patients with oral cancer, were recruited for this study. Allelic discrimination of rs17071138 T/C, rs3744941 C/T, and rs8089104 T/C of the SERPINB5 gene was assessed by a real-time PCR with a TaqMan assay. We found that individuals carrying the polymorphic rs17071138 and rs8089104 are more susceptible to oral cancer (OR, 1.57; 95% CI, 1.07~2.31 and OR, 1.58; 95% CI, 1.04~2.39, respectively). Among oral cancer-related risk factor exposures, the individuals carrying the polymorphic rs17071138 had 4.26- (95% CI: 1.65~11.01; p = 0.002), 2.34- (95% CI: 1.19~4.61; p = 0.01), and 2.34-fold (95% CI: 1.38~3.96; p = 0.001) higher risks of developing oral cancer. CONCLUSIONS:Heterozygous TC of the SERPINB5 rs17071138 polymorphism may be a factor that increases susceptibility to oral cancer. Interactions of gene-to-gene and gene-to-oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.

[This corrects the article DOI: 10.1371/journal.pone.0056661.].

Background/purpose: Wogonin (5,7-dihydroxy-8-methoxyflavone) is a bioactive compound from Scutellariae radix that has been shown to have anticancer effects. However, the effects of wogonin on oral cancer cells still remain speculative. Materials and methods: Cytotoxicity and invasion assays were used to investigate the effects of the human oral cancer cell line OC2 cells exposed to wogonin. To examine the precise involvement of wogonin in cancer metastasis, gelatin and casein zymography were performed to evaluate the impacts of wogonin on matrix metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (u-PA) secretion in OC2 cells. Results: Wogonin exhibited a dose-dependent inhibitory effect on the invasion of OC2 cells in the absence of cytotoxicity (P

Objective: To investigate the correlation of two important inflammatory biomarkers, plasma osteopontin and neutrophil gelatinase-associated lipocalin (NGAL), with the severity and outcome of pelvic inflammatory disease (PID). Materials and methods: Sixty-one patients with PID, including 25 patients with tubo-ovarian abscess (TOA), were consecutively recruited. Their blood samples were tested for the concentrations of plasma osteopontin and NGAL using enzyme-linked immunosorbent assay. The associations of these biomarkers with TOA, length of hospitalization, and incidence of surgery were also analyzed. Results: Plasma osteopontin level was significantly increased in PID patients with TOA compared to PID patients without TOA (median 107.77 ng/mL vs. 72.39 ng/mL, p = 0.004). However, there was no significant difference for plasma NGAL. If the cutoff level of plasma osteopontin was set at 81.1 ng/mL, there was a 76.0% sensitivity and a 24.0% false negative rate in predicting TOA in PID patients. Plasma osteopontin significantly correlated with length of hospital stay (r = 0.467, p

Background/purpose: Areca quid chewing is associated with periodontal diseases. Many of the undesirable effects of areca quid have been attributed to safrole, a major polyphenolic compound in the inflorescence of Piper betle. Matrix metalloproteinases (MMPs, a subclass of proteolytic enzymes), plasminogen activators (PAs), and cyclooxygenase-2 (COX-2) have been reported to play an important role in the pathogenesis of periodontitis. Therefore, this study was carried out to determine the biological effects of safrole on human gingival fibroblasts (HGFs). Materials and methods: Five HGF strains were established from crown lengthening surgery. The cytotoxicity of safrole was measured by H33258 fluorescence assay and the levels of gelatinolytic and caseinolytic activities were measured by gelatin and casein zymography, respectively. Western blot analysis was used to evaluate the expression of COX-2. Results: The study results show that safrole was cytotoxic to HGFs in a concentration-dependent manner (P

Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA’s increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA’s apoptotic effects on human osteosarcoma cells.

Fisetin, a natural flavonoid, is known to have anticarcinogenic effects against several cancers, but its role in mediating renal cell carcinoma (RCC) progression has not been delineated. Cell viability, cytotoxicity, and cell cycle distribution were measured using the 3‐(4,5‐cimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and propidium iodide staining with flow cytometry. The in vitro migration and invasion assay was used to examine in vivo cell migration and invasion. Human protease antibody array analysis was conducted with cell migration/invasion-related proteins. Western blotting and quantitative reverse transcription polymerase chain reaction were used for assessing protein expression related to the cell cycle, cell invasion, and mitogen-activated protein kinase (MAPK) signaling pathway. We found that fisetin significantly inhibited cell viability through cell cycle arrest in the G2/M phase, in addition to downregulating cyclin D1 and upregulating p21/p27. Fisetin inhibited the migration and invasion of human RCC cells through the downregulation of CTSS and a disintegrin and metalloproteinase 9 (ADAM9). Fisetin also upregulated ERK phosphorylation in 786-O and Caki-1 cells. Furthermore, treatment with a MEK inhibitor (UO126) reduced the inhibitory effects of fisetin on the metastasis of RCC cells through the ERK/CTSS/ADAM9 pathway. Fisetin inhibits proliferation and metastasis of RCC cells by downregulating CTSS and ADAM9 through the MEK/ERK signaling pathway. These findings indicate that fisetin is a promising antitumor agent against RCC.

BACKGROUND:The B-lymphocyte-activating factor (BAFF) is associated with B-cell functions, and gene polymorphisms of the BAFF have been linked to autoimmune diseases (AIDs). In this study, we explored possible associations of two BAFF single-nucleotide polymorphisms (SNPs), rs1041569 and rs2893321, with autoimmune thyroid diseases (AITDs) in an ethnic Chinese population. MATERIAL AND METHODS:In total, 319 Graves' disease (GD), 83 Hashimoto's thyroiditis (HT) patients, and 369 healthy controls were enrolled. Polymerase chain reaction-restriction fragment length polymorphism and direct sequencing were used to genotype rs2893321 and rs1041569. RESULTS:There was a significant difference in frequencies of the G allele and AG+GG genotype of rs2893321 between the GD and control groups (p = 0.013, odds ratio (OR) = 0.76, and p = 0.017, OR = 0.68, respectively) and between the AITD and control groups (p = 0.009, OR = 0.76, and, p = 0.014, OR = 0.69, respectively). The AA genotype of rs2893321 was associated with low titers of the thyroid-stimulating hormone receptor antibody (TSHRAb) (p = 0.015) in males but not in females. The AA genotype of rs2893321 was associated with the presence of two different types of thyroid autoantibody (TAb) (TSHRAb and Hashimoto's autoantibody (anti-thyroglobulin or anti-microsomal antibody)) in females and with that of one type in males. CONCLUSIONS:rs2893321 may be a susceptible genetic variant for the development of GD and AITDs. Associations of rs2893321 with susceptibility to GD and AITDs and the correlation between rs2893321 and TAb exhibit a dimorphic pattern. Additional studies with larger sample sizes are required to confirm our findings.

Oral cancer is a common malignancy that is shown to be causally associated with hereditary and acquired factors. ADAMTS14 is a member of the ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin motifs) metalloproteinase family that plays an important role in extracellular matrix (ECM) assembly and degradation. Elevation or deficiency of certain ADAMTS proteinases has been known to be implicated in a wide range of pathological processes including atherosclerosis, arthritis, and cancer. The present study aimed to explore the impact of ADAMTS14 gene polymorphisms, combined with environmental risks on the susceptibility to oral tumorigenesis.Four single-nucleotide polymorphisms (SNPs) of the ADAMTS14 gene, including rs10823607, rs12774070, rs4747096, and rs61573157 were evaluated from 1200 normal controls and 850 patients with oral cancer. We failed to detect a significant association of four individual SNPs with oral cancer between case and control group. However, while considering behavioral exposure of environmental carcinogens, the presence of four ADAMTS14 SNPs, combined with betel nut chewing and/or smoking, profoundly leveraged the risk of oral cancer. Moreover, we observed a significant association of rs12774070, which is predicted to alter the expression and function of ADAMTS14 by in silico and bioinformatics analyses, with poor tumor cell differentiation (AOR: 0.59; 95% CI: 0.38-0.92; p = 0.02) in patients who chewed betel nuts.These results implicate the interaction between ADAMTS14 gene polymorphisms and environmental mutagens as a risk factor of oral tumorigenesis and suggest a correlation of rs12774070 with the degree of oral tumor cell differentiation.

The purpose of this study was to identify gene polymorphisms of mammary serine protease inhibitor (Maspin) specific to patients with oral cancer susceptibility and clinicopathological status.Three single-nucleotide polymorphisms (SNPs) of the Maspin gene from 741 patients with oral cancer and 601 non-cancer controls were analyzed by real-time PCR. The participants with G/G homozygotes or with G/C heterozygotes of Maspin rs2289520 polymorphism had a 2.07-fold (p = 0.01) and a 2.01-fold (p = 0.02) risk of developing oral cancer compared to those with C/C homozygotes. Moreover, gene-gene interaction increased the risk of oral cancer susceptibility among subjects expose to oral cancer related risk factors, including areca, alcohol, and tobacco consumption.G allele of Maspin rs2289520 polymorphism may be a factor that increases the susceptibility to oral cancer. The interactions of gene to oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.

Cathepsin B (CTSB), a member of the cathepsin family, is a cysteine protease that is widely distributed in the lysosomes of cells in various tissues. It is overexpressed in several human cancers and may be related to tumorigenesis. The main purpose of this study was to analyze CTSB expression in oral squamous cell carcinoma (OSCC) and its correlation with patient prognosis.Tissue microarrays were used to detect CTSB expression in 280 patients and to examine the association between CTSB expression and clinicopathological parameters. In addition, the metastatic effects of the CTSB knockdown on two oral cancer cell lines were investigated by transwell migration assay. Cytoplasmic CTSB expression was detected in 34.6% (97/280) of patients. CTSB expression was correlated with positive lymph node metastasis (p = 0.007) and higher tumor grade (p = 0.008) but not with tumor size and distant metastasis. In addition, multivariate analysis using a Cox proportional hazards model revealed a higher hazard ratio, demonstrating that CTSB expression was an independent unfavorable prognostic factor in buccal mucosa carcinoma patients. Furthermore, the Kaplan-Meier curve revealed that buccal mucosa OSCC patients with positive CTSB expression had significantly shorter overall survival. Moreover, treatment with the CTSB siRNA exerted an inhibitory effect on migration in OC2 and CAL27 oral cancer cells.We conclude that CTSB expression may be useful for determining OSCC prognosis, particularly for patients with lymph node metastasis, and may function as a biomarker of the survival of OSCC patients in Taiwan.

Cysteinyl leukotrienes (CysLTs) play a major role in the pathogenic changes of airway inflammation in asthma treatment. The matrix metalloproteinase (MMP) family, especially MMP-9 and MMP-2 levels, can reflect the status of airway remodeling. This study was undertaken to determine the role of a specific CysLT receptor antagonist in inhibition of airway inflammation and reversal of airway remodeling. Methods: Ovalbumin (OVA)-sensitized BALB/c mice were fed with a specific leukotriene receptor antagonist (MK-679), prednisolone or placebo from Days 15 to 27. Airway hyperreactivity, bronchoalveolar lavage fluid (BALF), and sera were analyzed. Pulmonary histology was obtained, and the levels of MMP-2 and MMP-9 in BALF were measured. Results: The OVA-sensitized mice developed significant airway inflammatory responses, including extensive eosinophils trafficking into BALF and lung interstitium, goblet cell hyperplasia, mucus hypersecretion, elevated serum immunoglobulin (Ig) E, and decreased level of serum IgG2a. Administration of MK-679 could reduce airway inflammation but was not as effective as prednisolone. However, MK-679 was more effective than prednisolone for reversing subepithelial fibrotic and myofibrotic reactions of airway remodeling. The levels of MMP-2 and -9 in BALF were proportional to the extent of airway remodeling, which can reflect the effects of treatment. Both prednisolone and MK-679 reverse airway hyperresponsiveness induced by OVA-sensitized mice. Conclusion: Cysteinyl leukotriene receptor plays a more important role than CysLT in the pathogenesis of allergic airway inflammation. MMP-2 and -9 may be more sensitive indicators of airway remodeling.

Reference intervals of biochemical tests for screening for diabetes mellitus and liver and renal function among school children in Central Taiwan have never been documented. Therefore, this study aimed to establish the reference intervals for the above mentioned biochemical tests for pediatric populations. Methods: A total of 4326 subjects, including 2029 kindergarten children, 1624 elementary-school children, 325 junior-high-school children, and 348 teachers were selected randomly in Central Taiwan. All serum alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (Cr) and glucose levels were determined using a Beckman Synchron CX5 analyzer. The reference intervals reflected estimates of the 2.5th–97.5th percentiles of non-parametric distributions. Results: Adults had significantly higher biochemical analyte values [except for BUN/creatinine (B/C) ratio] than children had. Multiple logistic regression analysis showed that biochemical analyte values were significantly higher in male than in female subjects. The concentrations of glucose and Cr increased with age. On the contrary, the B/C ratio decreased with age. Conclusion: Our study provides new pediatric reference intervals (2.5th–97.5th percentiles) of 60–99 mg/dL for serum glucose concentrations, 8–38 IU/L for ALT, 0.4–1.1 mg/L for Cr, 8.7–18.0 mg/L for BUN, and 10–34 for B/C ratio. The B/C ratio in children was higher than those of adults, possibily due to that children had a higher intake of protein.

Oral lichen planus (OLP) is a chronic inflammatory disorder characterized by a T cell-mediated immune response against epithelial cells. Matrix metalloproteinases (MMPs) are an important group of zinc enzymes and are thought to play an important role in the degradation of components of the extracellular matrix. The aim of this study was to assess the expression of MMP-2 and MMP-9 in both tissue specimens and circulating plasma. Materials and methods: Twelve cases of OLP were collected. In addition, six cases with chronic inflammation and six normal subjects were also recruited. Oral tissue specimens were collected for measurement of MMPs by immunohistochemistry, and sera prepared from peripheral blood were used for gelatin zymography to determine MMP levels in plasma. Results: The level of MMP-2 in patients with OLP was significantly higher than that in the other two groups, both in tissues and sera (P < 0.0001). However, there was no difference in the expression of MMP-9 among these groups. Conclusion: MMP-2 overexpression in OLP is consistent with its upregulation in peripheral serum. This result also indicates that MMP-2 might play a role in the pathogenesis of OLP.

Heme oxygenase-1 (HO-1) is known as an oxidative stress responsive protein that is upregulated by various physiologic and endogenous stimuli. HO-1 has been proposed to provide an important cellular response that protects cells against oxidative damage. Areca quid chewing is a major risk factor in the development and further progression of oral squamous cell carcinoma (OSCC). The aim of the present study was to investigate the difference in HO-1 expression in normal human oral epithelium and OSCC, and further explore the potential mechanism that may lead to HO-1 expression. Methods: Thirty-five OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by clinicopathologic profiles. The oral epithelial GNM cell line was 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 were added to find the possible regulatory mechanisms. Results: HO-1 expression was significantly higher in OSCC specimens (p < 0.05). No significant difference in HO-1 expression was observed with respect to age, sex, T category, and stage (p > 0.05). The high HO-1 expression was associated with lymph node metastasis (p = 0.005). In addition, arecoline was found to elevate HO-1 mRNA in a dose-dependent manner (p < 0.05). The addition of BaP enhanced arecoline-induced HO-1 expression (p < 0.05). Moreover, addition of NAC markedly inhibited arecoline-induced HO-1 expression (p < 0.05). Conclusion: Taken together, these results suggest that HO-1 expression is significantly upregulated in OSCC from areca quid chewers, and arecoline may be responsible for enhanced HO-1 expression in vivo. The compounds of cigarette smoke may act synergistically in the pathogenesis of areca-quid-chewing-associated OSCC. The regulation of HO-1 expression induced by arecoline is critically dependent on intracellular GSH concentration.

The purpose of this study was to explore the combined effect of melatonin receptor type 1A (MTNR1A) gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of oral cancer.Three polymorphisms of the MTNR1A gene from 618 patients with oral cancer and 560 non-cancer controls were analyzed by real-time polymerase chain reaction (PCR). The CTA haplotype of the studied MTNR1A polymorphisms (rs2119882, rs13140012, rs6553010) was related to a higher risk of oral cancer. Moreover, MTNR1A gene polymorphisms exhibited synergistic effects of environmental factors (betel quid and tobacco use) on the susceptibility of oral cancer. Finally, oral-cancer patients with betel quid-chewing habit who had T/T allele of MTNR1A rs13140012 were at higher risk for developing an advanced clinical stage and lymph node metastasis.These results support gene-environment interactions of MTNR1A polymorphisms with smoking and betel quid-chewing habits possibly altering oral-cancer susceptibility and metastasis.

Fibroblast growth factor receptor 4 (FGFR4) polymorphisms are positively correlated with tumor progression in numerous malignant tumors. However, the association between FGFR4 genetic variants and the risk of hepatocellular carcinoma (HCC) has not yet been determined. In this study, we investigated the potential associations of FGFR4 single nucleotide polymorphisms (SNPs) with HCC susceptibility and its clinicopathological characteristics.Four SNPs in FGFR4 (rs1966265, rs351855, rs2011077, and rs7708357) were analyzed among 884 participants, including 595 controls and 289 patients with HCC. The samples were further analyzed to clarify the associations between these gene polymorphisms and the risk of HCC, and the impact of these SNPs on the susceptibility and clinicopathological characteristics of HCC. After adjusting for other covariants, HCC patients who carrying at least one A genotype (GA and AA) at rs351855 were observed to have a higher risk of liver cirrhosis compared with those carrying the wild-type genotype (GG) (OR: 2.113, 95% CI: 1.188-3.831). Moreover, the patients with at least one A genotype were particularly showed a high level of alpha-fetoprotein (AFP).Our findings suggest that genetic polymorphism in FGFR4 rs351855 may be associated with the risk of HCC coupled with liver cirrhosis and may markedly increase the AFP level in Taiwanese patients with HCC. In addition, this is the first study that evaluated the risk factors associated with FGFR4 polymorphism variants in Taiwanese patients with HCC.

BACKGROUND: Matrix metalloproteinase-11 (MMP-11) is reported to be overexpressed in several cancers and may contribute to tumorigenesis. The current study investigated the association between the clinicopathological characteristics and plasma level of MMP-11 in oral squamous cell carcinoma (OSCC) patients. METHODOLOGY AND PRINCIPAL FINDINGS: The plasma MMP-11 concentration was determined by ELISA on 330 male OSCC patients. In addition, the metastatic effects of the MMP-11 knockdown on the oral cancer cells were investigated by cell migration assay. Our results showed that the plasma MMP-11 levels were significantly higher in patients with advanced T status (p = 0.001), lymph node metastasis (p = 0.006) and higher TNM stages (p

Pterostilbene (PTER) is a dimethylated analog of the phenolic phytoalexin, resveratrol, with higher anticancer activity in various tumors. Herein, the molecular mechanisms by which PTER exerts its anticancer effects against acute myeloid leukemia (AML) cells were investigated.Results showed that PTER suppressed cell proliferation in various AML cell lines. PTER-induced G0/G1-phase arrest occurred when expressions of cyclin D3 and cyclin-dependent kinase (CDK)2/6 were inhibited. PTER-induced cell apoptosis occurred through activation of caspases-8-9/-3, and a mitochondrial membrane permeabilization (MMP)-dependent pathway. Moreover, treatment of HL-60 cells with PTER induced sustained activation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2, and inhibition of both MAPKs by their specific inhibitors significantly abolished the PTER-induced activation of caspases-8/-9/-3. Of note, PTER-induced cell growth inhibition was only partially reversed by the caspase-3-specific inhibitor, Z-DEVE-FMK, suggesting that this compound may also act through a caspase-independent pathway. Interestingly, we also found that PTER promoted disruption of lysosomal membrane permeabilization (LMP) and release of activated cathepsin B.Taken together, our results suggest that PTER induced HL-60 cell death via MAPKs-mediated mitochondria apoptosis pathway and loss of LMP might be another cause for cell apoptosis induced by PTER.

BACKGROUND: Glabridin, a prenylated isoflavonoid of G. glabra L. roots, has been associated with a wide range of biological properties such as regulation of energy metabolism, estrogenic, neuroprotective, anti-osteoporotic, and skin-whitening in previous studies. However, the effect of glabridin on tumor cells metastasis has not been clearly clarified. Here, the molecular mechanism by which glabridin anticancer effects in human promyelocytic leukemia cells was investigated. METHODOLOGY AND PRINCIPAL FINDINGS: The results showed that glabridin significantly inhibited cell proliferation of four AML cell lines (HL-60, MV4-11, U937, and THP-1). Furthermore, glabridin induced apoptosis of HL-60 cells through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner. Moreover, western blot analysis also showed that glabridin increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner. Inhibition of p38 MAPK and JNK1/2 by specific inhibitors significantly abolished the glabridin-induced activation of the caspase-3, -8 and -9. CONCLUSION: Taken together, our results suggest that glabridin induced HL-60 cell apoptosis through p38 MAPK and JNK1/2 pathways and could serve as a potential additional chemotherapeutic agent for treating AML.

Hepatocellular cell carcinoma (HCC) is one of the most commonly diagnosed cancers worldwide and in Taiwan. Chemoprevention of cancer with dietary bioactive compounds could potentially reverse, suppress, or prevent cancer progression. Licochalcone A (LicA) is a characteristic chalcone of licorice, which is the root of Glycyrrhiza inflate. It had been reported that LicA has anti-inflammatory, anti-microbial, and anti-tumor properties. However, the effects of LicA on the migration and invasion of human HCC cells have not yet been reported. In the present study, it was found that LicA inhibits the migratory and invasion ability of SK-Hep-1 and HA22T/VGH cells in a dose-dependent manner, as assessed by the cell migration and Matrigel cell invasion assay. Using casein zymography, Western blotting, reverse transcriptase polymerase chain reaction, and an immunofluorescence assay, it was found that LicA induces a dose-dependent inhibition of uPA activity and expression, as well as reduces mRNA levels in SK-Hep-1 and HA22T/VGH cells. LicA was also found to inhibit the expression of phosphor-JNK and phosphor-MKK4 in SK-Hep-1 cells. Furthermore, LicA significantly decreased uPA levels in SP600125-treated or si-MKK4-transfected cells alongside a marked reduction in cell migration and invasion, which supports the notion that an inhibition of MKK4/JNK results in anti-metastatic effects. Moreover, LicA inhibited the expression of nuclear NF-κB, as well as the binding ability of NF-κB to the uPA promoter. These findings further our understanding of the role of LicA in suppressing tumor metastasis and its underlying molecular mechanisms, as well as suggest that LicA may be a promising anti-metastatic agent.

This study aimed to investigate the relationships of chitinase 3-like 1 (CHI3L1) single nucleotide polymorphisms (SNPs) and haplotypes with the development of uterine cervical cancer in Taiwanese women. The SNPs frequencies and haplotypes were also correlated with the clinicopathologic variables of cervical cancer, cancer recurrence, and patient survival.Ninety-nine patients with invasive cancer and 61 with pre-cancerous lesions of the uterine cervix were compared to 310 healthy control subjects. Three SNPs rs6691378 (-1371, G/A), rs10399805 (-247, G/A) and rs4950928 (-131, C/G) in the promoter region, and one SNP rs880633 (+2950, T/C) in exon 5 were analyzed by real time polymerase chain reaction and genotyping. The results showed that the mutant homozygous genotype AA of CHI3L1 SNP rs6691378 and AA of rs10399805, and haplotypes AACC and AACT increased the risk of developing pre-cancerous lesions and invasive cancer. The patients with these risk haplotypes had higher than stage I tumors, larger tumors, and vaginal invasion. In logistic regression model, they also tended to have poor survival event [p = 0.078; odds ratio (OR): 2.99, 95% confidence interval (CI): 0.89-10.08] and a higher probability of recurrence event (p = 0.081; OR: 3.07, 95% CI: 0.87-10.81). There was a significant association between the CHI3L1 risk haplotypes and probability of recurrence (p = 0.002; hazard ratio: 6.21, 95% CI: 1.90-20.41), and a marginal association between the risk haplotypes and overall survival (p = 0.051; hazard ratio: 3.76, 95% CI: 0.99-14.29) in the patients with SCC, using Cox proportional hazard model.The CHI3L1 SNPs rs6691378 and rs10399805 and CHI3L1 haplotypes all correlated with the development of cervical pre-cancerous lesions and invasive cancer. The cervical cancer patients with the CHI3L1 haplotypes AACC or AACT had poor clinicopathologic characteristics and poor recurrence and survival events. These risk haplotypes were associated with higher recurrence, especially in the patients with SCC.

The gene EZH2, the polycomb group protein enhancer of zeste 2, encodes a transcriptional repressor that also serves as a histone methyltransferase that is associated with progression to more advanced disease in a variety of malignancies. EZH2 expression level in urothelial cell carcinoma (UCC) is highly correlated with tumor aggressiveness, but it has not been determined if specific EZH2 genetic variants are associated with UCC risk. This study investigated the potential associations of EZH2 single-nucleotide polymorphisms with UCC susceptibility and its clinicopathologic characteristics.A total of 233 UCC patients and 552 cancer-free controls, all of whom were from Taiwan, were analyzed for four EZH2 single-nucleotide polymorphisms (rs6950683, rs2302427, rs3757441, and rs41277434) using real-time PCR genotyping. After adjusting for other co-variants, we found that individuals carrying at least one C allele at EZH2 rs6950683 had a lower risk of developing UCC than did major allele carriers. The CCCA or TGTA haplotype among the four EZH2 sites was also associated with a reduced risk of UCC. Furthermore, UCC patients who carried at least one G allele at rs2302427 had a lower invasive tumor stage than did patients carrying the major allele.The rs6950683 SNPs of EZH2 might contribute to the prediction of UCC susceptibility. This is the first study to provide insight into risk factors associated with EZH2 variants in carcinogenesis of UCC in Taiwan.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the fourth leading cause of male cancer death in Taiwan. Exposure to environmental carcinogens is the primary risk factor for developing OSCC. CD44, a well-known tumor marker, plays a crucial role in tumor cell differentiation, invasion, and metastasis. This study investigated CD44 single-nucleotide polymorphisms (SNPs) with environmental risk factors to determine OSCC susceptibility and clinicopathological characteristics. METHODOLOGY/PRINCIPAL FINDINGS: Real-time polymerase chain reaction (PCR) was used to analyze 6 SNPs of CD44 in 599 patients with oral cancer and 561 cancer-free controls. We determined that the CD44 rs187115 polymorphism carriers with the genotype AG, GG, or AG+GG were associated with oral cancer susceptibility. Among 731 smokers, CD44 polymorphisms carriers with the betel-nut chewing habit had a 10.30-37.63-fold greater risk of having oral cancer compared to CD44 wild-type (WT) carriers without the betel-nut chewing habit. Among 552 betel-nut chewers, CD44 polymorphisms carriers who smoked had a 4.23-16.11-fold greater risk of having oral cancer compared to those who carried the WT but did not smoke. Finally, we also observed that the stage III and IV oral cancer patients had higher frequencies of CD44 rs187115 polymorphisms with the variant genotype (AG+GG) compared with the wild-type (WT) carriers. CONCLUSION: Our results suggest that gene-environment interactions between the CD44 polymorphisms and betel quid chewing and tobacco smoking increase the susceptibility to oral cancer development. Patients with CD44 rs187115 variant genotypes (AG+GG) were correlated with a higher risk of oral cancer development, and these patients may possess greater chemoresistance to advanced- to late-stage oral cancer than WT carriers do. The CD44 rs187115 polymorphism has potential predictive significance in oral carcinogenesis and also may be applied as factors to predict the clinical stage in OSCC patients.

BACKGROUND: To investigate the implication of ribonucleotide reductase M2 (RRM2) in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients. METHODOLOGY AND PRINCIPAL FINDINGS: The impact of RRM2 on cell viability was investigated in SiHa cervical cancer cells after RRM2 knockdown and the addition of cisplatin, which induces inter- and intra-strand DNA crosslinks. RRM2 immunoreactivity was evaluated by semi-quantitative H score among 29 normal, 30 low-grade dysplasia, 30 high-grade dysplasia and 103 invasive cancer tissue specimens of the uterine cervix, using tissue microarrays. RRM2 was then correlated with the clinicopathological variables of cervical cancer and patient survival. A greater toxic effect on cell viability using cisplatin was reflected by the greater reduction in RRM2 protein expression in SiHa cells. The RRM2 expression in cancer tissues was higher than that in high-grade dysplasia, low-grade dysplasia or normal cervical tissues. RRM2 upregulation was correlated with deep stromal invasion, large tumors and parametrial invasion and predicted poor survival. CONCLUSIONS: RRM2 is a new molecular marker for the diagnosis and clinical outcomes of cervical cancer. It is involved in cervical carcinogenesis and predicts poor survival, and may be a potential therapeutic target including in cisplatin treatment.

BACKGROUND: Vascular endothelial growth factor (VEGF)-C is associated with lymphangiogenesis, pelvic regional lymph node metastasis, and an antiapoptotic phenotype in urothelial cell carcinoma (UCC). Knowledge of potential roles of VEGF-C genetic polymorphisms in susceptibility to UCC is lacking. This study was designed to examine associations between VEGF-C gene variants and UCC susceptibility and evaluate whether they are modified by smoking. METHODOLOGY/PRINCIPAL FINDINGS: Five single-nucleotide polymorphisms (SNPs) of VEGF-C were analyzed by a TaqMan-based real-time polymerase chain reaction (PCR) in 233 patients with UCC and 520 cancer-free controls. A multivariate logistic regression was applied to model associations between genetic polymorphisms and UCC susceptibility, and to determine if the effect was modified by smoking. We found that after adjusting for other covariates, individuals within the entire population and the 476 non-smokers carrying at least one A allele at VEGF-C rs1485766 respectively had 1.742- and 1.834-fold risks of developing UCC than did wild-type (CC) carriers. Among the 277 smokers, we found that VEGF-C rs7664413 T (CT+TT) and rs2046463 G (AG+GG) allelic carriers were more prevalent in UCC patients than in non-cancer participants. Moreover, UCC patients with the smoking habit who had at least one T allele of VEGF-C rs7664413 were at higher risk of developing larger tumor sizes (p = 0.021), compared to those patients with CC homozygotes. CONCLUSIONS: Our results suggest that the involvement of VEGF-C genotypes in UCC risk differs among smokers compared to non-smokers among Taiwanese. The genetic polymorphism of VEGF-C rs7664413 might be a predictive factor for the tumor size of UCC patients who have a smoking habit.

BACKGROUND: Micro RNAs (miRNAs) are small RNA fragments that naturally exist in the human body. Through various physiological mechanisms, miRNAs can generate different functions for regulating RNA protein levels and balancing abnormalities. Abnormal miRNA expression has been reported to be highly related to several diseases and cancers. Single-nucleotide polymorphisms (SNPs) in miRNAs have been reported to increase patient susceptibility and affect patient prognosis and survival. We adopted a case-control research design to verify the relationship between miRNAs and hepatocellular carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: A total of 525 subjects, including 377 controls and 188 hepatocellular carcinoma patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR were used to analyze miRNA146a (rs2910164), miRNA149 (rs2292832), miRNA196 (rs11614913), and miRNA499 (rs3746444) genetic polymorphisms between the control group and the case group. The results indicate that people who carry the rs3746444 CT or CC genotypes may have a significantly increased susceptibility to hepatocellular carcinoma (adjusted odds ratio [AOR] = 2.84, 95% confidence interval [CI] = 1.88-4.30). In addition, when combined with environmental risk factors, such as smoking and alcohol consumption, interaction effects were observed between gene polymorphisms and environmental factors (odds ratio [OR] = 4.69, 95% CI = 2.52-8.70; AOR = 3.38, 95% CI = 1.68-6.80). CONCLUSIONS: These results suggest that a significant association exists between miRNA499 SNPs and hepatocellular carcinoma. Gene-environment interactions of miRNA499 polymorphisms, smoking, and alcohol consumption might alter hepatocellular carcinoma susceptibility.

BackgroundKaempferol has been proposed as a potential drug for cancer chemoprevention and treatment because it is a natural polyphenol contained in plant-based foods. Recent studies have demonstrated that kaempferol protects against cardiovascular disease and cancer. Based on this finding, we investigated the mechanisms by which kaempferol produces the anti-metastatic effect in human tongue squamous cell carcinoma SCC4 cells.Methodology/principal findingsIn this study, we provided molecular evidence associated with the anti-metastatic effect of kaempferol by demonstrating a substantial suppression of SCC4 cell migration and invasion. This effect was associated with reduced expressions of MMP-2 and TIMP-2 mRNA and protein levels. Analysis of the transcriptional regulation indicated that kaempferol inhibited MMP-2 transcription by suppressing c-Jun activity. Kaempferol also produced an inhibitory effect on the phosphorylation of ERK1/2.ConclusionsThese findings provide new insights into the molecular mechanisms involved in the anti-metastatic effect of kaempferol, and are valuable in the prevention of oral cancer metastasis.

Constitutive activation of nuclear factor (NF)-κB is frequently observed in hepatocellular carcinoma (HCC). The current study examined associations of polymorphisms within promoter regions of NFKB1 encoding NF-κB1 and NFKBIA encoding IκBα with the susceptibility of developing HCC and clinicopathological characteristics of the tumors.Genetic polymorphisms of NFKB1 and NFKBIA were analyzed by a real-time polymerase chain reaction (PCR) in 135 HCC patients and 520 healthy controls. The genotypic frequency of the NFKB1 -94 Ins polymorphism in HCC patients was significantly higher than that of the controls (adjusted odds ratio (AOR) = 2.23; 95% confidence interval (CI) 1.32∼3.77). No statistical significance was observed for the distribution frequency of the NFKBIA --519 C/T, -826 C/T, or -881 A/G genotype and haplotype polymorphisms between HCC patients and controls. Furthermore, female HCC patients carrying the NFKB1 -94 Ins polymorphism were associated with lower clinical stages and smaller tumor sizes.Our results indicate that the NFKB1 -94 Ins promoter polymorphism increased the risk of HCC, and may be applied as a predictive factor for the clinical stage and tumor size in female HCC patients.

BACKGROUND: In Taiwan, oral cancer has causally been associated with environmental carcinogens. Intercellular adhesion molecule (ICAM)-1, a cell adhesion molecule with a key role in inflammation and immunosurveillance, was implicated in carcinogenesis by facilitating instability in the tumor environment. The current study explored the combined effect of ICAM-1 gene polymorphisms and exposure to environmental carcinogens on the susceptibility of developing oral squamous cell carcinoma (OSCC) and the clinicopathological characteristics of the tumors. METHODOLOGY AND PRINCIPAL FINDINGS: Four single-nucleotide polymorphisms (SNPs) of the ICAM-1 gene from 595 patients with oral cancer and 561 non-cancer controls were analyzed by a real-time PCR. We found that the ICAM-1 rs5498 polymorphism and the TAGG or TACG haplotype of 4 ICAM-1 SNPs (rs3093030, rs5491, rs281432, and rs5498) combined were associated with oral-cancer susceptibility. Among 727 smokers, ICAM-1 polymorphisms carriers with the betel-nut chewing habit had a 27.49-36.23-fold greater risk of having oral cancer compared to ICAM-1 wild-type (WT) carriers without the betel-nut chewing habit. Among 549 betel-nut chewers, ICAM-1 polymorphisms carriers who smoked had a 9.93-14.27-fold greater risk of having oral cancer compared to those who carried the WT but did not smoke. Finally, patients with oral cancer who had at least 1 T allele of ICAM-1 rs5491 or 1 G allele of rs281432 were at lower risk of developing an advanced clinical stage (III/IV) (p

BACKGROUND: The purpose of this study was to identify gene polymorphisms of interleukin-18 (IL-18) -607A/C and -137G/C specific to patients with oral cancer susceptibility and clinicopathological status. METHODOLOGY AND PRINCIPAL FINDINGS: A total of 1,126 participants, including 559 healthy people and 567 patients with oral cancer, were recruited for this study. Allelic discrimination of -607A/C (rs1946518) and -137G/C (rs187238) polymorphisms of the IL-18 gene was assessed by a real-time PCR with the TaqMan assay. There was no significant association between IL-18 -607A/C polymorphism and oral cancer risk. However, among alcohol consumers, people with A/A homozygotes of IL-18 -607A/C polymorphism had a 2.38-fold (95% CI=1.17-4.86; p=0.01) increased risk of developing oral cancer compared with those with C/C homozygotes. The participants with G/C heterozygotes of IL-18 -137 polymorphism had a 1.64-fold (95% CI: 1.08-2.48; p=0.02) increased risk of developing oral cancer compared with those with G/G wild type homozygotes. Both sets of statistics were determined after adjusting for confounding factors. Among people who had exposure to oral cancer-related environmental risk factors such as areca, alcohol, and tobacco consumption, the adjusted odd ratios and 95% confidence intervals were increased to a 2.02-fold (95% CI=1.01-4.04; p=0.04), 4.04 (95% CI=1.65-9.87; p=0.002) and a 1.66-fold (95% CI=1.00-2.84; p=0.05) risk of developing oral cancer. However, patients with G/C alleles of IL-18 -137 were correlated with a lower clinical stage (AOR=0.59; 95% CI=0.39-0.89; p=0.01), smaller tumor size (AOR=0.56; 95% CI=0.35-0.87; p=0.01), and non-lymph node metastasis (AOR=0.51; 95% CI=0.32-0.80; p=0.003). CONCLUSION: IL-18 -137 G/C gene polymorphism may be a factor that increases the susceptibility to oral cancer, as well as a protective factor for oral cancer progression. The interactions of gene to oral cancer-related environmental risk factors have a synergetic effect that can further enhance oral cancer development.

The mortality and morbidity rates from cancer metastasis have not declined in Taiwan, especially because of hepatocellular carcinoma (HCC). Resveratrol has been shown to have benefits such as cardioprotection, providing antioxidative, anti-inflammatory, anti-cancer properties in previous studies. Therefore, HCC cells were subjected to treatment with resveratrol and then analyzed to determine the effects of resveratrol on the migration and invasion.Modified Boyden chamber assays revealed that resveratrol treatment significantly inhibited cell migration and invasion capacities of Huh7 cell lines that have low cytotoxicity in vitro, even at a high concentration of 100 µM. The results of casein zymography and western blotting revealed that the activities and protein levels of the urokinase-type plasminogen activator (u-PA) were inhibited by resveratrol. Western blot analysis also showed that resveratrol inhibits phosphorylation of JNK1/2. Tests of the mRNA level, real-time PCR, and promoter assays evaluated the inhibitory effects of resveratrol on u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay showed that reactive in transcription protein of nuclear factor SP-1 was inhibited by resveratrol.Resveratrol inhibits u-PA expression and the metastasis of HCC cells and is a powerful chemopreventive agent. The inhibitory effects were associated with the downregulation of the transcription factors of SP-1 signaling pathways.

Carbonic anhydrase 9 (CA9) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study explored the effect of CA9 gene polymorphisms on the susceptibility of developing urothelial cell carcinoma (UCC) and the clinicopathological status.A total of 442 participants, including 221 healthy people and 221 patients with UCC, were recruited for this study. Four single-nucleotide polymorphisms (SNPs) of the CA9 gene were assessed by a real-time PCR with the TaqMan assay. After adjusting for other co-variants, the individuals carrying at least one A allele at CA9 rs1048638 had a 2.303-fold risk of developing UCC than did wild-type (CC) carriers. Furthermore, UCC patients who carried at least one A allele at rs1048638 had a higher invasive stage risk (p< 0.05) than did patients carrying the wild-type allele. Moreover, among the UCC patients with smoker, people with at least one A allele of CA9 polymorphisms (rs1048638) had a 4.75-fold (95% CI = 1.204-18.746) increased risk of invasive cancer.The rs1048638 polymorphic genotypes of CA9 might contribute to the prediction of susceptibility to and pathological development of UCC. This is the first study to provide insight into risk factors associated with CA9 variants in carcinogenesis of UCC in Taiwan.

The enhancer of zeste 2 (EZH2) gene encodes the histone methyltransferase that is the catalytic component of the polycomb repressive complex-2, which initiates epigenetic silencing of genes. The expression level of EZH2 in hepatocellular carcinoma (HCC) is highly correlated with tumor progression; however, it has not been determined if specific EZH2 genetic variants are associated with the risk of HCC. This study investigated the potential associations of EZH2 single-nucleotide polymorphisms with HCC susceptibility and its clinicopathologic characteristics.A total of 220 HCC patients and 552 cancer-free controls were analyzed for four EZH2 single-nucleotide polymorphisms (rs6950683, rs2302427, rs3757441, and rs41277434) using real-time PCR genotyping. After adjusting for other co-variants, the individuals carrying at least one C allele at EZH2 rs6950683 and rs3757441 had a 0.611-fold and a 0.660-fold lower risk of developing HCC than did wild-type (TT) carriers, respectively. The CCCA or CCTA haplotype among the four EZH2 sites (rs6950683, rs2302427, rs3757441, and rs41277434), respectively, was also associated with a reduced risk of HCC. Furthermore, HCC patients who carried at least one C allele at rs6950683 or rs3757441 had a higher lymph-node-metastasis risk but a lower liver-cirrhosis risk than did patients carrying the wild-type allele.The rs6950683 and rs3757441 polymorphic genotypes of EZH2 might contribute to the prediction of susceptibility to and pathological development of HCC. This is the first study to provide insight into risk factors associated with EZH2 variants in carcinogenesis of HCC in Taiwan.

BACKGROUND: Oral cancer, which is the fourth most common male cancer, is associated with environmental carcinogens in Taiwan. Vascular endothelial growth factor (VEGF)-C, an angiogenic/lymphangiogenic factor with high expression levels in tumor tissues, plays important roles in the development of several malignancies. This study was designed to examine associations of five VEGF-C gene polymorphisms with the susceptibility to and clinicopathological characteristics of oral squamous cell carcinoma. METHODOLOGY/PRINCIPAL FINDINGS: Five single-nucleotide polymorphisms (SNPs) of VEGF-C were analyzed by a real-time polymerase chain reaction (PCR) in 470 male patients with oral cancer and 426 cancer-free controls. In this study, we found that the VEGF-C rs7664413 and rs2046463 polymorphisms were associated with oral-cancer susceptibility but not with any clinicopathological parameters. The GGACA or GACTG haplotype of five VEGF-C SNPs (rs3775194, rs11947611, rs1485766, rs7664413, and rs2046463) combined was also related to the risk of oral cancer. Among 611 male smokers, VEGF-C polymorphism carriers who also chewed betel quid were found to have a 14.5-24.2-fold risk of having oral cancer compared to the VEGF-C wild-type carrier who did not chew betel quid. Among 461 male betel-quid chewers, VEGF-C polymorphism carriers who also smoked had a 2.7-18.1-fold risk of having oral cancer compared to those who carried the wild type but did not smoke. CONCLUSIONS: Our results suggest that the two SNPs of VEGF-C (rs7664413 and rs2046463) and either of two haplotypes of five SNPs combined have potential predictive significance in oral carcinogenesis. Gene-environmental interactions among VEGF-C polymorphisms, smoking, and betel-quid chewing might alter one's susceptibility to oral cancer.

BACKGROUND: The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) down-regulation has been confirmed in numerous human cancers and is clinically associated with metastasis. This study investigates the potential associations of RECK single-nucleotide polymorphisms (SNPs) with hepatocellular carcinoma (HCC) susceptibility and its clinicopathologic characteristics. METHODOLOGY/PRINCIPAL FINDINGS: A total of 135 HCC cancer patients and 501 cancer-free controls were analyzed for four RECK SNPs (rs10814325, rs16932912, rs11788747, and rs10972727) using real-time PCR and PCR-RFLP genotyping analysis. After adjusting for other co-variants, the individuals carrying RECK promoter rs10814325 inheriting at least one C allele had a 1.85-fold [95% confidence interval (CI), 1.03-3.36] risk of developing HCC compared to TT wild type carriers. The HCC patients, who carried rs11788747 with at least one G allele, had a higher distant metastasis risk than wild type probands. CONCLUSIONS: RECK gene polymorphisms might be a risk factor increasing HCC susceptibility and distant metastasis in Taiwan.

Oral cancer, which is the fourth most common cancer in Taiwanese men, is associated with environmental carcinogens. The possibility that genetic predisposition in nuclear factor-kappa B (NF-κB)-signaling pathways activation is linked to the development of oral squamous cell carcinoma (OSCC) requires investigation. The current study examines associations between polymorphisms within promoter regions of NFKB1 encoding NF-κB1 and NFKBIA encoding IkappaBalpha (IκBα) with both the susceptibility to develop OSCC and the clinicopathological characteristics of the tumors.Genetic polymorphisms of NFKB1 and NFKBIA were analyzed by a real-time polymerase chain reaction (real-time PCR) for 462 patients with oral cancer and 520 non-cancer controls. We found that NFKB1 -94 ATGG1/ATGG2, -94 ATGG2/ATGG2, and the combination of -94 ATGG1/ATGG2 and ATGG2/ATGG2 genotypes NFKBIA -826 T (CT+TT) and -881 G (AG+GG) allelic carriages, were more prevalent in OSCC patients than in non-cancer participants. Moreover, we found that NFKB1 or NFKBIA gene polymorphisms seem to be related to susceptibility to develop oral cancer linked to betel nut and tobacco consumption. Finally, patients with oral cancer who had at least one -519 T allele of the NFKBIA gene were at higher risk for developing distant metastasis (P

BACKGROUND: MicroRNAs (miRNAs) have been regarded as a critical factor in targeting oncogenes or tumor suppressor genes in tumorigenesis. The genetic predisposition of miRNAs-signaling pathways related to the development of oral squamous cell carcinoma (OSCC) remains unresolved. This study examined the associations of polymorphisms with four miRNAs with the susceptibility and clinicopathological characteristics of OSCC. METHODOLOGY/PRINCIPAL FINDINGS: A total of 895 male subjects, including 425 controls and 470 male oral cancer patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR were used to analyze miRNA146a, miRNA196, miRNA499 and miRNA149 genetic polymorphisms between the control group and the case group. This study determined that a significant association of miRNA499 with CC genotype, as compared to the subjects with TT genotype, had a higher risk (AOR = 4.52, 95% CI = 1.24-16.48) of OSCC. Moreover, an impact of those four miRNAs gene polymorphism on the susceptibility of betel nut and tobacco consumption leading to oral cancer was also revealed. We found a protective effect between clinical stage development (AOR = 0.58, 95% CI = 0.36-0.94) and the tumor size growth (AOR = 0.47, 95% CI = 0.28-0.79) in younger patients (age

In Taiwan, oral cancer has causally been associated with environmental carcinogens. Carbonic anhydrase 9 (CA9) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study explored the combined effect of CA9 gene polymorphisms and exposure to environmental carcinogens on the susceptibility of developing oral squamous cell carcinoma (OSCC) and the clinicopathological characteristics of the tumors.Four single-nucleotide polymorphisms (SNPs) of the CA9 gene from 462 patients with oral cancer and 519 non-cancer controls were analyzed by a real-time polymerase chain reaction (PCR). While the studied SNPs (CA9 rs2071676, rs3829078, rs1048638 and +376 Del) were not associated with susceptibility to oral cancer, the GAA haplotype of 3 CA9 SNPs (rs2071676, rs3829078, and rs1048638) was related to a higher risk of oral cancer. Moreover, the four CA9 SNPs combined with betel quid chewing and/or tobacco consumption could robustly elevate susceptibility to oral cancer. Finally, patients with oral cancer who had at least one G allele of CA9 rs2071676 were at higher risk for developing lymph-node metastasis (p = 0.022), compared to those patients homozygous for AA.Our results suggest that the haplotype of rs2071676, rs3829078, and rs1048638 combined has potential predictive significance in oral carcinogenesis. Gene-environment interactions of CA9 polymorphisms, smoking, and betel-quid chewing might alter oral cancer susceptibility and metastasis.

The rate of morbidity and mortality of hepatocellular carcinoma (HCC) in Taiwan has not lessened because of difficulty in treating tumor metastasis. Norcantharidin (NCTD) is currently used as an anticancer drug for hepatoma, breast cancer, and colorectal adenocarcinoma. NCTD possesses various biological anticancer activities, including apoptosis. However, detailed effects and molecular mechanisms of NCTD on metastasis are unclear. Thus, HCC cells were subjected to treatment with NCTD and then analyzed to determine the effects of NCTD on cell metastasis.Modified Boyden chamber assays revealed that NCTD treatment inhibited cell migration and invasion capacities of HCC cells substantially. Results of zymography and western blotting showed that activities and protein levels of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (u-PA) were inhibited by NCTD. Western blot analysis showed that NCTD inhibits phosphorylation of ERK1/2. Testing of mRNA level, quantitative real-time PCR, and promoter assays evaluated the inhibitory effects of NCTD on MMP-9 and u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay for analyzing the genomic DNA sequences bound to these proteins was reactive to the transcription protein nuclear factor (NF)-kappaB, which was inhibited by NCTD. The expression of NF-kappa B was measured by western blot analysis, which revealed decreased nuclear-factor DNA-binding activity after NCTD treatment.NCTD inhibited MMP-9 and u-PA expression through the phosphorylation of ERK1/2 and NF-kappaB signaling pathway which serves as a powerful chemopreventive agent in HCC cell metastasis.