Chee-Yin Chai

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.

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.

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.

Immunoexpression and prognostic role of hTERT and cyclin D1 in urothelial carcinoma

The aim was to investigate the expression of human telomerase reverse transcriptase (hTERT) and cyclin D1 in correlation with clinicopathologic features of urothelial carcinoma (UC). Tissue microarrays (TMA) were constructed from paraffin‐embedded specimens of 94 UC patients and immunohistochemical staining was used. High hTERT expression was found in 50 (53%) of the 94 tumors and was significantly associated with tumor invasiveness and tumor grade (P=0.008 and 0.0190, respectively). High cyclin D1 expression was found in 69 (73%) of the 94 tumors and was significantly associated with non‐invasiveness and smaller tumor size, but there was no correlation with tumor grade. Kaplan–Meier analysis indicated that patients with low hTERT and high cyclin D1 levels had longer local recurrence‐free survival (P=0.0482 and 0.0123, respectively). In addition, patients with high cyclin D1 levels had longer disease‐free survival (P=0.0195). In conclusion, this study demonstrated that hTERT and cyclin D1 may be of recurrence predictive value for UC, thus providing clinicians with ancillary information when deciding on suitable therapeutic strategies in UC.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.