Original Paper
Urologia
Received: March 18, 2014 Accepted after revision: May 28, 2014 Published online: August 7, 2014
Urol Int DOI: 10.1159/000364907
Internationalis
Soluble CD138/Syndecan-1 Increases in the Sera of Patients with Moderately Differentiated Bladder Cancer Mohammad Nabi Sanaee a Mahyar Malekzadeh a Abdolaziz Khezri b Abbas Ghaderi a, c Mehrnoosh Doroudchi a, c
Institute for Cancer Research and Departments of b Urology and c Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Key Words Bladder cancer · CD138/Syndecan-1 · Grade · Serum
Abstract Background: CD138/Syndecan-1 (Sdc-1) is expressed on the tumor and stromal cells of invasive bladder carcinoma. CD138/Sdc-1 shedding from the cell surface is associated with the invasive phenotype in lung and breast cancers. Patients and Methods: Soluble CD138/Sdc-1 was measured in the sera of 86 bladder cancer patients and 57 healthy individuals by a commercial ELISA assay. Results: Soluble Sdc-1 was increased in the sera of patients with bladder cancer (138.42 ± 81.85 vs. 86.48 ± 82.58 ng/ml, p = 0.0003). Patients aged over 70 years had higher levels of CD138/Sdc-1 in their sera (159.7 ± 15.77 vs. 124.5 ± 9.99 ng/ml, p = 0.025), and soluble Sdc-1 levels were higher in the sera of patients with moderately differentiated tumors compared to poorly differentiated ones (170.47 ± 85.06 vs. 101.79 ± 68.24 ng/ml, p = 0.01). The soluble Sdc-1 level was higher in muscle-invasive (154.45 ± 83.60 vs. 89.9 ± 55.02 ng/ml) but not lymphatic-invasive (106.25 ± 52.10 vs. 123.43 ± 63.76 ng/ml) tumors (p = 0.027 and 0.45, respectively). A non-significant trend of soluble Sdc-1 increase in the sera of male patients compared to female patients was observed (145.38 ± 85.47 vs. 110.20 ±
© 2014 S. Karger AG, Basel 0042–1138/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/uin
59.04 ng/ml, p = 0.054). Conclusion: The elevated levels of soluble CD138/Sdc-1 in older bladder cancer patients and those with muscular invasion sheds some light on the mechanisms of the disease invasion. © 2014 S. Karger AG, Basel
Introduction
Bladder carcinoma ranks as the second most common malignancy of the genitourinary tract and the fourth most prevalent cancer among all cancers in the world [1, 2]. Moreover, bladder cancer is one of the five common cancers among Iranian men [3]. Although the mechanisms that lead to urothelial neoplastic transformation is poorly understood, it has been shown that several genes are upregulated during the early events of neoplastic transformation, among which Syndecan-1 (Sdc-1) is of interest [4]. Syndecans are a family of proteoglycans that contain heparin sulfate chains. Heparin sulfate chains help Sdc-1 to bind to different growth factors and regulate cellular adhesion and migration, as well as growth factor signaling and control of cell proliferation and differentiation [5–7]. Heparin sulfate proteoglycans are shown to be exDr. Mehrnoosh Doroudchi Department of Immunology School of Medicine, Shiraz University of Medical Sciences PO Box 71345-3119, Shiraz (Iran) E-Mail mdoroud @ sums.ac.ir
Downloaded by: Univ. of California Davis 128.120.194.195 - 12/29/2014 2:05:12 PM
a
Subjects and Methods The study population included 86 bladder cancer patients (mean age ± SD = 64.36 ± 12.04 years) and 57 healthy individuals (mean age ± SD = 61.79 ± 14.72 years). The patients were entered into this study by the convenient method of sampling from among the cancer registry database of Shiraz Institute for Cancer Research. The inclusion criteria for the patients were the diagnosis of bladder cancer by the collaborating physician based on clinical
2
Urol Int DOI: 10.1159/000364907
examination and a positive pathological report. The exclusion criteria for the patients were lack of complete clinical and pathological data and/or lack of a serum sample. The inclusion of healthy control individuals was based on the absence of a history of cancer and autoimmune diseases, as well as their age and gender. The demographical and clinical characteristics of patients are shown in table 1. Serum samples from patients with bladder cancer were gathered after the consideration of inclusion and exclusion criteria. The patients were referred to our laboratory by our clinical coinvestigator based on clinical and pathological data. The demographic and clinical information gathered included: name, age, gender, history of autoimmune diseases, familial history of cancer and other diseases, tumor type, tumor size, lymph node involvement, metastasis and histological grade. Clinical and pathological data for each patient were extracted from patients’ files and at the time of sampling using a questionnaire. The questionnaire included the demographic criteria as well as risk factors, history of treatment, history of personal and familial cancer, and pathological and histological reports. After obtaining informed consent, 5 ml of blood was collected by venipuncture method. After coagulation, the serum was separated, aliquoted in 50-μl volumes and stored at –70 ° C until used. The Sdc-1 ELISA was performed according to the manufacturer’s instructions (Gen-Probe Diaclone SAS, Besancon, France). The optical density values were transformed to concentration levels by the standard curve. The results were then analyzed using the ANOVA and t test, and Mann-Whitney, Pearson and Spearman tests were used to evaluate the correlations by SPSS 11.5 software, and are discussed accordingly.
Results
By comparing the serum levels of CD138/Sdc-1 between 86 patients with bladder cancer and 57 healthy controls, the soluble CD138/Sdc-1 was found to be elevated in the patient group (138.42 ± 81.85 vs. 86.48 ± 82.58 ng/ ml, p = 0.0003; fig. 1). We also observed a significant increase (p = 0.025; fig. 2a) in the level of CD138/Sdc-1 in the sera of patients older than 70 years (159.7 ± 15.77 ng/ ml, n = 34) compared to those who were younger than 70 years (124.5 ± 9.99 ng/ml, n = 52). The lowest level of soluble CD138/Sdc-1 was observed in the group aged below 50 years. There was a significantly higher level of CD138/Sdc-1 in the patients aged between 70 and 80 years compared to healthy controls in the same age group (p = 0.0038; fig. 2b). There was also a significant decrease in the level of CD138/Sdc-1 in the sera of patients with poorly differentiated tumors (101.79 ± 68.24 ng/ml) compared to those with moderately differentiated tumors (170.47 ± 85.06 ng/ml, p = 0.01). Interestingly, the CD138/Sdc-1 levels were lower in the sera of patients with well-differentiated and anaplastic tumors compared to patients with moderSanaee/Malekzadeh/Khezri/Ghaderi/ Doroudchi
Downloaded by: Univ. of California Davis 128.120.194.195 - 12/29/2014 2:05:12 PM
pressed by bladder cell lines and mediate the binding of adhesion factor/angiomodulin to these cells [8]. CD138/ Sdc-1 is expressed in normal epithelial, endothelial and vascular smooth muscle cells, but it is overexpressed in the stromal cells of tumors [9, 10]. The molecule is also expressed in the tumor cells of invasive bladder carcinoma [11]. By its presence on the cells of extracellular matrix, Sdc-1 may be involved in the degradation of the ECM, stromal-epithelial interaction and cancer progression [5, 10, 11]. It is shown that CD138/Sdc-1 plays a key role in the survival of urothelial carcinoma cells as silencing of Sdc-1 by siRNA transfection induces apoptosis in the urothelial carcinoma cell lines [12]. The expression level of Sdc-1 is reported to correlate with other markers of migration and invasiveness in bladder urothelial carcinoma [13, 14]. High expression of Sdc-1 in aggressive plasmacytoid urothelial carcinoma is also reported [15, 16]. However, the expression of Sdc-1 decreases within epithelial tumor cells during tumor progression, which is accompanied by a shift in the expression of this molecule from tumor to stromal cells [9, 10]. Sdc-1 has been shown to be shed from cell surfaces through degradation of its extracellular domain by heparanases [17]. This is considered as one of the mechanisms involved in progressive loss of its expression during tumor development [17, 18]. In other tumor types, loss of membranous expression of CD138/Sdc-1 has been shown to be associated with an increase in its expression by stromal cells [9, 10, 19]. Moreover, this shift from a membranous to soluble form of the molecule marks a turning point in the tumor phenotype from a proliferative to an invasive type [20]. Soluble CD138/Sdc-1 exerts tumor growth-promoting effects and elevated Sdc-1 levels in the sera of lung cancer patients correlates with a poor prognosis [21]. However, to the best of our knowledge there is no report concerning the level of the soluble Sdc-1 in the sera of bladder cancer patients. Thus, we measured soluble CD138/Sdc-1 in the sera of patients with bladder cancer and evaluated its correlation with the type, location and the stage of the tumor.
Table 1. Clinicopathological information of patients with bladder cancer
Bladder cancer patients
Tumor type Adenocarcinoma Squamous cell carcinoma Transitional cell carcinoma Low malignant potential neoplasms Papillary and urothelial carcinoma Other Histological grade Well differentiated (I) Moderately differentiated (II) Poorly differentiated (III) Anaplastic (IV) Missing Tumor size >3 cm 50 years (n = 63)
age 50 years (n = 14)
age
Urologia
Received: March 18, 2014 Accepted after revision: May 28, 2014 Published online: August 7, 2014
Urol Int DOI: 10.1159/000364907
Internationalis
Soluble CD138/Syndecan-1 Increases in the Sera of Patients with Moderately Differentiated Bladder Cancer Mohammad Nabi Sanaee a Mahyar Malekzadeh a Abdolaziz Khezri b Abbas Ghaderi a, c Mehrnoosh Doroudchi a, c
Institute for Cancer Research and Departments of b Urology and c Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Key Words Bladder cancer · CD138/Syndecan-1 · Grade · Serum
Abstract Background: CD138/Syndecan-1 (Sdc-1) is expressed on the tumor and stromal cells of invasive bladder carcinoma. CD138/Sdc-1 shedding from the cell surface is associated with the invasive phenotype in lung and breast cancers. Patients and Methods: Soluble CD138/Sdc-1 was measured in the sera of 86 bladder cancer patients and 57 healthy individuals by a commercial ELISA assay. Results: Soluble Sdc-1 was increased in the sera of patients with bladder cancer (138.42 ± 81.85 vs. 86.48 ± 82.58 ng/ml, p = 0.0003). Patients aged over 70 years had higher levels of CD138/Sdc-1 in their sera (159.7 ± 15.77 vs. 124.5 ± 9.99 ng/ml, p = 0.025), and soluble Sdc-1 levels were higher in the sera of patients with moderately differentiated tumors compared to poorly differentiated ones (170.47 ± 85.06 vs. 101.79 ± 68.24 ng/ml, p = 0.01). The soluble Sdc-1 level was higher in muscle-invasive (154.45 ± 83.60 vs. 89.9 ± 55.02 ng/ml) but not lymphatic-invasive (106.25 ± 52.10 vs. 123.43 ± 63.76 ng/ml) tumors (p = 0.027 and 0.45, respectively). A non-significant trend of soluble Sdc-1 increase in the sera of male patients compared to female patients was observed (145.38 ± 85.47 vs. 110.20 ±
© 2014 S. Karger AG, Basel 0042–1138/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/uin
59.04 ng/ml, p = 0.054). Conclusion: The elevated levels of soluble CD138/Sdc-1 in older bladder cancer patients and those with muscular invasion sheds some light on the mechanisms of the disease invasion. © 2014 S. Karger AG, Basel
Introduction
Bladder carcinoma ranks as the second most common malignancy of the genitourinary tract and the fourth most prevalent cancer among all cancers in the world [1, 2]. Moreover, bladder cancer is one of the five common cancers among Iranian men [3]. Although the mechanisms that lead to urothelial neoplastic transformation is poorly understood, it has been shown that several genes are upregulated during the early events of neoplastic transformation, among which Syndecan-1 (Sdc-1) is of interest [4]. Syndecans are a family of proteoglycans that contain heparin sulfate chains. Heparin sulfate chains help Sdc-1 to bind to different growth factors and regulate cellular adhesion and migration, as well as growth factor signaling and control of cell proliferation and differentiation [5–7]. Heparin sulfate proteoglycans are shown to be exDr. Mehrnoosh Doroudchi Department of Immunology School of Medicine, Shiraz University of Medical Sciences PO Box 71345-3119, Shiraz (Iran) E-Mail mdoroud @ sums.ac.ir
Downloaded by: Univ. of California Davis 128.120.194.195 - 12/29/2014 2:05:12 PM
a
Subjects and Methods The study population included 86 bladder cancer patients (mean age ± SD = 64.36 ± 12.04 years) and 57 healthy individuals (mean age ± SD = 61.79 ± 14.72 years). The patients were entered into this study by the convenient method of sampling from among the cancer registry database of Shiraz Institute for Cancer Research. The inclusion criteria for the patients were the diagnosis of bladder cancer by the collaborating physician based on clinical
2
Urol Int DOI: 10.1159/000364907
examination and a positive pathological report. The exclusion criteria for the patients were lack of complete clinical and pathological data and/or lack of a serum sample. The inclusion of healthy control individuals was based on the absence of a history of cancer and autoimmune diseases, as well as their age and gender. The demographical and clinical characteristics of patients are shown in table 1. Serum samples from patients with bladder cancer were gathered after the consideration of inclusion and exclusion criteria. The patients were referred to our laboratory by our clinical coinvestigator based on clinical and pathological data. The demographic and clinical information gathered included: name, age, gender, history of autoimmune diseases, familial history of cancer and other diseases, tumor type, tumor size, lymph node involvement, metastasis and histological grade. Clinical and pathological data for each patient were extracted from patients’ files and at the time of sampling using a questionnaire. The questionnaire included the demographic criteria as well as risk factors, history of treatment, history of personal and familial cancer, and pathological and histological reports. After obtaining informed consent, 5 ml of blood was collected by venipuncture method. After coagulation, the serum was separated, aliquoted in 50-μl volumes and stored at –70 ° C until used. The Sdc-1 ELISA was performed according to the manufacturer’s instructions (Gen-Probe Diaclone SAS, Besancon, France). The optical density values were transformed to concentration levels by the standard curve. The results were then analyzed using the ANOVA and t test, and Mann-Whitney, Pearson and Spearman tests were used to evaluate the correlations by SPSS 11.5 software, and are discussed accordingly.
Results
By comparing the serum levels of CD138/Sdc-1 between 86 patients with bladder cancer and 57 healthy controls, the soluble CD138/Sdc-1 was found to be elevated in the patient group (138.42 ± 81.85 vs. 86.48 ± 82.58 ng/ ml, p = 0.0003; fig. 1). We also observed a significant increase (p = 0.025; fig. 2a) in the level of CD138/Sdc-1 in the sera of patients older than 70 years (159.7 ± 15.77 ng/ ml, n = 34) compared to those who were younger than 70 years (124.5 ± 9.99 ng/ml, n = 52). The lowest level of soluble CD138/Sdc-1 was observed in the group aged below 50 years. There was a significantly higher level of CD138/Sdc-1 in the patients aged between 70 and 80 years compared to healthy controls in the same age group (p = 0.0038; fig. 2b). There was also a significant decrease in the level of CD138/Sdc-1 in the sera of patients with poorly differentiated tumors (101.79 ± 68.24 ng/ml) compared to those with moderately differentiated tumors (170.47 ± 85.06 ng/ml, p = 0.01). Interestingly, the CD138/Sdc-1 levels were lower in the sera of patients with well-differentiated and anaplastic tumors compared to patients with moderSanaee/Malekzadeh/Khezri/Ghaderi/ Doroudchi
Downloaded by: Univ. of California Davis 128.120.194.195 - 12/29/2014 2:05:12 PM
pressed by bladder cell lines and mediate the binding of adhesion factor/angiomodulin to these cells [8]. CD138/ Sdc-1 is expressed in normal epithelial, endothelial and vascular smooth muscle cells, but it is overexpressed in the stromal cells of tumors [9, 10]. The molecule is also expressed in the tumor cells of invasive bladder carcinoma [11]. By its presence on the cells of extracellular matrix, Sdc-1 may be involved in the degradation of the ECM, stromal-epithelial interaction and cancer progression [5, 10, 11]. It is shown that CD138/Sdc-1 plays a key role in the survival of urothelial carcinoma cells as silencing of Sdc-1 by siRNA transfection induces apoptosis in the urothelial carcinoma cell lines [12]. The expression level of Sdc-1 is reported to correlate with other markers of migration and invasiveness in bladder urothelial carcinoma [13, 14]. High expression of Sdc-1 in aggressive plasmacytoid urothelial carcinoma is also reported [15, 16]. However, the expression of Sdc-1 decreases within epithelial tumor cells during tumor progression, which is accompanied by a shift in the expression of this molecule from tumor to stromal cells [9, 10]. Sdc-1 has been shown to be shed from cell surfaces through degradation of its extracellular domain by heparanases [17]. This is considered as one of the mechanisms involved in progressive loss of its expression during tumor development [17, 18]. In other tumor types, loss of membranous expression of CD138/Sdc-1 has been shown to be associated with an increase in its expression by stromal cells [9, 10, 19]. Moreover, this shift from a membranous to soluble form of the molecule marks a turning point in the tumor phenotype from a proliferative to an invasive type [20]. Soluble CD138/Sdc-1 exerts tumor growth-promoting effects and elevated Sdc-1 levels in the sera of lung cancer patients correlates with a poor prognosis [21]. However, to the best of our knowledge there is no report concerning the level of the soluble Sdc-1 in the sera of bladder cancer patients. Thus, we measured soluble CD138/Sdc-1 in the sera of patients with bladder cancer and evaluated its correlation with the type, location and the stage of the tumor.
Table 1. Clinicopathological information of patients with bladder cancer
Bladder cancer patients
Tumor type Adenocarcinoma Squamous cell carcinoma Transitional cell carcinoma Low malignant potential neoplasms Papillary and urothelial carcinoma Other Histological grade Well differentiated (I) Moderately differentiated (II) Poorly differentiated (III) Anaplastic (IV) Missing Tumor size >3 cm 50 years (n = 63)
age 50 years (n = 14)
age
