Hui-Hui Lin

Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine.

OBJECTIVES To identify a better set of DNA methylation markers to detect superficial, low grade cancer cell in urine sediment for improving cancer treatment, morbidity, and mortality. MATERIALS AND METHODS Methylation-specific PCR (MSP) assay was used to detect promoter hypermethylation in 4 genes (E-cadherin, p16, p14, and RASSF1A) to identify reliable biomarkers for bladder cancer diagnosis in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. Urine DNA was compared with 20 healthy controls. RESULTS Fifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder cancer patients revealed hypermethylation in at least 1 of the 4 analyzed genes, whereas all urine samples from normal controls were negative. The sensitivity of MSP assay for detecting E-cadherin, p16, p14 and RASSF1A in tumor cells in voided urine was 35%, 35%, 33%, and 65%, respectively. Diagnostic sensitivity was 75% for combining RASSF1A and p14, and 83% for RASSF1A, p14 and E-cadherin. Urine cytology, however, detect only 13 (28%) cases of cancer or suspicious cancer. For detecting superficial and invasive bladder tumor, urine cytology revealed a sensitivity of 23% (6/26) and 35% (7/20), respectively. In contrast, MSP detected hypermethylation in the urine of 80% (37/46) bladder cancer patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) of low grade, 75% (15/20) of invasive and 79% (26/33) of high grade bladder cancers. Importantly, hypermethylation was detected in the urine DNA of 90% (18/20) superficial tumors with negative or atypia cytology. CONCLUSIONS Hypermethylation of E-cadherin, p14 or RASSF1A in urine sediment DNA is a potential biomarker for detecting superficial, low grade cancer. Besides, hypermethylation of these 3 genes is a valuable adjunct diagnostic marker to urine cytology, which can enhance the diagnostic accuracy and follow-up treatment of bladder cancer patients.

The associations among eNOS G894T gene polymorphism, erectile dysfunction and related risk factors

To investigate the possible correlations among eNOS G894T polymorphism, erectile dysfunction (ED) and related risk factors in a Taiwanese population.

Osteopontin overexpression predicts poor prognosis of upper urinary tract urothelial carcinoma

OBJECTIVES Studies indicate overexpression of osteopontin (OPN) promotes carcinogenesis, progression and metastasis of multiple human malignancies. However, the function of OPN in urothelial carcinoma (UC) of the upper urinary tract has not been investigated. This study evaluates the clinical significance of OPN expression in upper urinary tract UC. MATERIALS AND METHODS One hundred and ten cases (median age = 64, range = 24-84 years) of renal pelvic or ureter UC were retrospectively reviewed in this study. OPN expression were evaluated by immunohistochemistry staining on paraffin-embedded section of the tumor and scored by two qualified pathologists. RESULTS High OPN expression was found in 54 (49.1%) of the cancer specimens. OPN expression was not significantly correlated with tumor T stage (P = 0.761), N stage (P = 0.339) or grade (P = 0.349). However, OPN expression was differently expressed by gender (P = 0.012) and cancer location (P = 0.026). OPN expression did not correlate with bladder recurrence-free (P = 0.661) or extra-bladder recurrence-free (P = 0.787) survival, but high OPN expression was a significant predictor for cancer-specific survival (P = 0.014). CONCLUSION Our findings indicated that higher OPN expression is a potential biomarker to predict patient survival. Further study is necessary to investigate the role of OPN in the carcinogenesis of upper urinary tract UC.

Nuclear factor‐κB activation predicts an unfavourable outcome in human upper urinary tract urothelial carcinoma

To examine the hypothesis that nuclear factor‐κB (NF‐κB), a transcription factor involved in anti‐apoptosis, invasion, and angiogenesis, plays a role in the carcinogenesis of upper urinary tract urothelial carcinoma (UUT‐UC) and has prognostic value for survival.

Hypermethylation of E-cadherin, p16, p14, and RASSF1A genes in pathologically normal urothelium predict bladder recurrence of bladder cancer after transurethral resection

OBJECTIVES This study investigated the hypermethylation of E-cadherin, p16, p14, and RASSF1A in pathologically normal urothelium to predict recurrence of bladder cancer after transurethral resection. MATERIALS AND METHODS Samples of bladder tumor and paired pathologically normal urothelium were obtained from 50 bladder cancer patients. The status of promoter hypermethylation in these four genes was investigated by methylation-specific polymerase chain reaction. The clinicopathologic data in these patients were also analyzed in order to evaluate the clinical implication of aberrant methylation in bladder cancer recurrence. RESULTS Hypermethylation of E-cadherin (30%), p16 (16%), p14 (14%), and RASSF1A (36%) was detected in the pathologically normal urothelium samples. Promoter hypermethylation occurred frequently in both pathologically normal urothelium and tumor samples from bladder cancer patients, and increased with progression from normal to bladder cancer at E-cadherin (P = 0.067), p16 (P < 0.001), p14 (P = 0.01), and RASSF1A (P = 0.01). No significant correlation was observed between hypermethylation in any genes and muscle/organ invasion and stage/grade, except p14. However, p14 hypermethylation in pathologically normal urothelium samples was associated with shorter recurrence-free interval (P = 0.019). CONCLUSIONS p14 hypermethylation could be involved in early stage of bladder carcinogenesis, and p14 hypermethylation in pathologically normal urothelium samples should be considered a predictor of bladder cancer recurrence.

ORIGINAL RESEARCHORIGINAL RESEARCH—BASIC SCIENCE: The Associations among GNB3 C825T Polymorphism, Erectile Dysfunction, and Related Risk Factors

INTRODUCTION Vascular etiologies are the most common risk factors for erectile dysfunction (ED). Published studies have reported the associations of GNB3 C825T polymorphism with many vascular diseases. However, there are few reports about the association between this gene polymorphism and ED. AIM To investigate the associations among GNB3 C825T polymorphism, ED, and related risk factors in Taiwanese subjects. METHODS A total of 155 patients with ED and 81 healthy controls were enrolled. All men had complete clinical histories taken. The 5-item International Index of Erectile Function (IIEF-5) was used to assess erectile conditions. The GNB3 C825T polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism method. MAIN OUTCOME MEASURES Patients with ED were defined as those having an IIEF-5 of <21. RESULTS Two hundred thirty-six men were enrolled with a mean (standard deviation) age of 59.0 (10.2) years. Diabetes mellitus (DM), hypertension, and age were the three most significant independent risk factors for ED in a multiple logistic regression analysis (P = 0.008, 0.003, and 0.007, respectively). The prevalence of DM, hypertension, and body mass index (BMI) was significantly higher in GNB3 825T allele (CT/TT) carriers (P = 0.023, 0.049, and 0.035, respectively). There was no significant difference of ED prevalence between T and C allele carriers (69.1% vs. 56.2%, P = 0.07). However, the T allele carriers had significantly lower IIEF-5 scores (P = 0.02) associated with an increment of the T allele number (16.4[CC] vs. 14.4[CT] vs. 13.2[TT], P = 0.04). CONCLUSIONS In the present study, DM, hypertension, and BMI had significant associations with GNB3 825T allele carriers. Our results failed to show a significant association of the GNB3 C825T polymorphisms with ED prevalence. However, we cannot exclude that the presence of the T allele might influence the risk for ED severity indirectly through an increased risk for some vascular diseases.

ORIGINAL RESEARCH—BASIC SCIENCE: The Associations among GNB3 C825T Polymorphism, Erectile Dysfunction, and Related Risk Factors

INTRODUCTION Vascular etiologies are the most common risk factors for erectile dysfunction (ED). Published studies have reported the associations of GNB3 C825T polymorphism with many vascular diseases. However, there are few reports about the association between this gene polymorphism and ED. AIM To investigate the associations among GNB3 C825T polymorphism, ED, and related risk factors in Taiwanese subjects. METHODS A total of 155 patients with ED and 81 healthy controls were enrolled. All men had complete clinical histories taken. The 5-item International Index of Erectile Function (IIEF-5) was used to assess erectile conditions. The GNB3 C825T polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism method. MAIN OUTCOME MEASURES Patients with ED were defined as those having an IIEF-5 of <21. RESULTS Two hundred thirty-six men were enrolled with a mean (standard deviation) age of 59.0 (10.2) years. Diabetes mellitus (DM), hypertension, and age were the three most significant independent risk factors for ED in a multiple logistic regression analysis (P = 0.008, 0.003, and 0.007, respectively). The prevalence of DM, hypertension, and body mass index (BMI) was significantly higher in GNB3 825T allele (CT/TT) carriers (P = 0.023, 0.049, and 0.035, respectively). There was no significant difference of ED prevalence between T and C allele carriers (69.1% vs. 56.2%, P = 0.07). However, the T allele carriers had significantly lower IIEF-5 scores (P = 0.02) associated with an increment of the T allele number (16.4[CC] vs. 14.4[CT] vs. 13.2[TT], P = 0.04). CONCLUSIONS In the present study, DM, hypertension, and BMI had significant associations with GNB3 825T allele carriers. Our results failed to show a significant association of the GNB3 C825T polymorphisms with ED prevalence. However, we cannot exclude that the presence of the T allele might influence the risk for ED severity indirectly through an increased risk for some vascular diseases.

MiR‐193b Mediates CEBPD‐Induced Cisplatin Sensitization Through Targeting ETS1 and Cyclin D1 in Human Urothelial Carcinoma Cells

Transcription factor CCAAT/enhancer‐binding protein delta (CEBPD) plays multiple roles in tumor progression. Studies have demonstrated that cisplatin (CDDP) induced CEBPD expression and had led to chemotherapeutic drug resistance. However, the underlying molecular mechanisms of CDDP‐regulated CEBPD expression and its relevant roles in CDDP responses remain elusive. MicroRNAs (miRNAs) are small non‐coding RNAs that negatively regulate gene expression in a sequence‐specific manner. Abnormal miRNAs expression is associated with tumor progression. In current study, a large‐scale PCR‐based miRNA screening was performed to identify CEBPD‐associated miRNAs in urothelial carcinoma cell line NTUB1. Eleven miRNAs were selected with more than twofold changes. MiR‐193b‐3p, a known tumor suppressor, down‐regulated proto‐oncogenes Cyclin D1, and ETS1 expression and led to cell cycle arrest, cell invasion, and migration inhibition. The expression of miR‐193b‐3p was associated with the DNA binding ability of CEBPD in CDDP response. CEBPD knocking‐down approach provided a strong evidence of the positive correlation between CEBPD and miR‐193b‐3p. CDDP‐induced CEBPD trans‐activated miR‐193b‐3p expression and it directly targeted the 3′‐UTR of Cyclin D1 and ETS1 mRNA, and silenced the protein expression. In addition, miR‐193b‐3p also inhibited cell migration activity, arrested cell at G1 phase, and sensitized NTUB1 to CDDP treatment. In conclusion, this study indicates that CEBPD exhibits an anti‐tumorigenic function through transcriptionally activating miR‐193b‐3p expression upon CDDP treatment. This study provides a new direction for managing human urothelial carcinoma. J. Cell. Biochem. 118: 1563–1573, 2017.

Glutathione S-Transferase Expression in Upper Urinary Tract Urothelial Carcinomas: a Taiwan Study

OBJECTIVES Glutathione S-transferase (GST) isoenzymes play important roles in resistance to cell apoptosis and carcinogenesis. We aimed to establish the relationship between GST expression and the prognosis of upper urinary tract urothelial carcinoma (UTT-UC) in Taiwan. METHODS This study retrospectively reviewed 46 patients with pathologically confirmed UUT-UC at Kaohsiung Medical University Hospital. In each patient, expression of GSTT1 and GSTP1 was compared between urothelial carcinoma and normal urothelial cells by Western blotting. RESULTS GSTP1 expression in the UUT-UC cells was significantly higher than that in normal urothelial cells (1.6 fold, p<0.001). Expression of GSTT1 was significantly associated with the invasiveness of the carcinoma (p=0.006). CONCLUSIONS In UUT-UC, GSTP1 might be a potential tumor marker, whereas high GSTT1 expression could be used as an indicator of cancer progression. This study is the first to demonstrate potential applications of different GST isoenzymes for biomolecular analysis of UUT-UCs in Taiwan.

Hypoxia-regulated MicroRNA-210 Overexpression is Associated with Tumor Development and Progression in Upper Tract Urothelial Carcinoma

Background: Hypoxia has been shown to facilitate tumor progression. Hypoxia-regulated microRNA-210 (miR-210) may play an important role in carcinogenesis and tumor progression. In this study, we evaluated the clinical significance of miR-210 expression in upper tract urothelial carcinoma (UTUC). Methods: Eighty-three UTUC patients participated in this study. All of them provided cancer tissue samples and 50 of them provided non-cancerous urothelium samples. Clinicopathologic data were collected by reviewing medical records. The expression of miR-210 and hypoxia-inducible factor-1α (HIF-1α) was determined by quantitative real-time polymerase chain reaction. The relationship between clinicopathologic variables and the expression of miR-210 and HIF-1α was analyzed statistically. Results: MiR-210 is overexpressed in UTUC compared to non-cancerous urothelium (p < 0.001); it is also upregulated in high-stage and high-grade tumors (p = 0.020 and 0.049, respectively). HIF-1α is overexpressed in UTUC and correlates positively with miR-210 expression (r = 0.442, p = 0.001). Conclusion: Both miR-210 and HIF-1α are involved in promoting UTUC carcinogenesis. MiR-210 is also correlated with tumor progression. Further studies are needed to clarify the underlying mechanism.