Pathology – Research and Practice 210 (2014) 799–803
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Clinical significance of the reduction of UT-B expression in urothelial carcinoma of the bladder Chun Li a , Haogang Xue a , Yanming Lei b , Jianqiang Zhu a , Baoxue Yang c , Xiaodong Gai a,∗ a
Department of Immunology, School of Basic Medical Sciences, Beihua University, Jilin, China Department of Pathology, The General Hospital of CNPC in Jilin, Jilin, China Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing, China b c
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 9 July 2014 Accepted 24 September 2014 Keywords: Bladder cancer Urea transporter B Urothelial carcinoma Immunohistochemistry
a b s t r a c t Urea transporter B (UT-B) is a membrane protein and plays an important role in regulating urea concentration in bladder urothelial cells. It has been reported that UT-B gene mutations were related to bladder carcinogenesis, and UT-B deletion could induce DNA damage and apoptosis in bladder urothelium. However, the functions and clinical significance of UT-B in human bladder cancer remain unknown. The most common type of bladder cancer is urothelial carcinoma (UC). We hypothesized that UT-B expression was related to bladder UC progress. In this study, UT-B was detected using immunohistochemistry in 52 paraffin-embedded specimens of bladder UC and 10 normal urothelium specimens. The results showed that UT-B protein expression in UC tumor cells was significantly lower as compared with normal urothelial cells (P = 0.021). UT-B protein expression was significantly reduced with increasing histological grade (P = 0.010). UT-B protein expression in muscle-invasive stage was significantly lower than in non-muscle-invasive stage (P = 0.014). Taken together, our data suggest that the reduction or loss of UT-B expression may be related to the incidence, progression and invasiveness of bladder UC. UT-B may be a novel diagnostic or prognostic biomarker, as well as a potential therapeutic target in UC of the bladder. © 2014 Elsevier GmbH. All rights reserved.
Introduction Bladder cancer is the most common malignancy of the urinary tract, with about 40 million new cases and 15 million deaths each year worldwide [1,2]. In addition, the incidence rates of bladder cancer in the countries of Europe and America are higher than Asia and Africa [3]. Over 90% of epithelial tumors in bladder cancer are urothelial carcinoma (UC), the remaining types include adenocarcinoma, squamous cell carcinoma and other rare histological types [4]. Despite most noninvasive UC patients can be successfully treated by surgery, 70% of patients suffered from tumor recurrence, and 15% of those recurrent tumors will become invasive [5]. Conventional clinical and pathologic parameters, such as tumor grade and stage, are limited to predict the clinical outcome of UC patients. There is an urgent need for new and more effective biomarkers
∗ Corresponding author at: Department of Pathology, School of Basic Medical Sciences, Beihua University, 3999 Huashan Road, Jilin 132013, China. Tel.: +86 432 64608104. E-mail addresses: [email protected], [email protected] (X. Gai). http://dx.doi.org/10.1016/j.prp.2014.09.012 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
for UC, not only for clinical early detection, accurate evaluation of tumor recurrence and progression, but also as a target for anticancer therapy. Studies suggest that bladder cancer has a genetic predisposition. Individuals who have a family history with bladder cancer have an almost 2-fold risk of developing bladder cancer than the normal population. Recently, three genome-wide association studies in Europe and the USA have reported that the occurrence of bladder cancer was significantly associated with the gene mutations of urea transporter B (UT-B) [6,7]. UT-B is a member of the urea transporters (UTs) family. UTs belong to a family of membrane proteins that selectively transport urea driven by urea gradient across cell membranes. Humans and other mammals have two UT genes, SLC14A1 (UT-B) and SLC14A2 (UT-A) [8]. UT-B has a wide tissue distribution, including erythrocyte, kidney, brain, spleen, liver, heart, colon, spleen, lung, stomach, testis, ureter, bladder, etc. [9–13], and its expression in the bladder is much higher than in other tissues. The functions of UT-B are closely related to urinary concentration, and its related mechanism has been verified by UT-B functional knockout studies in mice [13–15]. In addition, mutations in human UT-B might significantly decrease urea permeability in erythrocytes [16,17] and result in mild urinary concentrating defects [18].
800
C. Li et al. / Pathology – Research and Practice 210 (2014) 799–803
Recent studies showed that UT-B also played an important role in regulating urea concentration in bladder urothelial cells. UTB deletion could cause a marked urea accumulation and induce DNA damage and apoptosis in mouse bladder urothelium, and this biological process might lead to an increased risk of bladder carcinogenesis [19]. Up to now, the UT-B expression in bladder UC is unclear, and the functions and clinical significance of UT-B in bladder UC also remain unknown. The aim of this study was to better clarify the expression and functions of UT-B in bladder UC. We therefore examined the UT-B expression in 52 resected tumor specimens of bladder UC by immunohistochemistry (IHC) and analyzed the correlation between this expression and the clinicopathologic parameters to evaluate UT-B expression in relation to bladder UC development and clinical significance.
Materials and methods Patients and tissue specimens Formalin-fixed, paraffin-embedded (FFPE) samples from 52 patients with bladder UC and 10 FFPE normal urothelium samples were included in this study. UC tissues were collected from the General Hospital of CNPC Jilin from 2010 to 2012. The patients with bladder UC were treated by transurethral resection and radical cystectomy. This study was approved by the Ethics Committee of CNPC. All patients were classified according to the 1973 World Health Organization classification for histological grading and the 1997 UICC TNM classification for pathological staging. Immunohistochemistry Immunohistochemical staining for rabbit polyclonal antibody against UT-B (against a C-terminal peptide, DNRIFYLQNKKRMVESPL) was performed using Ready-to-Use Immunohistochemistry Hypersensitivity UltraSensitiveTM S-P kit (Maxim, Fuzhou, China) according to the manufacturer’s instructions. Briefly, tissue sections were first deparaffinized in xylene and rehydrated in a series of graded alcohols then treated in citrate buffer pH 6.0 for heat-induced antigen retrieval. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide. Sections were subsequently incubated overnight at 4 ◦ C in a humidified chamber with polyclonal antibody against UT-B (1:500). After washing, sections were incubated with a biotin-labeled secondary antibody and streptomycin anti-biotin peroxidase. Diaminobenzidine (DAB) was used as a chromogen. Sections were counterstained with hematoxylin solution, dehydrated and mounted. Negative controls were performed without the primary antibody instead of using PBS. Internal positive control was used for quality assurance. Histology scoring All slides were assessed using a double-blind method. For each section, 10 high-power fields (400× magnification) were selected and 100 tumor cells were counted in each field. The expression of UT-B was scored in 5 categories according to their percentages of positive staining: 0 for ≤5%, 1 for 6–25%, 2 for 26–50%, 3 for 51–75% and 4 for >75%. The expression was also scored according to IHC staining intensities into 4 grades as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. Values for positive area and staining intensity were multiplied to give the final score. A score of 0–2 was considered negative, 3–4 was weakly
Table 1 Patient and bladder cancer characteristics. Factor
Value
Number of patients (%) Age (years) Range Mean ≥62
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Clinical significance of the reduction of UT-B expression in urothelial carcinoma of the bladder Chun Li a , Haogang Xue a , Yanming Lei b , Jianqiang Zhu a , Baoxue Yang c , Xiaodong Gai a,∗ a
Department of Immunology, School of Basic Medical Sciences, Beihua University, Jilin, China Department of Pathology, The General Hospital of CNPC in Jilin, Jilin, China Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Beijing, China b c
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 9 July 2014 Accepted 24 September 2014 Keywords: Bladder cancer Urea transporter B Urothelial carcinoma Immunohistochemistry
a b s t r a c t Urea transporter B (UT-B) is a membrane protein and plays an important role in regulating urea concentration in bladder urothelial cells. It has been reported that UT-B gene mutations were related to bladder carcinogenesis, and UT-B deletion could induce DNA damage and apoptosis in bladder urothelium. However, the functions and clinical significance of UT-B in human bladder cancer remain unknown. The most common type of bladder cancer is urothelial carcinoma (UC). We hypothesized that UT-B expression was related to bladder UC progress. In this study, UT-B was detected using immunohistochemistry in 52 paraffin-embedded specimens of bladder UC and 10 normal urothelium specimens. The results showed that UT-B protein expression in UC tumor cells was significantly lower as compared with normal urothelial cells (P = 0.021). UT-B protein expression was significantly reduced with increasing histological grade (P = 0.010). UT-B protein expression in muscle-invasive stage was significantly lower than in non-muscle-invasive stage (P = 0.014). Taken together, our data suggest that the reduction or loss of UT-B expression may be related to the incidence, progression and invasiveness of bladder UC. UT-B may be a novel diagnostic or prognostic biomarker, as well as a potential therapeutic target in UC of the bladder. © 2014 Elsevier GmbH. All rights reserved.
Introduction Bladder cancer is the most common malignancy of the urinary tract, with about 40 million new cases and 15 million deaths each year worldwide [1,2]. In addition, the incidence rates of bladder cancer in the countries of Europe and America are higher than Asia and Africa [3]. Over 90% of epithelial tumors in bladder cancer are urothelial carcinoma (UC), the remaining types include adenocarcinoma, squamous cell carcinoma and other rare histological types [4]. Despite most noninvasive UC patients can be successfully treated by surgery, 70% of patients suffered from tumor recurrence, and 15% of those recurrent tumors will become invasive [5]. Conventional clinical and pathologic parameters, such as tumor grade and stage, are limited to predict the clinical outcome of UC patients. There is an urgent need for new and more effective biomarkers
∗ Corresponding author at: Department of Pathology, School of Basic Medical Sciences, Beihua University, 3999 Huashan Road, Jilin 132013, China. Tel.: +86 432 64608104. E-mail addresses: [email protected], [email protected] (X. Gai). http://dx.doi.org/10.1016/j.prp.2014.09.012 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
for UC, not only for clinical early detection, accurate evaluation of tumor recurrence and progression, but also as a target for anticancer therapy. Studies suggest that bladder cancer has a genetic predisposition. Individuals who have a family history with bladder cancer have an almost 2-fold risk of developing bladder cancer than the normal population. Recently, three genome-wide association studies in Europe and the USA have reported that the occurrence of bladder cancer was significantly associated with the gene mutations of urea transporter B (UT-B) [6,7]. UT-B is a member of the urea transporters (UTs) family. UTs belong to a family of membrane proteins that selectively transport urea driven by urea gradient across cell membranes. Humans and other mammals have two UT genes, SLC14A1 (UT-B) and SLC14A2 (UT-A) [8]. UT-B has a wide tissue distribution, including erythrocyte, kidney, brain, spleen, liver, heart, colon, spleen, lung, stomach, testis, ureter, bladder, etc. [9–13], and its expression in the bladder is much higher than in other tissues. The functions of UT-B are closely related to urinary concentration, and its related mechanism has been verified by UT-B functional knockout studies in mice [13–15]. In addition, mutations in human UT-B might significantly decrease urea permeability in erythrocytes [16,17] and result in mild urinary concentrating defects [18].
800
C. Li et al. / Pathology – Research and Practice 210 (2014) 799–803
Recent studies showed that UT-B also played an important role in regulating urea concentration in bladder urothelial cells. UTB deletion could cause a marked urea accumulation and induce DNA damage and apoptosis in mouse bladder urothelium, and this biological process might lead to an increased risk of bladder carcinogenesis [19]. Up to now, the UT-B expression in bladder UC is unclear, and the functions and clinical significance of UT-B in bladder UC also remain unknown. The aim of this study was to better clarify the expression and functions of UT-B in bladder UC. We therefore examined the UT-B expression in 52 resected tumor specimens of bladder UC by immunohistochemistry (IHC) and analyzed the correlation between this expression and the clinicopathologic parameters to evaluate UT-B expression in relation to bladder UC development and clinical significance.
Materials and methods Patients and tissue specimens Formalin-fixed, paraffin-embedded (FFPE) samples from 52 patients with bladder UC and 10 FFPE normal urothelium samples were included in this study. UC tissues were collected from the General Hospital of CNPC Jilin from 2010 to 2012. The patients with bladder UC were treated by transurethral resection and radical cystectomy. This study was approved by the Ethics Committee of CNPC. All patients were classified according to the 1973 World Health Organization classification for histological grading and the 1997 UICC TNM classification for pathological staging. Immunohistochemistry Immunohistochemical staining for rabbit polyclonal antibody against UT-B (against a C-terminal peptide, DNRIFYLQNKKRMVESPL) was performed using Ready-to-Use Immunohistochemistry Hypersensitivity UltraSensitiveTM S-P kit (Maxim, Fuzhou, China) according to the manufacturer’s instructions. Briefly, tissue sections were first deparaffinized in xylene and rehydrated in a series of graded alcohols then treated in citrate buffer pH 6.0 for heat-induced antigen retrieval. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide. Sections were subsequently incubated overnight at 4 ◦ C in a humidified chamber with polyclonal antibody against UT-B (1:500). After washing, sections were incubated with a biotin-labeled secondary antibody and streptomycin anti-biotin peroxidase. Diaminobenzidine (DAB) was used as a chromogen. Sections were counterstained with hematoxylin solution, dehydrated and mounted. Negative controls were performed without the primary antibody instead of using PBS. Internal positive control was used for quality assurance. Histology scoring All slides were assessed using a double-blind method. For each section, 10 high-power fields (400× magnification) were selected and 100 tumor cells were counted in each field. The expression of UT-B was scored in 5 categories according to their percentages of positive staining: 0 for ≤5%, 1 for 6–25%, 2 for 26–50%, 3 for 51–75% and 4 for >75%. The expression was also scored according to IHC staining intensities into 4 grades as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining. Values for positive area and staining intensity were multiplied to give the final score. A score of 0–2 was considered negative, 3–4 was weakly
Table 1 Patient and bladder cancer characteristics. Factor
Value
Number of patients (%) Age (years) Range Mean ≥62
