Ya-Chun Huang

Hypoxia-inducible factor-1α expression correlates with focal macrophage infiltration, angiogenesis and unfavourable prognosis in urothelial carcinoma

Background: Hypoxia inducible factor (HIF)-1α is a critical regulatory protein of cellular response to hypoxia and is closely related to angiogenic process. Aims: To explore the potential role and the prognostic value of HIF-1α in urothelial carcinoma (UC). Methods: Clinicopathological and follow-up data on 99 UC cases were reviewed and immunostained for HIF-1α, CD68, vascular endothelial growth factor (VEGF) and CD34 antigen. Tumour-associated macrophage (TAM) counts and HIF-1α expression were compared with clinicopathologic characteristics, overall survival (OS) and disease-free survival rates (DFS). Results: High expression of HIF-1α was detected in 55 of 99 (55.6%) tumours. HIF-1α expression was correlated with tumour size, histological grade, tumour invasiveness and recurrence. VEGF and microvessel density (MVD) demonstrated their positive correlation with HIF-1α overexpression, supporting the correlation of HIF-1α up-regulation with tumour angiogenesis. Higher TAM infiltration was identified in high expression of HIF-1α cases rather than HIF-1α low expression cases (p = 0.002). Kaplan–Meier analysis found that HIF-1α overexpression and high TAM count was only associated with worse DFS (p = 0.009, p = 0.023) but was not associated with OS (p = 0.696, p = 0.141). Multivariate analyses indicated only tumour size (p = 0.038) to be an independently significant prognostic factor for OS, in addition, HIF-1α expression (p = 0.011), as well as histological grade (p = 0.038), and MVD (p = 0.004), to be independently significant prognostic factors for DFS. Conclusions: Our results indicate that HIF-1α is a key regulator of the angiogenic cascade. We show that HIF-1α is an independent prognostic factor for disease-free survival.

Urothelial carcinomas arising in arsenic-contaminated areas are associated with hypermethylation of the gene promoter of the death-associated protein kinase

Aims:  The mechanisms of urothelial carcinogenesis in areas highly contaminated with arsenic remain unclear. The aim was to determine whether hypermethylation of death‐associated protein kinase (DAPK) gene is associated with chronic arsenic exposure.

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.

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.

EXPRESSION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 AND SUPPRESSOR OF CYTOKINE SIGNALING 3 IN UROTHELIAL CARCINOMA

In this study, we investigated the expression of phosphorylated signal transducer and activator of transcription 3 (p‐STAT3) Tyr705 and suppressor of cytokine signaling 3 (SOCS3) in urothelial carcinoma (UC). p‐STAT3 (Tyr705) and SOCS3 were analyzed by immunohistochemistry using tissue microarray and Western blotting. Our results showed that p‐STAT3 (Tyr705) was frequently detected in high‐grade and infiltrating UC. However, there was no difference in p‐STAT3 (Tyr705) staining between UC of the upper and lower urinary tracts. In addition, there was no significant correlation between expression of SOCS3 and histological differentiation and invasiveness of UC. These findings suggest that overexpression of p‐STAT3 (Tyr705) occurs in UC, and that pathways other than SOCS3 may contribute to its activation in this cancer.

Effects of MEK and DNMT inhibitors on arsenic-treated human uroepithelial cells in relation to Cyclin-D1 and p16.

Arsenic compounds are well-known toxic and carcinogenic agents, and they are widely distributed throughout the earths crust. These compounds are associated with various human malignancies. It has been reported that there is an elevated risk of bladder cancer in an area highly contaminated with arsenic on the southwest coast of Taiwan. However, the underlying mechanisms of arsenic-associated carcinogenesis are still unclear. The cell cycle regulatory proteins are important indicators in control of cell cycle progression. Moreover, the high expression of Cyclin-D1 and loss of p16 has been associated with a worse prognosis in a variety of human cancers. Therefore, we investigated the effect of arsenic on Cyclin-D1 and p16 expression and evaluated the role of the ERK signaling pathway and DNA methylation in arsenic carcinogenesis. Our study results showed that Cyclin-D1 high expression was found in 56.3% (9/16) of urothelial carcinomas (UC) from a blackfoot disease (BFD) area and 6.3% (1/16) of UC from a non-BFD area (p=0.002). The p16 low expression in 81.2% (13/16) of UC from BFD areas was significantly lower than in non-BFD areas (25.0%; 4/16) (p=0.001). In addition, the Cyclin-D1 increased expression but decreased p16 expression in arsenite-treated SV-HUC-1 cells. However, when cells were pretreated with inhibitors (5-aza-CdR or U0126), the effects of arsenite on Cyclin-D1 and p16 expression were suppressed. Finally, these results indicated that Cyclin-D1 and p16 both might play important roles in carcinogenesis as a result of arsenic.

Melanin bleaching with dilute hydrogen peroxide: a simple and rapid method.

Melanins are naturally occurring pigments in both normal and pathologic tissues. Two common bleaching processes are potassium permanganate followed by oxalic acid treatment and dilute hydrogen peroxide (H2O2) process. The potassium permanganate/oxalic acid method is faster and more easily incorporated in conventional daily immunostaining protocols, whereas the dilute H2O2 method requires 24 hours. This study aimed to reduce melanin bleaching time by using a 10% H2O2 dilution. First, reaction time was reduced to 30 minutes by raising the temperature to 65°C. Second, containers with high thermal conductivity were used to improve bleaching effectiveness. Experimental comparisons of melanin treatments with H2O2 contained in an iron jar, a glass coplin jar, and a plastic steel jar obtained bleaching time of 20, 30, and 40 minutes, respectively. These modifications of the conventional bleaching method significantly improve the speed and efficiency of the procedure and are recommended when performing immunohistochemical studies.