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Up-regulation of glucosylceramide synthase in urinary bladder neoplasms
Urologic Oncology: Seminars and Original Investigations 30 (2012) 444 – 449
Original article
Up-regulation of glucosylceramide synthase in urinary bladder neoplasms Chang-cheng Sun, M.D., Zhe Zhang, Ph.D., Si-Yang Zhang, Ph.D., Jun Li, M.D., Ze-liang Li, M.D., Chui-ze Kong, M.D.* Department of Urology, the First Affiliated Hospital of China Medical University, Shenyang, China Received 17 February 2010; received in revised form 26 April 2010; accepted 27 April 2010
Abstract Objective: To investigate the relationships between the clinicopathologic features and the expression of GCS in bladder cancer. Methods and materials: Using immunohistochemistry and Western blotting method, 75 bladder cancer specimens were tested for expression of GCS. The correlation of GCS with clinicopathologic features of the patients was analyzed in combination with clinical data. Statistics analyses were done with SPSS 13.0 software, 2 test, Fisher’s exact test, Kaplan-Meier method, Log-rank test. Results: High and low level expression of GCS explored by immunohistochemistry were 61.3 (46/75) and 39.6 (29/75), respectively. The high expression group (n ⫽ 46) showed a significant correlation with high histologic grade (P ⫽ 0.021) and tended to show (P ⫽ 0.045) that up-expression of GCS was positive related to BNs with lymph node metastasis among the various clinicopathologic characteristics. The overall 5-year survival and disease-free survival rates were 39.5% and 18.4%, respectively. Mean overall survival time was 60.3 months for the low expression group and 45.1 months for the high expression group. Mean disease-free survival was 36.2 months for the low-expression group and 27.3 months for the high-expression group. Conclusion: Our study suggested that up-regulation of GCS might make an aggressive choice of surgical therapy. A high expression of GCS seemed to be an indicator of poor prognosis. © 2012 Elsevier Inc. All rights reserved. Keywords: Glucosylceramide synthase (GCS); Bladder cancer; Bladder neoplasms; Clinicopathologic features; Prognosis
1. Introduction Bladder cancer is the second most common cancer of the genitourinary tract with approximately 350,000 new cases worldwide annually [1]. Seventy to 80% of patients with newly-diagnosed bladder cancer will present with superficial tumors (Ta, Tis, or T1). There is, however, a continuum between superficial and muscle-invasive cancer, with the advanced cases usually associated with less-differentiated histology and aneuploidy. Tumors of the bladder can be classified depending on their depth of invasion. Although radical cystectomy, with continent diversion or neobladder construction in selected cases, remains the standard of care in China for patients with muscle-invasive bladder cancer, radical cystectomy may cause important changes in the lives of patients, not only in urinary and sexual function, but also * Corresponding author. Tel.: ⫹86-024-83283433; fax: ⫹86-02483283433. E-mail address: [email protected] (C.-Z. Kong). 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2010.04.012
in social function, daily living activities, and satisfaction with body image [2– 4]; several groups have explored therapeutic strategies that aim at bladder preservation. However, the options of methods of therapy are still obscure. Ceramide, now recognized as a second messenger in cellular apoptotic signaling events, has been shown to play a role in chemotherapy and radiotherapy of cancer [5,6]. Loss of ceramide production is one cause of cellular resistance to apoptosis induced by either ionizing radiation or tumor necrosis factor-␣ and adriamycin [7–9]. Accumulation of glucosylceramide (GC), a simple glycosylated form of ceramide, is a characteristic of some multidrug-resistant cancer cells and tumors derived from patients who are less responsive to chemotherapy [10,11]. Nevertheless, the key enzyme of GC metabolism is the glucosylceramide synthase (GCS). GCS catalyzes the first glycosylation step in the biosynthesis of glycosphingolipids [12], which is a key regulatory factor controlling intracellular levels of ceramide. Some reports have demonstrated that GCS plays an
C.-c. Sun et al. / Urologic Oncology: Seminars and Original Investigations 30 (2012) 444 – 449
important role in cancer multidrug resistance [13–15]. To our knowledge, there are no reports related to the expression of GCS and clinical significance in bladder neoplasms up to now. So the aim of this study is to investigate the relationships between the clinicopathologic features and the expressions of GCS.
2. Materials and methods 2.1. Patients and samples This study was approved by the Ethics Committee of China Medical University. Clinical and pathologic data and the specimens used for Western blotting and immunohistochemical analysis were obtained through a detailed retrospective review of the medical records of 75 patients of urinary bladder neoplasms who had undergone initial surgical resection with preservation of the bladder between 2002 and 2003 at the First Affiliated Hospital of China Medical University. Most of the patients with nodules had received chemotherapy and radiation therapy. All of the patients were studied in order to evaluate the clinicopathologic correlation of the prevalence of GCS positive cells with specific variables. Tumors were classified according to the International Union against Cancer tumor-node-metastasis (TNM) classification. If the patients had multiple nodules in the bladder, we selected the nodule showing the most advanced histologic grade for our study. All patients had complete medical records and had been followed by the tumor registries for survival and outcome. Follow-up was available in all cases and ranged from 10.5 to 60 months (mean value is 51.02 months). The latest survival data were collected on April 30, 2006. The overall survival rate at 5 years and the disease-free survival rate were 39.5% and 18.4%, respectively. The clinicopathologic features of the patients are summarized in Table 1. 2.2. Immunohistochemical analysis Immunohistochemistry was done on the formalin-fixed, paraffin- embedded tissue sections . We reacted 4-m-thick Table 1 Clinicopathologic features of the patients Variables
Results
Characteristics of the patients with BN (75 cases) Age, y (median, range) Sex (male/female) Depth of tumor invasiveness (Tis-T1/T2/T3/T4) Sentinel node (positive/negative) Histologic type (tcc/others) Differentiation (well/moderate/poor) Style of surgery (Tur-B/PR)
54, 45–82 58/17 45/28/2/0 16/59 72/3 64/8/3 51/24
BN ⫽ bladder neoplasm; tcc ⫽ transitional cell carcinoma; Tur-B ⫽ transurethral resection of bladder tumor; PR ⫽ partial resection.
445
sections of representative blocks with monoclonal antibodies against GCS (1:50) from Sigma Ltd, CA. Briefly, the sections were deparaffinized and rehydrated. After blocking of endogenous peroxidase with methanol containing 0.3% H2O2, the sections were autoclaved at 121°C for 10 minutes in citrate buffer (10 mmol/l sodium citrate; pH 6.0) for antigen retrieval. After blocking with normal goat serum, the sections were reacted overnight with appropriately diluted primary antibodies. The sections were then reacted sequentially with biotin-conjugated anti-mouse IgG antibodies (Vector Laboratories, Burlingame, CA) and Vectastain Elite ABC reagent (Vector Laboratories). Diaminobenzidine was used as the chromogen, and the nuclei were counterstained with hematoxylin. Brown particles appearing in cytoplasm was regarded as positive cells. The intensity of GCS immunostaining (1 ⫽ weak, 2 ⫽ moderate, and 3 ⫽ intense) and the percentage of positive tumor cells (0% ⫽ negative, 1%–50% ⫽ 1, 51%–75% ⫽ 2, ⱖ76% ⫽ 3) were assessed in at least 5 high power fields (⫻400 magnification). The scores of each tumorous sample were multiplied to give a final score of 0, 1, 2, 3, 4, 6, or 9, and the tumors were finally determined as negative: score 0; lower expression: score ⱕ 4; or higher expression: score ⱖ 6. Scoring was performed by three pathologists. 2.3. Western blotting Frozen tissues (including tumor and non-tumorous portion) or cells were washed twice with ice-cold phosphatebuffered saline (PBS), homogenized on ice in 10 volumes (wt/vol) of lysis buffer containing 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1% Triton-X100, 1 mM PMSF, and phosphatase inhibitor using a homogenizer (Heidolph, DLA ⫻ 900; Heidolph Instruments GmbH, Elk Grove Village, IL). The homogenate was centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was collected and stored at ⫺70°C. Protein content was determined by the BCA assay (BCA protein assay kit-23227; Pierce Biotechnology, Rockford, IL). From each sample preparation, 80 g of total protein was separated by 8% SDS-PAGE and then transferred to PVDF blotting membranes. The total protein extracts were analyzed by immunoblotting with indicated antibodies following SDS-PAGE analysis. Immunoblots were performed using rabbit polyclonal primary antibodies specific for GCS and -actin (a housekeeping protein used as a loading control to assure equal amounts of protein in all lanes). After blocking nonspecific binding with 5% BSA in TBS (pH 7.5) containing 0.05% Tween-20 (TBST), primary antibodies were incubated on the membranes for GCS and -actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C in TBST. Following three times washes in TBST, the membranes were incubated for 2 h at 37°C with secondary goat anti-rabbit IgG antibodies (1:2000, ZDR-5306) and goat anti-mouse IgG antibody (1:2000, ZDR-5307) labeled with horseradish peroxidase (all from Zhongshan Biotech-
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nology, Peking City, China). Immunoreactive straps were identified using the DAB system (DAB kit-0031; Maixin Biotechnology, Fuzhou City, China), as directed by the manufacturer. Specific bands for GCS and -actin were identified by prestained protein molecular weight marker (SM0441; MBI Fermentas, Glen Burnie, MD). The EC3 Imaging System (UVP Inc., Upland, CA) was used to catch up the specific bands, and the optical density of each band was measured using Image J software, Shenyang city, China. The ratio between the optical density of interest proteins and -actin of the same sample was calculated as relative content and expressed graphically. 2.4. Statistical analysis Values were expressed as mean ⫾ SD. Statistical analyses were done with SPSS 13.0 software, Shenyang city, China. Associations among the variables were assessed by the 2 test and Student’s t-test. Survival rates were calculated by the Kaplan-Meier method. Differences between survival curves were analyzed by the log-rank test. Differences were considered significant at P ⬍ 0.05.
3. Results 3.1. GCS expression and localization in bladder neoplasms by immunohistochemistry GCS immunoreactivity was detected in both normal and tumorous bladder cells. In normal bladder tissues, GCS expression was observed, while GCS immunostaining was observed in every neoplastic tissue as shown in Fig. 1. We considered that 46 of the bladder neoplasms (61%) were higher expression (scores of 6 or 9); 29 cases (39%) were lower expression (scores of 0, 1, 2, 3, or 4), as described above in the Materials and Methods section. Fig. 1 shows that the expression of GCS located in the cytoplasm and stroma. 3.2. GCS expressions in BNs by Western blotting Western blotting was used to evaluate GCS expression in 24 BNs and paired non-tumorous bladder tissues distant from the primary tumor of the same case. Increased GCS expression was found in 15 BNs samples in comparison with the non-tumorous counterparts. Western blotting of 8 samples is shown in Fig. 2A, and the optical densities of the tumorous (T) and non-tumorous (N) tissues of the same patient were measured and expressed graphically (Fig. 2B). Comparison of GCS expression was made between BNs with and without lymph node metastasis statistically. GCS expression was significantly higher in node-positive BNs (Fig. 2B).
Fig. 1. GCS expression by immunohistochemistry. (A) GCS immunostaining in the normal epithelial cells of bladder. (B) GCS expression in the transitional carcinoma cells with sentinel node negative. (Color version of figure is available online).
3.3. Clinicopathologic features of BNs and the prevalence of GCS high expressions We analyzed the correlation between clinicopathologic features of BNs and the prevalence of GCS expressions in BNs (Table 2). Patients with BNs were divided into 2 groups by the median value for the prevalence of GCS expressions. The high expression group (n ⫽ 46) showed a significant correlation with high histologic grade (P ⫽ 0.021) and tended to show (P ⫽ 0.045) that up-expression of GCS was positive related to BNs with lymph node metastasis among the various clinicopathologic characteristics.
C.-c. Sun et al. / Urologic Oncology: Seminars and Original Investigations 30 (2012) 444 – 449
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Fig. 2. (A) Expression of GCS by Western blotting in matched tumorous (T) and non-tumorous (N) tissues from 8 of 24 BN patients, and 4 of which were accompanied with lymph node metastasis (the lower panel). Band intensities indicate significant GCS up-regulation in tumorous in comparison with the non-tumorous tissue of the same patient. Furthermore, patients with positive nodes expressed higher level of GCS in tumor. -Actin was used as a loading control to assure equal amounts of protein in all lanes. (B) The ratio between the optical density of GCS and -actin of the same patient was calculated and expressed graphically. Significant differences of GCS expression between tumorous (T) and non-tumorous (N) tissues as well as neoplastic samples with positive and negative nodes were analyzed statistically. GCS immunoreactivity is greater in neoplastic tissues (P ⫽ 0.000) and cases with positive nodes (P ⫽ 0.018).
3.4. Prognostic significance of the expressions of GCS
4. Discussion
Overall and disease-free survivals were analyzed in these patients. The overall 5-year survival and disease-free survival rates were 39.5% and 18.4%, respectively. The lowexpression of GCS group showed significantly better overall survival than the high-expression group (log-rank test, P ⫽ 0.007; Fig. 3A). Mean overall survival was 60.3 (F33.8) months for the low-expression group and 45.1 (F38.7) months for the high-expression group. The low-expression group also showed significantly better disease-free survival than the high-expression group (log-rank test, P ⫽ 0.015; Fig. 3B). Mean disease-free survival was 36.2 (F31.7) months for the low-expression group and 27.3 (F32.9) months for the high-expression group.
GCS is a transmembrane protein with the C-terminal catalytic domain located in the cytoplasm [16]. GCS transfers a glucose residue from UDP-glucose to ceramide for the synthesis of GC. This mainly occurs on the cytoplasmic surface of the Golgi [17]. In the Golgi lumen, GC is further modified by a series of glycosyltransferases that produce higher order glycosphingolipids. Glycosphingolipids are composed of a group of membrane lipids in which the lipid portion is embedded in the outer leaflet of the plasma membrane with the sugar chain extending to the extracellular space. Glycosphingolipids are integral components of plasma membrane microdomains known as rafts, caveolae, and glycosignaling domains that are rich in sphingolipids and cholesterol [18,19]. These lipid domains assemble receptors and glycosyl-phosphatidylinositol anchored proteins on their external surface and signaling molecules, including Src family kinases, G proteins, and nitric oxide synthase, on their internal surface. Some studies showed that overexpression of GCS by gene transfection conferred cellular resistance to chemotherapy and to tumor necrosis factor-␣ [13,20]. Moreover, Inhibition of GCS activity is being evaluated as a possible treatment for several lipid-storage diseases and some types of cancer [21,22]. This study evaluated GCS expression in BNs, with regard to the depth of tumor invasion, histologic type, differentiation, stage, and lymph node status of BNs, to determine the clinical significance of GCS for the advanced BNs. We examined 75 tumors by means of immunohistochemistry, 24 of which were also analyzed by Western blotting, and found a statistical evidence of GCS up-regulation in BNs. Weak-moderate GCS immunostaining was observed in the normal transitional cells, as well as part of BNs. We also found that overexpression of GCS was common in BNs, regardless of the histologic type. However, patients with higher GCS expression had a significant metastatic phenotype. The immunohistochemical obser-
Table 2 Correlation between clinicopathologic findings and the prevalence of the expression of GCS Variables
Age, y (mean ⫾ SD) Sex (male/female) Depth of tumor invasiveness (Tis-T1/T2/T3/T4) Sentinel node (positive/negative) Histologic type (tcc/others) Differentiation (well/moderate/poor) Style of surgery (Tur-B/PR)
Expressions of GCS High
Low
62.4 ⫾ 5.9 42/4 28/16/2/0
70.5 ⫾ 9.2 16/13 17/12/0/0
P
0.514** 0.907* 0.105*
13/33
3/26
0.045*
44/2 36/7/3
28/1 28/1/0
0.524* 0.021*
26/20
25/4
0.075*
BN ⫽ bladder neoplasm; tcc ⫽ transitional cell carcinoma; Tur-B ⫽ transurethral resection of bladder tumor; PR ⫽ partial resection. * 2 test or Fisher’s exact test. ** Student’s t-test.
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Fig. 3. Overall survival curve (A) and disease-free survival curve (B) are shown. The prognosis was significantly worse in the high expression group than in the low expression group [log-rank test, P ⫽ 0.007 (A) and P ⫽ 0.015 (B)]. (Color version of figure is available online.)
vations might be further supported by our semiquantitative Western blotting evaluations of GCS expression in 24 tumorous and paired non-tumorous counterparts. GCS expressions were significantly higher in the neoplastic than the non-neoplastic tissues. Furthermore, in the present study, we investigated the relationship between clinicopathologic features of BNs and the prevalence of GCS expressions in BNs. Patients with BNs in the high-expression of GCS group showed a significantly lower survival ratio. Both overall survival (log-rank test, P ⫽ 0.007) and disease-free survival (log-rank test, P ⫽ 0.015) were lower than for patients with BNs belonging to the low-
expression group. These findings suggest that high expression of GCS was closely correlated with the progression of neoplastic cells in BNs; moreover, the options of surgical styles should be decided more aggressively for cases with GCS high expression. To our knowledge, this is the first report to show that high expression of GCS is closely correlated with the development and progression of BNs, and may be a useful prognostic factor in patients with BNs. In conclusion, our data suggested that besides correlating with multidrug resistance, GCS also played a role in controlling the progression of neoplastic cells in BNs, and that up-regulation of GCS might make an aggressive choice of
C.-c. Sun et al. / Urologic Oncology: Seminars and Original Investigations 30 (2012) 444 – 449
surgical therapy. A high expression of GCS seemed to be an indicator of poor prognosis.
References [1] Parkin DM, Pisani P, Ferlay J. Global Cancer Statistics. CA Cancer J Clin 1999;49:33– 64. [2] Shipley WU, Kaufman DS, Tester WJ, et al. An overview of bladder cancer trials in the Radiation Therapy Oncology Group (RTOG). Cancer 2003;97(8 Suppl):2115–9. [3] Shipley WU, Kaufman DS, Zehr E, et al. Selective bladder preservation by combined modality protocol treatment: Long-term outcomes of 190 patients with invasive bladder cancer. Urol 2002;60:62–8. [4] Zietman AL, Grocela J, Zehr E, et al. Selective bladder conservation using transurethral resection, chemotherapy and radiation: The management and consequences of Ta, T1, Tis recurrence within the retained bladder. Urology 2001;58:380 –5. [5] Carpinteiro A, Dumitru C, Schenck M, et al. Ceramide-induced cell death in malignant cells. Cancer Lett 2008;264:1–10. [6] Alphonse G, Bionda C, Aloy MT, et al. Overcoming resistance to ␥-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels. Oncogene 2004;23:2703–15. [7] Liu YY, Yu JY, Yin D, et al. A role for ceramide in driving cancer cell resistance to doxorubicin. FASEB J 2008;22:2541–51. [8] Gouazé-Andersson V, Yu JY, Kreitenberg AJ, et al. Ceramide and glucosylceramide up-regulate expression of the multidrug resistance gene MDR1 in cancer cells. Biochim Biophys Acta 2007;1771:1407–17. [9] Xie P, Shen YF, Shi YP, et al. Overexpression of glucosylceramide synthase in associated with multidrug resistance of leukemia cells. Leuk Res 2008;32:475– 80. [10] Liu YY, Han TY, Giuliano AE, et al. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. J Biol Chem 1999;274:1140–6.
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[11] Lucci A, Cho WI, Han TY, et al. Glucosylceramide: a marker for multiple-drug resistant cancers. Anticancer Res 1998;18:475– 80. [12] Jeckel D, Karrenbauer A, Burger KN, et al. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions. J Cell Biol 1992;117:259 – 67. [13] Yong-yu Liu, Tie-yan Han, Armando E, et al. Ceramide glycosylation potentiates cellular multidrug resistance. The FASEB J 2001;15:719 –30. [14] Gouaze V, Liu YY, Prickett CS, et al. Glucosylceramide synthase blockade down-regulates P-glycoprotein and resensitizes multidrugresistant breast cancer cells to anticancer drugs. Cancer Res 2005;65: 3861–7. [15] Gouazé-Andersson V, Yu JY, Kreitenberg AJ, et al. Ceramide and glucosylceramide up-regulate expression of the multidrug resistance gene MDR1 in cancer cells. Biochim Biophys Acta 2007;1771:1407–17. [16] Marks DL, Wu K, Paul PY, et al. Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase. J Biol Chem 1999; 274:451– 6. [17] Jeckel D, Karrenbauer A, Burger KN, et al. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions. J Cell Biol 1992;117:259 – 67. [18] Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997;387:569 –72. [19] Hakomori S, Handa K, Iwabuchi K, et al. New insights in glycosphingolipid function: “glycosignaling domain,” a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in cell adhesion coupled with signaling. Glycobiology 1998;8:11–9. [20] Liu YY, Han TY, Giuliano AE, et al. Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-␣ induced apoptosis. Exp Cell Res 1999;252:464 –70. [21] Lachmann RH. Miglustat Oxford GlycoSciences/Actelion. Curr Opin Invest Drugs 2003;4:472–9. [22] Deng W, Li R, Ladisch S. Influence of cellular ganglioside depletion on tumor formation. J Natl Cancer Inst 2000;92:912–7.
Original article
Up-regulation of glucosylceramide synthase in urinary bladder neoplasms Chang-cheng Sun, M.D., Zhe Zhang, Ph.D., Si-Yang Zhang, Ph.D., Jun Li, M.D., Ze-liang Li, M.D., Chui-ze Kong, M.D.* Department of Urology, the First Affiliated Hospital of China Medical University, Shenyang, China Received 17 February 2010; received in revised form 26 April 2010; accepted 27 April 2010
Abstract Objective: To investigate the relationships between the clinicopathologic features and the expression of GCS in bladder cancer. Methods and materials: Using immunohistochemistry and Western blotting method, 75 bladder cancer specimens were tested for expression of GCS. The correlation of GCS with clinicopathologic features of the patients was analyzed in combination with clinical data. Statistics analyses were done with SPSS 13.0 software, 2 test, Fisher’s exact test, Kaplan-Meier method, Log-rank test. Results: High and low level expression of GCS explored by immunohistochemistry were 61.3 (46/75) and 39.6 (29/75), respectively. The high expression group (n ⫽ 46) showed a significant correlation with high histologic grade (P ⫽ 0.021) and tended to show (P ⫽ 0.045) that up-expression of GCS was positive related to BNs with lymph node metastasis among the various clinicopathologic characteristics. The overall 5-year survival and disease-free survival rates were 39.5% and 18.4%, respectively. Mean overall survival time was 60.3 months for the low expression group and 45.1 months for the high expression group. Mean disease-free survival was 36.2 months for the low-expression group and 27.3 months for the high-expression group. Conclusion: Our study suggested that up-regulation of GCS might make an aggressive choice of surgical therapy. A high expression of GCS seemed to be an indicator of poor prognosis. © 2012 Elsevier Inc. All rights reserved. Keywords: Glucosylceramide synthase (GCS); Bladder cancer; Bladder neoplasms; Clinicopathologic features; Prognosis
1. Introduction Bladder cancer is the second most common cancer of the genitourinary tract with approximately 350,000 new cases worldwide annually [1]. Seventy to 80% of patients with newly-diagnosed bladder cancer will present with superficial tumors (Ta, Tis, or T1). There is, however, a continuum between superficial and muscle-invasive cancer, with the advanced cases usually associated with less-differentiated histology and aneuploidy. Tumors of the bladder can be classified depending on their depth of invasion. Although radical cystectomy, with continent diversion or neobladder construction in selected cases, remains the standard of care in China for patients with muscle-invasive bladder cancer, radical cystectomy may cause important changes in the lives of patients, not only in urinary and sexual function, but also * Corresponding author. Tel.: ⫹86-024-83283433; fax: ⫹86-02483283433. E-mail address: [email protected] (C.-Z. Kong). 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2010.04.012
in social function, daily living activities, and satisfaction with body image [2– 4]; several groups have explored therapeutic strategies that aim at bladder preservation. However, the options of methods of therapy are still obscure. Ceramide, now recognized as a second messenger in cellular apoptotic signaling events, has been shown to play a role in chemotherapy and radiotherapy of cancer [5,6]. Loss of ceramide production is one cause of cellular resistance to apoptosis induced by either ionizing radiation or tumor necrosis factor-␣ and adriamycin [7–9]. Accumulation of glucosylceramide (GC), a simple glycosylated form of ceramide, is a characteristic of some multidrug-resistant cancer cells and tumors derived from patients who are less responsive to chemotherapy [10,11]. Nevertheless, the key enzyme of GC metabolism is the glucosylceramide synthase (GCS). GCS catalyzes the first glycosylation step in the biosynthesis of glycosphingolipids [12], which is a key regulatory factor controlling intracellular levels of ceramide. Some reports have demonstrated that GCS plays an
C.-c. Sun et al. / Urologic Oncology: Seminars and Original Investigations 30 (2012) 444 – 449
important role in cancer multidrug resistance [13–15]. To our knowledge, there are no reports related to the expression of GCS and clinical significance in bladder neoplasms up to now. So the aim of this study is to investigate the relationships between the clinicopathologic features and the expressions of GCS.
2. Materials and methods 2.1. Patients and samples This study was approved by the Ethics Committee of China Medical University. Clinical and pathologic data and the specimens used for Western blotting and immunohistochemical analysis were obtained through a detailed retrospective review of the medical records of 75 patients of urinary bladder neoplasms who had undergone initial surgical resection with preservation of the bladder between 2002 and 2003 at the First Affiliated Hospital of China Medical University. Most of the patients with nodules had received chemotherapy and radiation therapy. All of the patients were studied in order to evaluate the clinicopathologic correlation of the prevalence of GCS positive cells with specific variables. Tumors were classified according to the International Union against Cancer tumor-node-metastasis (TNM) classification. If the patients had multiple nodules in the bladder, we selected the nodule showing the most advanced histologic grade for our study. All patients had complete medical records and had been followed by the tumor registries for survival and outcome. Follow-up was available in all cases and ranged from 10.5 to 60 months (mean value is 51.02 months). The latest survival data were collected on April 30, 2006. The overall survival rate at 5 years and the disease-free survival rate were 39.5% and 18.4%, respectively. The clinicopathologic features of the patients are summarized in Table 1. 2.2. Immunohistochemical analysis Immunohistochemistry was done on the formalin-fixed, paraffin- embedded tissue sections . We reacted 4-m-thick Table 1 Clinicopathologic features of the patients Variables
Results
Characteristics of the patients with BN (75 cases) Age, y (median, range) Sex (male/female) Depth of tumor invasiveness (Tis-T1/T2/T3/T4) Sentinel node (positive/negative) Histologic type (tcc/others) Differentiation (well/moderate/poor) Style of surgery (Tur-B/PR)
54, 45–82 58/17 45/28/2/0 16/59 72/3 64/8/3 51/24
BN ⫽ bladder neoplasm; tcc ⫽ transitional cell carcinoma; Tur-B ⫽ transurethral resection of bladder tumor; PR ⫽ partial resection.
445
sections of representative blocks with monoclonal antibodies against GCS (1:50) from Sigma Ltd, CA. Briefly, the sections were deparaffinized and rehydrated. After blocking of endogenous peroxidase with methanol containing 0.3% H2O2, the sections were autoclaved at 121°C for 10 minutes in citrate buffer (10 mmol/l sodium citrate; pH 6.0) for antigen retrieval. After blocking with normal goat serum, the sections were reacted overnight with appropriately diluted primary antibodies. The sections were then reacted sequentially with biotin-conjugated anti-mouse IgG antibodies (Vector Laboratories, Burlingame, CA) and Vectastain Elite ABC reagent (Vector Laboratories). Diaminobenzidine was used as the chromogen, and the nuclei were counterstained with hematoxylin. Brown particles appearing in cytoplasm was regarded as positive cells. The intensity of GCS immunostaining (1 ⫽ weak, 2 ⫽ moderate, and 3 ⫽ intense) and the percentage of positive tumor cells (0% ⫽ negative, 1%–50% ⫽ 1, 51%–75% ⫽ 2, ⱖ76% ⫽ 3) were assessed in at least 5 high power fields (⫻400 magnification). The scores of each tumorous sample were multiplied to give a final score of 0, 1, 2, 3, 4, 6, or 9, and the tumors were finally determined as negative: score 0; lower expression: score ⱕ 4; or higher expression: score ⱖ 6. Scoring was performed by three pathologists. 2.3. Western blotting Frozen tissues (including tumor and non-tumorous portion) or cells were washed twice with ice-cold phosphatebuffered saline (PBS), homogenized on ice in 10 volumes (wt/vol) of lysis buffer containing 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1% Triton-X100, 1 mM PMSF, and phosphatase inhibitor using a homogenizer (Heidolph, DLA ⫻ 900; Heidolph Instruments GmbH, Elk Grove Village, IL). The homogenate was centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was collected and stored at ⫺70°C. Protein content was determined by the BCA assay (BCA protein assay kit-23227; Pierce Biotechnology, Rockford, IL). From each sample preparation, 80 g of total protein was separated by 8% SDS-PAGE and then transferred to PVDF blotting membranes. The total protein extracts were analyzed by immunoblotting with indicated antibodies following SDS-PAGE analysis. Immunoblots were performed using rabbit polyclonal primary antibodies specific for GCS and -actin (a housekeeping protein used as a loading control to assure equal amounts of protein in all lanes). After blocking nonspecific binding with 5% BSA in TBS (pH 7.5) containing 0.05% Tween-20 (TBST), primary antibodies were incubated on the membranes for GCS and -actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C in TBST. Following three times washes in TBST, the membranes were incubated for 2 h at 37°C with secondary goat anti-rabbit IgG antibodies (1:2000, ZDR-5306) and goat anti-mouse IgG antibody (1:2000, ZDR-5307) labeled with horseradish peroxidase (all from Zhongshan Biotech-
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nology, Peking City, China). Immunoreactive straps were identified using the DAB system (DAB kit-0031; Maixin Biotechnology, Fuzhou City, China), as directed by the manufacturer. Specific bands for GCS and -actin were identified by prestained protein molecular weight marker (SM0441; MBI Fermentas, Glen Burnie, MD). The EC3 Imaging System (UVP Inc., Upland, CA) was used to catch up the specific bands, and the optical density of each band was measured using Image J software, Shenyang city, China. The ratio between the optical density of interest proteins and -actin of the same sample was calculated as relative content and expressed graphically. 2.4. Statistical analysis Values were expressed as mean ⫾ SD. Statistical analyses were done with SPSS 13.0 software, Shenyang city, China. Associations among the variables were assessed by the 2 test and Student’s t-test. Survival rates were calculated by the Kaplan-Meier method. Differences between survival curves were analyzed by the log-rank test. Differences were considered significant at P ⬍ 0.05.
3. Results 3.1. GCS expression and localization in bladder neoplasms by immunohistochemistry GCS immunoreactivity was detected in both normal and tumorous bladder cells. In normal bladder tissues, GCS expression was observed, while GCS immunostaining was observed in every neoplastic tissue as shown in Fig. 1. We considered that 46 of the bladder neoplasms (61%) were higher expression (scores of 6 or 9); 29 cases (39%) were lower expression (scores of 0, 1, 2, 3, or 4), as described above in the Materials and Methods section. Fig. 1 shows that the expression of GCS located in the cytoplasm and stroma. 3.2. GCS expressions in BNs by Western blotting Western blotting was used to evaluate GCS expression in 24 BNs and paired non-tumorous bladder tissues distant from the primary tumor of the same case. Increased GCS expression was found in 15 BNs samples in comparison with the non-tumorous counterparts. Western blotting of 8 samples is shown in Fig. 2A, and the optical densities of the tumorous (T) and non-tumorous (N) tissues of the same patient were measured and expressed graphically (Fig. 2B). Comparison of GCS expression was made between BNs with and without lymph node metastasis statistically. GCS expression was significantly higher in node-positive BNs (Fig. 2B).
Fig. 1. GCS expression by immunohistochemistry. (A) GCS immunostaining in the normal epithelial cells of bladder. (B) GCS expression in the transitional carcinoma cells with sentinel node negative. (Color version of figure is available online).
3.3. Clinicopathologic features of BNs and the prevalence of GCS high expressions We analyzed the correlation between clinicopathologic features of BNs and the prevalence of GCS expressions in BNs (Table 2). Patients with BNs were divided into 2 groups by the median value for the prevalence of GCS expressions. The high expression group (n ⫽ 46) showed a significant correlation with high histologic grade (P ⫽ 0.021) and tended to show (P ⫽ 0.045) that up-expression of GCS was positive related to BNs with lymph node metastasis among the various clinicopathologic characteristics.
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Fig. 2. (A) Expression of GCS by Western blotting in matched tumorous (T) and non-tumorous (N) tissues from 8 of 24 BN patients, and 4 of which were accompanied with lymph node metastasis (the lower panel). Band intensities indicate significant GCS up-regulation in tumorous in comparison with the non-tumorous tissue of the same patient. Furthermore, patients with positive nodes expressed higher level of GCS in tumor. -Actin was used as a loading control to assure equal amounts of protein in all lanes. (B) The ratio between the optical density of GCS and -actin of the same patient was calculated and expressed graphically. Significant differences of GCS expression between tumorous (T) and non-tumorous (N) tissues as well as neoplastic samples with positive and negative nodes were analyzed statistically. GCS immunoreactivity is greater in neoplastic tissues (P ⫽ 0.000) and cases with positive nodes (P ⫽ 0.018).
3.4. Prognostic significance of the expressions of GCS
4. Discussion
Overall and disease-free survivals were analyzed in these patients. The overall 5-year survival and disease-free survival rates were 39.5% and 18.4%, respectively. The lowexpression of GCS group showed significantly better overall survival than the high-expression group (log-rank test, P ⫽ 0.007; Fig. 3A). Mean overall survival was 60.3 (F33.8) months for the low-expression group and 45.1 (F38.7) months for the high-expression group. The low-expression group also showed significantly better disease-free survival than the high-expression group (log-rank test, P ⫽ 0.015; Fig. 3B). Mean disease-free survival was 36.2 (F31.7) months for the low-expression group and 27.3 (F32.9) months for the high-expression group.
GCS is a transmembrane protein with the C-terminal catalytic domain located in the cytoplasm [16]. GCS transfers a glucose residue from UDP-glucose to ceramide for the synthesis of GC. This mainly occurs on the cytoplasmic surface of the Golgi [17]. In the Golgi lumen, GC is further modified by a series of glycosyltransferases that produce higher order glycosphingolipids. Glycosphingolipids are composed of a group of membrane lipids in which the lipid portion is embedded in the outer leaflet of the plasma membrane with the sugar chain extending to the extracellular space. Glycosphingolipids are integral components of plasma membrane microdomains known as rafts, caveolae, and glycosignaling domains that are rich in sphingolipids and cholesterol [18,19]. These lipid domains assemble receptors and glycosyl-phosphatidylinositol anchored proteins on their external surface and signaling molecules, including Src family kinases, G proteins, and nitric oxide synthase, on their internal surface. Some studies showed that overexpression of GCS by gene transfection conferred cellular resistance to chemotherapy and to tumor necrosis factor-␣ [13,20]. Moreover, Inhibition of GCS activity is being evaluated as a possible treatment for several lipid-storage diseases and some types of cancer [21,22]. This study evaluated GCS expression in BNs, with regard to the depth of tumor invasion, histologic type, differentiation, stage, and lymph node status of BNs, to determine the clinical significance of GCS for the advanced BNs. We examined 75 tumors by means of immunohistochemistry, 24 of which were also analyzed by Western blotting, and found a statistical evidence of GCS up-regulation in BNs. Weak-moderate GCS immunostaining was observed in the normal transitional cells, as well as part of BNs. We also found that overexpression of GCS was common in BNs, regardless of the histologic type. However, patients with higher GCS expression had a significant metastatic phenotype. The immunohistochemical obser-
Table 2 Correlation between clinicopathologic findings and the prevalence of the expression of GCS Variables
Age, y (mean ⫾ SD) Sex (male/female) Depth of tumor invasiveness (Tis-T1/T2/T3/T4) Sentinel node (positive/negative) Histologic type (tcc/others) Differentiation (well/moderate/poor) Style of surgery (Tur-B/PR)
Expressions of GCS High
Low
62.4 ⫾ 5.9 42/4 28/16/2/0
70.5 ⫾ 9.2 16/13 17/12/0/0
P
0.514** 0.907* 0.105*
13/33
3/26
0.045*
44/2 36/7/3
28/1 28/1/0
0.524* 0.021*
26/20
25/4
0.075*
BN ⫽ bladder neoplasm; tcc ⫽ transitional cell carcinoma; Tur-B ⫽ transurethral resection of bladder tumor; PR ⫽ partial resection. * 2 test or Fisher’s exact test. ** Student’s t-test.
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Fig. 3. Overall survival curve (A) and disease-free survival curve (B) are shown. The prognosis was significantly worse in the high expression group than in the low expression group [log-rank test, P ⫽ 0.007 (A) and P ⫽ 0.015 (B)]. (Color version of figure is available online.)
vations might be further supported by our semiquantitative Western blotting evaluations of GCS expression in 24 tumorous and paired non-tumorous counterparts. GCS expressions were significantly higher in the neoplastic than the non-neoplastic tissues. Furthermore, in the present study, we investigated the relationship between clinicopathologic features of BNs and the prevalence of GCS expressions in BNs. Patients with BNs in the high-expression of GCS group showed a significantly lower survival ratio. Both overall survival (log-rank test, P ⫽ 0.007) and disease-free survival (log-rank test, P ⫽ 0.015) were lower than for patients with BNs belonging to the low-
expression group. These findings suggest that high expression of GCS was closely correlated with the progression of neoplastic cells in BNs; moreover, the options of surgical styles should be decided more aggressively for cases with GCS high expression. To our knowledge, this is the first report to show that high expression of GCS is closely correlated with the development and progression of BNs, and may be a useful prognostic factor in patients with BNs. In conclusion, our data suggested that besides correlating with multidrug resistance, GCS also played a role in controlling the progression of neoplastic cells in BNs, and that up-regulation of GCS might make an aggressive choice of
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surgical therapy. A high expression of GCS seemed to be an indicator of poor prognosis.
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