Wan-Tzu Chen

Oral administration of Lactobacillus reuteri GMNL-263 improves insulin resistance and ameliorates hepatic steatosis in high fructose-fed rats.

BackgroundType 2 diabetes mellitus (DM), characterized by peripheral insulin resistance, is the most common form of diabetes. Probiotics are live micro-organisms that, when administered in adequate amounts, confer delaying effect on DM development. In this study, the effects Lactobacillus reuteri GMNL-263 (Lr263), a new probiotic strain developed by our laboratory, on insulin resistance and the development of hepatic steatosis in high-fructose fed rats were explored. Furthermore, the relevant regulatory pathways involved were also investigated.MethodMale Sprague–Dawley rats were fed a high-fructose diet with or without Lr263 administration for 14 weeks. The composition of fecal microbiota, oral glucose tolerance, glycated haemoglobin, insulin, leptin, C-peptide, and incretins were measured. The markers of liver injury, serum and hepatic lipids profile, activity of hepatic antioxidant enzyme, and proinflammatory cytokines in adipose tissue were investigated. Additionally, the expression of hepatic lipogenic genes and insulin signaling related genes in adipose tissue were also studied. Liver sections were examined for hepatic steatosis using hematoxylin-eosin staining.ResultsThe levels of serum glucose, insulin, leptin, C-peptide, glycated hemoglobin, GLP-1, liver injury markers, lipid profile in serum and liver were significantly increased in high-fructose-fed rats. However, after Lr263 administration, the elevation of these parameters was significantly suppressed. Feeding of Lr263 reversed the decreased number of bifidobacterium species and lactobacillus species and increased number of clostridium species induced by high fructose treatment. The decreased activities of hepatic antioxidant enzymes in HFD rats were dramatically reversed by Lr263 treatment. Concentrations of IL-6 and TNF-α in adipose tissue which were elevated in high fructose treatment were markedly decreased after Lr263 feeding. Decreased levels of PPAR-γ and GLUT4 mRNA after high fructose treatment were significantly enhanced by Lr263 administration. Lr263 consumption normalized the increased lipogenic gene (Srebp-1c, FAS, and Elvol6) expressions stimulated by high fructose. Administration of Lr263 significantly ameliorated hepatic steatosis observed in high fructose treated rats.ConclusionOur study provided evidences clarifying the effectiveness of Lr263 on reducing insulin resistance as well as hepatic steatosis formation in high-fructose-fed rats and suggested that Lr263 may be a promising therapeutic agent in treating type 2 diabetes.

Potential prognostic value of leptin receptor in hepatocellular carcinoma

Background: Obesity is associated with several human malignancies, including hepatocellular carcinoma (HCC). This association may result from the deregulated expression of adipokines. Aims: To explore the potential role and the prognostic value of leptin receptor (Ob-R) in HCC. Methods: 66 patients with pathologically confirmed HCC were included in this study. Immunohistochemistry was used to evaluate the expression of Ob-R, microvessel density (MVD) and Ki-67 index in these patients. Eventually, the profiles of Ob-R expression, obtained by a semiquantitative scoring system, were further correlated with Ki-67 expression, intratumour MVD, clinicopathological characteristics and overall survival. Results: High Ob-R expression was seen in 53% of patients with HCC and was significantly correlated with intratumour MVD (high v low; 59.4 ?3.2) v 44.7 ?3.7); p = 0.004), but not with Ki-67 expression. In addition, Ob-R expression was inversely correlated with vascular invasion (p = 0.037), but not with other known clinicopathological characteristics. The Kaplan–Meier survival curve showed that high Ob-R expression was associated with a better overall survival (p = 0.027). Meanwhile, multivariate analysis showed that Ob-R expression was a significant determinant for HCC (odds ratio 0.02, 95% confidence interval 0.01 to 0.85; p = 0.041). Conclusion: Ob-R expression may have a potential role in the carcinogenesis of HCC. The positive association of Ob-R expression in the cancerous lesions of HCC with the survival outcome can be explained by its inverse correlation with vascular invasion, and may have prognostic value in HCC.

Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.

Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells.

It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (MSP). Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas (p=0.006, 0.007, 0.003, <0.001 and 0.001 respectively), as detected by immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.

The clinical significance of a positive test for direct MPO-ANCA ELISA or capture MPO-ANCA ELISA when using international units

This study examines whether the expression of cyclooxgenase‐2 (COX‐2) in urothelial carcinoma (UC) is associated with macrophage infiltration, hypoxia‐inducible factor‐1α (HIF‐1α) expression and angiogenesis. We investigated the expression of COX‐2 associated with HIF‐1α and performed double immunohistochemical analysis of 216 UCs for COX‐2 expression and the correlation with tumor‐associated‐macrophage (TAM) density and microvessel density (MVD) in situ. A high expression of COX‐2 was positively correlated with tumor invasiveness, histologic grade and HIF‐1α expression in UC (p<0.0001, p=0.003, p<0.0001, respectively). Quantification of double staining of COX‐2/CD34 and COX‐2/CD68 showed that a higher MVD and TAM density was found in COX‐2 high‐expression than in COX‐2 low‐expression tumor fields (p<0.0001). Adjacent to the principal of COX‐2 expression areas, MVD value and TAM density were significantly increased in HIF‐1α high‐expression specimens compared with HIF‐1α low‐expression ones (p<0.0001). Interestingly, our data revealed that high COX‐2 expression (p=0.002), high HIF‐1α expression (p<0.0001) and TAM density (p<0.0001) were all associated with high MVD value. Our results suggest that COX‐2 may produce a cooperative effect in promoting tumor progression and may be involved in the process of angiogenesis through increasing TAM infiltration or HIF‐1α regulation by hypoxia.

Jab1 is overexpressed in human breast cancer and is a downstream target for HER-2/neu

Jab1 is a coactivator of AP-1 transcription factor and the fifth subunit of the COP9 signalosome. This protein is a potential oncogene and is involved in the mediation of nuclear exportation and degradation of the tumor suppressor p27Kip1. However, control of Jab1 gene expression and its de-regulation in cancer cells are largely unknown. In this study, we demonstrated that Jab1 is overexpressed in 53 (80.3%) of a series of 66 human breast tumor tissues. In addition, its expression is significantly correlated with HER-2/neu overexpression (P=0.0318). HER-2/neu-overexpressing MDA-MB-453 human breast cancer cells exhibited higher expression of Jab1 than that of MCF-7 breast cancer cells. Promoter activity assay suggested that HER-2/neu oncogene upregulated Jab1 via transcriptional activation. Inhibition of HER-2/neu activity by Herceptin or AG825 significantly attenuated Jab1 expression in HER-2/neu-overexpressing MDA-MB-453 cells. On the contrary, ectopic expression of HER-2/neu stimulated Jab1 expression in MCF-7 cells. Knockdown of Jab1 expression by siRNA resulted in p27Kip1 upregulation and G1 growth arrest in Jab1-overexpressing MDA-MB-453 cells. Taken together, our results suggest that Jab1 is a downstream target for HER-2/neu and its overexpression is linked with HER-2/neu expression in breast cancer.

SOCS-3 is associated with vascular invasion and overall survival in hepatocellular carcinoma

Aims: Alteration of the suppressor of cytokine signalling‐3 (SOCS‐3) has been observed in certain human cancers. However, the clinical role of this short‐lived protein in hepatocellular carcinoma (HCC) is not well established. Therefore, we aimed to explore the potential role of SOCS‐3 proteins in HCC. Methods: Paraffin embedded sections from 87 HCC patients were included in this study. The expression patterns of SOCS‐3 proteins were analysed using immunohistochemistry and the results were correlated with clinicopathological characteristics and overall survival of the HCC patients. Results: The SOCS‐3 expression of HCC lesions and the adjacent non‐tumourous liver tissues was significantly correlated (p = 0.035), while the SOCS‐3 expression in HCC lesions was significantly and positively correlated with vascular invasion and histological grading (p = 0.034 and 0.032, respectively). The Kaplan–Meier survival curve showed that the HCC patients with high SOCS‐3 expression were associated with a poor overall survival rate in the HCC subgroup with positive vascular invasion (p = 0.014). Furthermore, a multivariate Cox regression model showed that SOCS‐3 expression was also a significant determinant of the overall survival for HCC (p = 0.006). Conclusions: Our results indicate that altered SOCS‐3 expression is associated with the overall survival in a subset of HCC patients with positive vascular invasion. Constitutive and altered SOCS‐3 expression may have potential roles in a subset of HCC patients.

Targeted next-generation sequencing for molecular diagnosis of endometriosis-associated ovarian cancer

Recent molecular and pathological studies suggest that endometriosis may serve as a precursor of ovarian cancer (endometriosis-associated ovarian cancer, EAOC), especially of the endometrioid and clear cell subtypes. Accordingly, this study had two cardinal aims: first, to obtain mutation profiles of EAOC from Taiwanese patients; and second, to determine whether somatic mutations present in EAOC can be detected in preneoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from ten endometriosis patients with malignant transformation. Macrodissection was performed to separate four different types of cells from FFPE sections in six patients. The four types of samples included normal endometrium, ectopic endometriotic lesion, atypical endometriosis, and carcinoma. Ultra-deep (>1000×) targeted sequencing was performed on 409 cancer-related genes to identify pathogenic mutations associated with EAOC. The most frequently mutated genes were PIK3CA (6/10) and ARID1A (5/10). Other recurrently mutated genes included ETS1, MLH1, PRKDC (3/10 each), and AMER1, ARID2, BCL11A, CREBBP, ERBB2, EXT1, FANCD2, MSH6, NF1, NOTCH1, NUMA1, PDE4DIP, PPP2R1A, RNF213, and SYNE1 (2/10 each). Importantly, in five of the six patients, identical somatic mutations were detected in atypical endometriosis and tumor lesions. In two patients, genetic alterations were also detected in ectopic endometriotic lesions, indicating the presence of genetic alterations in preneoplastic lesion. Genetic analysis in preneoplastic lesions may help to identify high-risk patients at early stage of malignant transformation and also shed new light on fundamental aspects of the molecular pathogenesis of EAOC.Key messagesMolecular characterization of endometriosis-associated ovarian cancer genes by targeted NGS.Candidate genes predictive of malignant transformation were identified.Chromatin remodeling, PI3K-AKT-mTOR, Notch signaling, and Wnt/β-catenin pathway may promote cell malignant transformation.

The expression and significance of WWOX and β-catenin in hepatocellular carcinoma

WW domain‐containing oxidoreductase (WWOX) is a novel tumor suppressor gene, and its expression is reduced in various cancers, including hepatocellular carcinoma (HCC). WWOX has been reported to be downregulated in HCC cell lines as well as in primary HCC tissues. It has been suggested that WWOX is implicated in Wnt/β‐catenin pathway, which is frequently affected in HCC. The aim of this study was to evaluate the expression of WWOX, β‐catenin and T‐cell factor 4 (TCF4) in HCC. Our result showed that downregulation of WWOX in HCC was correlated with cytoplasmic accumulation of β‐catenin. In addition, strong nuclear TCF4 expression was associated with tumor grade and stage in HCC. In conclusion, our result implied that downregulation of WWOX might lead to accumulation of cytoplasmic β‐catenin and the subsequent activation of Wnt/β‐catenin signaling pathway in HCC.

para-Phenylenediamine-induced autophagy in human uroepithelial cell line mediated mutant p53 and activation of ERK signaling pathway.

para-Phenylenediamine (p-PD) is a major aromatic amine that is a widely used commercial oxidative-type hair dye. Some epidemiologic studies have suggested that the use of p-PD-based hair dyes might be related to increased risk of human malignant tumors including bladder cancer. However, the effects of p-PD on autophagy in human uroepithelial cells (SV-HUC-1) is still unclear. In this study, we demonstrate that p-PD can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway in SV-HUC-1 cells. In addition, we observed that autophagosomes increased in p-PD-treated SV-HUC-1 cells as shown by electron microscopy. Our results showed incremental increase of the concentrations, Beclin-1 and microtubule-associated protein light chain 3B (LC3B), which are important regulators of autophagosomes. In contrast, the MEK inhibitor (U0126) was suppressed autophagy and the effect of p-PD on ERK1/2, Beclin-1 and LC3B proteins expression, except for mutant p53. In this study, we demonstrated that inactivation of p53 induces a potent autophagy response. Finally, our results suggest that p-PD can activate the ERK1/2 signaling pathway and mutant p53, leading to the stimulation of autophagy in SV-HUC-1 cells. These results provide us with new insights for the understanding of the mechanism of p-PD-induced cell death in urothelial cells.