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786 SIMULTANEOUS SIRNA-MEDIATED KNOCKDOWN OF FOUR ANTIAPOPTOTIC GENES IN BLADDER CANCER CELLS
Vol. 183, No. 4, Supplement, Monday, May 31, 2010
CONCLUSIONS: Our data suggest that miR-133a might function as a tumor suppressor through repression of oncogenic LASP1 expression in BC. These pathways may play a critical role in BC development and serve as a novel therapeutic target in BC. Source of Funding: the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B)20390427 and (C) 20591861, 2008.
784 SYNERGISTIC THERAPEUTIC EFFECT OF NF-B AND AP-1 FUNCTION INHIBITORS ON SUPPRESSION OF CELL GROWTH AGAINST HIGHLY AGGRESSIVE BLADDER CANCER CELLS Eriko Suzuki*, Eiji Kikuchi, Yutaka Horiguchi, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: NF-B is often constitutively activated in bladder carcinoma cells, which may cause difficulty in treatment. AP-1 may also increase malignant character. In the present study we assessed the cytotoxic effect of a novel NF-B inhibitor (dehydroxymethyl epoxyquinomycin, DHMEQ) and/or a strong AP-1 inhibitor (DTCM-glutarimide, DTCM-G) against bladder cancer cells with various levels of NF-B and AP-1 activity. METHODS: Activation of either NF-B or AP-1 was examined by EMSA in 7 bladder cancer cell lines (KU-7, KU-19-19, T24, 5637, UMUC-3, RT-4 and MBT-2). NF-B and AP-1 activities were evaluated by phosphorylation of p65 and c-Jun, respectively by Western blotting analysis. Cell proliferation was assessed by MTT assay. RESULTS: Among 7 bladder cancer cell lines tested, KU-7 and KU-1919 cells revealed constitutive activation of NF-B, while 3 including KU-1, KU-7 and T24 cells revealed constitutive activation of AP-1. Especially KU-7 cells showed potent NF-B and AP-1 activities. The binding of NF-B to DNA was completely inhibited after exposure to 10 g/mL of DHMEQ for 2 to 6 hr in KU-7 and KU-19-19 cells. These cell viabilities were significantly inhibited by DHMEQ in a dose-dependent manner. DTCM-G inhibited the DNA binding activity of AP-1 for 3 to 12 hr in KU-7 and T24 cells. The combination treatment of DHMEQ (10 g/ml) with DTCM-G (10 g/ml) resulted in a significantly higher (56%) inhibition of cell proliferation compared to treatment with DHMEQ (30%) alone or DTCM-G (32.4%) alone (p⬍0.001 for both). The synergistic effects were not observed in either KU-19-19 or T24 cells. Significant level of apoptosis was induced by both DHMEQ and DTCM-g when cells were treated in combination with Docetaxel. Compared to normal tissues, clinical samples of cancer tissues from patients with invasive bladder cancer revealed higher levels of AP-1 and NF-B activity. CONCLUSIONS: Aggressive bladder carcinoma tissues were found to show high NF-B and AP-1 activities. Combination of DHMEQ and newly discovered AP-1 inhibitor DTCM-G synergistically inhibited the growth of KU-7 bladder carcinoma cells. Dual blockade of cellular NF-B and AP-1 activities would be a novel approach for the treatment against highly aggressive bladder cancer. Source of Funding: None
785 COMPENSATORY TUMOR DEFENSES IN UROTHELIUM DURING TUMOR SUPPRESSOR DEFICIENCY: IMPLICATION ON THE REQUIREMENT OF MULTI-GENE DEFECTS IN INVASIVE CARCINOMA FORMATION Feng He, Lan Mo, New York, NY; Eva Lee, Irvine, CA; Herbert Lepor, Tung-Tien Sun, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Urothelium, one of the slowest proliferating epithelia, is under the tight control of multiple growth inhibitors that act as tumor barriers. Overcoming these barriers is considered a prerequisite to transform urothelial cells into full-fledged carcinomas. However, the identity of the barriers and precisely how they operate during tumorigenesis have not been elucidated.
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METHODS: We assessed the expression of key tumor suppressors in normal mouse urothelia using real-time PCR, in situ hybridization, immunohistochemical staining and Western blotting. We also examined the urothelial responses to tumor suppressor loss using urothelium-specific knockout mice. Additionally, we studied whether multi-gene defects were necessary for invasive urothelial tumor initiation. Finally, we determined whether the combinatorial molecular defects identified in mice have correlates in humans. RESULTS: Unlike p53 which was undetectable in normal mouse urothelium, all pRb family proteins (pRb, p107 and p130) were highly expressed in all urothelial layers. E2F1, a potent growth stimulator whose activity is suppressed by pRb, was expressed at a very low level. Functionally ablating pRb in mouse urothelium provoked a marked increase of p107 as well as p53 pathway components (p53 itself, p19, p21, Bak and Bax). Instead of proliferation, pRb-deficient urothelial cells underwent apoptosis, due to dual (p107 and p53) compensatory tumor defenses. Removal of p53 from pRb-deficient cells bypassed p53-pathway activation, leading to late-onset hyperplasia. Treating mice lacking both pRb and p53 with a bladder-specific carcinogen elicited high-frequency invasive urothelial carcinomas, where p107 was markedly down-regulated. These results suggest that combined deficiency of pRb, p107 and p53 is necessary for invasive urothelial tumorigenesis. A follow-up immunohistochemical survey identified human muscle-invasive urothelial carcinomas that had the same molecular defects as in mice. CONCLUSIONS: Our data reveal important mechanisms of urothelial cells in tumor defense and demonstrate a new concept for invasive urothelial tumorigenesis. They also suggest that a combination of pRb, p107 and p53 may be more accurate prognostic predictors for patients with muscle-invasive bladder cancer. Source of Funding: NIH, VA
786 SIMULTANEOUS SIRNA-MEDIATED KNOCKDOWN OF FOUR ANTIAPOPTOTIC GENES IN BLADDER CANCER CELLS Doreen Kunze*, Kai Kraemer, Susanne Fuessel, Marc-Oliver Grimm, Manfred P. Wirth, Dresden, Germany INTRODUCTION AND OBJECTIVES: An important event in tumor development is the deregulation of the apoptotic pathway which is mainly characterized by overexpression of the antiapoptotic genes BCL2, BCL2L1, BIRC5 and XIAP. The knockdown of these genes, e.g. by using siRNAs, may represent a promising approach in the treatment of bladder cancer (BCa). METHODS: Two siRNAs were selected against each target mentioned. EJ28 and J82 BCa cells were transfected lipid-mediated with 40nM of one siRNA or with combinations of four (10nM per siRNA, one against each target) or all eight (5nM each) siRNAs. Treatments with target-directed siRNAs were normalized to non-silencing-siRNA control. Cell count, cell cycle distribution (PI staining), apoptosis induction (annexin V/PI staining) as well as mRNA and protein expression levels of the targets (quantitative PCR, western blot) were examined 48h after transfection. Cellular viability was analyzed by WST-1 assay 96h after transfection. Furthermore, the effects of siRNA (mono- and multitarget) pretreatment followed by chemotherapy with mitomycin C or cisplatin were examined by WST 1 assay. RESULTS: Single siRNA treatment potently decreased mRNA expression levels of the targets in both cell lines. In EJ28 cells, BCL2 was reduced down to 26%, BCL2L1 down to 36%, BIRC5 down to 24% and XIAP down to 42%. The simultaneous inhibition of all four antiapoptotic genes resulted in mRNA downregulation of all targets. Using a combination of all eight siRNAs, BCL2 was reduced down to 51%, BCL2L1 down to 48%, BIRC5 down to 37% and XIAP down to 67% in EJ28 cells. Knockdown of BIRC5, alone and in combination treatments, induced G2/M cell cycle arrest and formation of multi-nucleic cells, consequently leading to a significant reduction in cell count. Strongest inhibition of cellular viability (approx. 30%) was observed in combina-
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tion treatments. Furthermore, simultaneous inhibition of all four targets sensitized EJ28 cells to subsequent chemotherapy. Similar results were obtained in J82 cells. CONCLUSIONS: Since tumor cells may bypass the inhibition of one gene the combined knockdown of antiapoptotic genes may be more suitable for BCa therapy. Multi-target inhibition of BCL2, BCL2L1, BIRC5 and XIAP reduced expression levels of all target genes, inhibited BCa cell proliferation and sensitized the BCa cells to subsequent chemotherapy. Source of Funding: Funding provided by Else Kro¨ ner Fresenius-Stiftung.
787 CIS-DICHLORODIAMMINEPLATINUM UPREGULATES ANGIOTENSIN TYPE 1 RECEPTOR AND ENHANCES THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCED BY ANGIOTENSIN II Nobuyuki Tanaka*, Akira Miyajima, Masanori Hasegawa, Suguru Shirotake, Takeo Kosaka, Eiji Kikuchi, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Cis-dichlorodiammineplatinum (CDDP) is commonly considered the most effective agent for the treatment of advanced urothelial cancer. However, despite a continuous search for an effective chemotherapy regimen using CDDP in combination with another drugs, the results thus far have not been satisfactory. We previously reported that angiotensin II type 1 receptor (AT1R) antagonist suppressed tumor growth and angiogenesis in bladder cancer through the prevention of vascular endothelial growth factor (VEGF) production induced by angiotensin II (AII). Furthermore, the combination therapy with CDDP and AT1R antagonist has been shown to enhance CDDP–induced cytotoxity in a mouse xenograft model. In the present study, we investigated whether the combination of CDDP and AT1R antagonist exhibits any synergic action. METHODS: The invasive bladder cancer cell lines T24, KU-1919, KU-1, and 5637 were used. Cytotoxicity in tumor cells was determined by WST assay and protein expression levels were evaluated by Western blotting and ELISA. RESULTS: The IC50 values of CDDP for 24 hours were 19.4M in T24, 29.6M in KU-19-19, 26.4M in KU-1, and 30.6M in 5637. AII and AT1R antagonist administration showed no direct cytotoxicity in any cell line, and AII or/and AT1R antagonist administration showed no synergic actions in CDDP-induced cytotoxicity. Exposure to 10M CDDP for 24 hours induced the upregulation of expression of AT1R in T24 (1.9 times), KU-19-19 (1.5 times), and KU-1 (1.3 times), however, no marked change was observed in 5637. Next, we investigated the expression levels of VEGF production in T24. Although AII-treated cells without CDDP showed no significant stimulation of VEGF production, VEGF production was significantly upregulated (8.7⫾1.1 pg/103 cells) with the combination of CDDP and AII compared with cells treated with CDDP only (6.3⫾0.8 pg/103 cells). The AT1R antagonist significantly inhibited AII-induced VEGF production in T24 cells (7.6⫾1.2 pg/103 cells). CONCLUSIONS: CDDP upregulated the expression of AT1R and enhanced VEGF production induced by AII. These results may provide clues to one of the mechanisms of CDDP resistance, and suggest that combination therapy consisting of CDDP and an AT1R antagonist may become a new modality for the treatment of CDDPresistant bladder cancer. Source of Funding: None
Vol. 183, No. 4, Supplement, Monday, May 31, 2010
788 PROTEOMIC AND EPIGENETIC CHARACTERISATION OF TUMOUR ASSOCIATED FIBROBLASTS (TAF) IN URINARY BLADDER CARCINOMA Astrid Enkelmann*, Joana Heinzelmann, Beatrice Stubendorff, Daniel Steinbach, Heiko Wunderlich, Kerstin Junker, Jena, Germany INTRODUCTION AND OBJECTIVES: Interaction of tumour cells and tumour stroma has a high impact on tumour growth and progression due to different mechanisms in which they are involved in e.g. cell proliferation and invasion. TAF as a part of the tumour stroma are known to be supportive for tumour genesis. First aim of this study is the proteomic characterisation of TAF in comparison to non-tumour fibroblasts. Further it is well known that microRNAs (miRNA) are crucial for regulation of translational gene expression. DNA methylation of CpG sites in the promoter region of genes are involved in regulation of tumour suppressor genes. Based on this knowledge second aim of the present work is the epigenetic characterisation of TAF of urinary bladder cancer. METHODS: TAF were isolated from cultured urinary bladder tumour specimen by treatment with EDTA and differential detachment steps. Non-tumour fibroblasts were isolated from foreskin and normal urinary bladder tissue. Analyses of protein patterns were carried out by SELDI-TOF-MS and bioinformatics. Furthermore total RNA was isolated (MirVana) to analyse the miRNA expression profile by miRNA array analysis (Milteny; Genepix Pro). After DNA isolation and enrichment by MethylCollector Ultra-Kit (Activemotif) CpG methylation array analysis was performed by using Agilent Microarrays. RESULTS: We developed a cell culture routine to isolate and subsequently cultivate TAF from primary material of urinary bladder carcinoma. SELDI-TOF-MS measurement reveals specific proteomic expression patterns of TAF compared to non-tumour fibroblasts. Microarray analyses indicated a significant down regulation of the expression levels of several miRNAs in TAF in comparison to non-tumour fibroblasts. Determining the methylation level of CpG sites of gene promoter regions revealed a specific methylation signature of TAF. CONCLUSIONS: The ability to obtain TAF from primary tumour material leads us to analyse the proteomic and epigenetic characteristics of TAF. Proteomic characterisation shows specific protein expression patterns in TAF. Furthermore differential miRNA expression profiles and specific methylation pattern were observed in TAF. Therefore, epigenetic regulation of protein expression in TAF seems more likely than genomic alterations. These results facilitate to understand the specific regulation mechanisms of the interaction of TAF and tumour cells. Source of Funding: IZKF University Hospitals Jena
789 NO SYNTHASE (ENOS4A/B) GENE POLYMORPHISM IS ASSOCIATED WITH TUMOR RECURRENCE AND PROGRESSION IN SUPERFICIAL BLADDER CANCER Canan Ku¨c¸u¨kgergin, Akin S. Amasyali*, Oner Sanli, Selcuk Erdem, Faruk Ozcan, Sule Seckin, Istanbul, Turkey INTRODUCTION AND OBJECTIVES: The aim of this study is to investigate the relationship between the distribution of endothelial NO synthase (eNOS4a/b) gene polymorphism and clinical features of superficial bladder cancer (SBC). METHODS: The present study includes healthy controls (n⫽ 143, mean age⫽ 63.1 years) and patients with a diagnosis of histopathologically confirmed SBC (n⫽ 111, mean age⫽ 64.5 years). Genotypes (aa, bb, ab) for eNOS4a/b gene polymorphisms were identified by polymerase chain reaction (PCR) analysis. Meanwhile, plasma nitrate and nitrite levels (NOx) were used to estimate the amounts of endogeneous NO formation for both groups of patients. Chi-square and Student’s t tests, and Mann-Whitney U tests were used as statistical analyses where appropriate.
CONCLUSIONS: Our data suggest that miR-133a might function as a tumor suppressor through repression of oncogenic LASP1 expression in BC. These pathways may play a critical role in BC development and serve as a novel therapeutic target in BC. Source of Funding: the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B)20390427 and (C) 20591861, 2008.
784 SYNERGISTIC THERAPEUTIC EFFECT OF NF-B AND AP-1 FUNCTION INHIBITORS ON SUPPRESSION OF CELL GROWTH AGAINST HIGHLY AGGRESSIVE BLADDER CANCER CELLS Eriko Suzuki*, Eiji Kikuchi, Yutaka Horiguchi, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: NF-B is often constitutively activated in bladder carcinoma cells, which may cause difficulty in treatment. AP-1 may also increase malignant character. In the present study we assessed the cytotoxic effect of a novel NF-B inhibitor (dehydroxymethyl epoxyquinomycin, DHMEQ) and/or a strong AP-1 inhibitor (DTCM-glutarimide, DTCM-G) against bladder cancer cells with various levels of NF-B and AP-1 activity. METHODS: Activation of either NF-B or AP-1 was examined by EMSA in 7 bladder cancer cell lines (KU-7, KU-19-19, T24, 5637, UMUC-3, RT-4 and MBT-2). NF-B and AP-1 activities were evaluated by phosphorylation of p65 and c-Jun, respectively by Western blotting analysis. Cell proliferation was assessed by MTT assay. RESULTS: Among 7 bladder cancer cell lines tested, KU-7 and KU-1919 cells revealed constitutive activation of NF-B, while 3 including KU-1, KU-7 and T24 cells revealed constitutive activation of AP-1. Especially KU-7 cells showed potent NF-B and AP-1 activities. The binding of NF-B to DNA was completely inhibited after exposure to 10 g/mL of DHMEQ for 2 to 6 hr in KU-7 and KU-19-19 cells. These cell viabilities were significantly inhibited by DHMEQ in a dose-dependent manner. DTCM-G inhibited the DNA binding activity of AP-1 for 3 to 12 hr in KU-7 and T24 cells. The combination treatment of DHMEQ (10 g/ml) with DTCM-G (10 g/ml) resulted in a significantly higher (56%) inhibition of cell proliferation compared to treatment with DHMEQ (30%) alone or DTCM-G (32.4%) alone (p⬍0.001 for both). The synergistic effects were not observed in either KU-19-19 or T24 cells. Significant level of apoptosis was induced by both DHMEQ and DTCM-g when cells were treated in combination with Docetaxel. Compared to normal tissues, clinical samples of cancer tissues from patients with invasive bladder cancer revealed higher levels of AP-1 and NF-B activity. CONCLUSIONS: Aggressive bladder carcinoma tissues were found to show high NF-B and AP-1 activities. Combination of DHMEQ and newly discovered AP-1 inhibitor DTCM-G synergistically inhibited the growth of KU-7 bladder carcinoma cells. Dual blockade of cellular NF-B and AP-1 activities would be a novel approach for the treatment against highly aggressive bladder cancer. Source of Funding: None
785 COMPENSATORY TUMOR DEFENSES IN UROTHELIUM DURING TUMOR SUPPRESSOR DEFICIENCY: IMPLICATION ON THE REQUIREMENT OF MULTI-GENE DEFECTS IN INVASIVE CARCINOMA FORMATION Feng He, Lan Mo, New York, NY; Eva Lee, Irvine, CA; Herbert Lepor, Tung-Tien Sun, Xue-Ru Wu*, New York, NY INTRODUCTION AND OBJECTIVES: Urothelium, one of the slowest proliferating epithelia, is under the tight control of multiple growth inhibitors that act as tumor barriers. Overcoming these barriers is considered a prerequisite to transform urothelial cells into full-fledged carcinomas. However, the identity of the barriers and precisely how they operate during tumorigenesis have not been elucidated.
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METHODS: We assessed the expression of key tumor suppressors in normal mouse urothelia using real-time PCR, in situ hybridization, immunohistochemical staining and Western blotting. We also examined the urothelial responses to tumor suppressor loss using urothelium-specific knockout mice. Additionally, we studied whether multi-gene defects were necessary for invasive urothelial tumor initiation. Finally, we determined whether the combinatorial molecular defects identified in mice have correlates in humans. RESULTS: Unlike p53 which was undetectable in normal mouse urothelium, all pRb family proteins (pRb, p107 and p130) were highly expressed in all urothelial layers. E2F1, a potent growth stimulator whose activity is suppressed by pRb, was expressed at a very low level. Functionally ablating pRb in mouse urothelium provoked a marked increase of p107 as well as p53 pathway components (p53 itself, p19, p21, Bak and Bax). Instead of proliferation, pRb-deficient urothelial cells underwent apoptosis, due to dual (p107 and p53) compensatory tumor defenses. Removal of p53 from pRb-deficient cells bypassed p53-pathway activation, leading to late-onset hyperplasia. Treating mice lacking both pRb and p53 with a bladder-specific carcinogen elicited high-frequency invasive urothelial carcinomas, where p107 was markedly down-regulated. These results suggest that combined deficiency of pRb, p107 and p53 is necessary for invasive urothelial tumorigenesis. A follow-up immunohistochemical survey identified human muscle-invasive urothelial carcinomas that had the same molecular defects as in mice. CONCLUSIONS: Our data reveal important mechanisms of urothelial cells in tumor defense and demonstrate a new concept for invasive urothelial tumorigenesis. They also suggest that a combination of pRb, p107 and p53 may be more accurate prognostic predictors for patients with muscle-invasive bladder cancer. Source of Funding: NIH, VA
786 SIMULTANEOUS SIRNA-MEDIATED KNOCKDOWN OF FOUR ANTIAPOPTOTIC GENES IN BLADDER CANCER CELLS Doreen Kunze*, Kai Kraemer, Susanne Fuessel, Marc-Oliver Grimm, Manfred P. Wirth, Dresden, Germany INTRODUCTION AND OBJECTIVES: An important event in tumor development is the deregulation of the apoptotic pathway which is mainly characterized by overexpression of the antiapoptotic genes BCL2, BCL2L1, BIRC5 and XIAP. The knockdown of these genes, e.g. by using siRNAs, may represent a promising approach in the treatment of bladder cancer (BCa). METHODS: Two siRNAs were selected against each target mentioned. EJ28 and J82 BCa cells were transfected lipid-mediated with 40nM of one siRNA or with combinations of four (10nM per siRNA, one against each target) or all eight (5nM each) siRNAs. Treatments with target-directed siRNAs were normalized to non-silencing-siRNA control. Cell count, cell cycle distribution (PI staining), apoptosis induction (annexin V/PI staining) as well as mRNA and protein expression levels of the targets (quantitative PCR, western blot) were examined 48h after transfection. Cellular viability was analyzed by WST-1 assay 96h after transfection. Furthermore, the effects of siRNA (mono- and multitarget) pretreatment followed by chemotherapy with mitomycin C or cisplatin were examined by WST 1 assay. RESULTS: Single siRNA treatment potently decreased mRNA expression levels of the targets in both cell lines. In EJ28 cells, BCL2 was reduced down to 26%, BCL2L1 down to 36%, BIRC5 down to 24% and XIAP down to 42%. The simultaneous inhibition of all four antiapoptotic genes resulted in mRNA downregulation of all targets. Using a combination of all eight siRNAs, BCL2 was reduced down to 51%, BCL2L1 down to 48%, BIRC5 down to 37% and XIAP down to 67% in EJ28 cells. Knockdown of BIRC5, alone and in combination treatments, induced G2/M cell cycle arrest and formation of multi-nucleic cells, consequently leading to a significant reduction in cell count. Strongest inhibition of cellular viability (approx. 30%) was observed in combina-
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tion treatments. Furthermore, simultaneous inhibition of all four targets sensitized EJ28 cells to subsequent chemotherapy. Similar results were obtained in J82 cells. CONCLUSIONS: Since tumor cells may bypass the inhibition of one gene the combined knockdown of antiapoptotic genes may be more suitable for BCa therapy. Multi-target inhibition of BCL2, BCL2L1, BIRC5 and XIAP reduced expression levels of all target genes, inhibited BCa cell proliferation and sensitized the BCa cells to subsequent chemotherapy. Source of Funding: Funding provided by Else Kro¨ ner Fresenius-Stiftung.
787 CIS-DICHLORODIAMMINEPLATINUM UPREGULATES ANGIOTENSIN TYPE 1 RECEPTOR AND ENHANCES THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCED BY ANGIOTENSIN II Nobuyuki Tanaka*, Akira Miyajima, Masanori Hasegawa, Suguru Shirotake, Takeo Kosaka, Eiji Kikuchi, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Cis-dichlorodiammineplatinum (CDDP) is commonly considered the most effective agent for the treatment of advanced urothelial cancer. However, despite a continuous search for an effective chemotherapy regimen using CDDP in combination with another drugs, the results thus far have not been satisfactory. We previously reported that angiotensin II type 1 receptor (AT1R) antagonist suppressed tumor growth and angiogenesis in bladder cancer through the prevention of vascular endothelial growth factor (VEGF) production induced by angiotensin II (AII). Furthermore, the combination therapy with CDDP and AT1R antagonist has been shown to enhance CDDP–induced cytotoxity in a mouse xenograft model. In the present study, we investigated whether the combination of CDDP and AT1R antagonist exhibits any synergic action. METHODS: The invasive bladder cancer cell lines T24, KU-1919, KU-1, and 5637 were used. Cytotoxicity in tumor cells was determined by WST assay and protein expression levels were evaluated by Western blotting and ELISA. RESULTS: The IC50 values of CDDP for 24 hours were 19.4M in T24, 29.6M in KU-19-19, 26.4M in KU-1, and 30.6M in 5637. AII and AT1R antagonist administration showed no direct cytotoxicity in any cell line, and AII or/and AT1R antagonist administration showed no synergic actions in CDDP-induced cytotoxicity. Exposure to 10M CDDP for 24 hours induced the upregulation of expression of AT1R in T24 (1.9 times), KU-19-19 (1.5 times), and KU-1 (1.3 times), however, no marked change was observed in 5637. Next, we investigated the expression levels of VEGF production in T24. Although AII-treated cells without CDDP showed no significant stimulation of VEGF production, VEGF production was significantly upregulated (8.7⫾1.1 pg/103 cells) with the combination of CDDP and AII compared with cells treated with CDDP only (6.3⫾0.8 pg/103 cells). The AT1R antagonist significantly inhibited AII-induced VEGF production in T24 cells (7.6⫾1.2 pg/103 cells). CONCLUSIONS: CDDP upregulated the expression of AT1R and enhanced VEGF production induced by AII. These results may provide clues to one of the mechanisms of CDDP resistance, and suggest that combination therapy consisting of CDDP and an AT1R antagonist may become a new modality for the treatment of CDDPresistant bladder cancer. Source of Funding: None
Vol. 183, No. 4, Supplement, Monday, May 31, 2010
788 PROTEOMIC AND EPIGENETIC CHARACTERISATION OF TUMOUR ASSOCIATED FIBROBLASTS (TAF) IN URINARY BLADDER CARCINOMA Astrid Enkelmann*, Joana Heinzelmann, Beatrice Stubendorff, Daniel Steinbach, Heiko Wunderlich, Kerstin Junker, Jena, Germany INTRODUCTION AND OBJECTIVES: Interaction of tumour cells and tumour stroma has a high impact on tumour growth and progression due to different mechanisms in which they are involved in e.g. cell proliferation and invasion. TAF as a part of the tumour stroma are known to be supportive for tumour genesis. First aim of this study is the proteomic characterisation of TAF in comparison to non-tumour fibroblasts. Further it is well known that microRNAs (miRNA) are crucial for regulation of translational gene expression. DNA methylation of CpG sites in the promoter region of genes are involved in regulation of tumour suppressor genes. Based on this knowledge second aim of the present work is the epigenetic characterisation of TAF of urinary bladder cancer. METHODS: TAF were isolated from cultured urinary bladder tumour specimen by treatment with EDTA and differential detachment steps. Non-tumour fibroblasts were isolated from foreskin and normal urinary bladder tissue. Analyses of protein patterns were carried out by SELDI-TOF-MS and bioinformatics. Furthermore total RNA was isolated (MirVana) to analyse the miRNA expression profile by miRNA array analysis (Milteny; Genepix Pro). After DNA isolation and enrichment by MethylCollector Ultra-Kit (Activemotif) CpG methylation array analysis was performed by using Agilent Microarrays. RESULTS: We developed a cell culture routine to isolate and subsequently cultivate TAF from primary material of urinary bladder carcinoma. SELDI-TOF-MS measurement reveals specific proteomic expression patterns of TAF compared to non-tumour fibroblasts. Microarray analyses indicated a significant down regulation of the expression levels of several miRNAs in TAF in comparison to non-tumour fibroblasts. Determining the methylation level of CpG sites of gene promoter regions revealed a specific methylation signature of TAF. CONCLUSIONS: The ability to obtain TAF from primary tumour material leads us to analyse the proteomic and epigenetic characteristics of TAF. Proteomic characterisation shows specific protein expression patterns in TAF. Furthermore differential miRNA expression profiles and specific methylation pattern were observed in TAF. Therefore, epigenetic regulation of protein expression in TAF seems more likely than genomic alterations. These results facilitate to understand the specific regulation mechanisms of the interaction of TAF and tumour cells. Source of Funding: IZKF University Hospitals Jena
789 NO SYNTHASE (ENOS4A/B) GENE POLYMORPHISM IS ASSOCIATED WITH TUMOR RECURRENCE AND PROGRESSION IN SUPERFICIAL BLADDER CANCER Canan Ku¨c¸u¨kgergin, Akin S. Amasyali*, Oner Sanli, Selcuk Erdem, Faruk Ozcan, Sule Seckin, Istanbul, Turkey INTRODUCTION AND OBJECTIVES: The aim of this study is to investigate the relationship between the distribution of endothelial NO synthase (eNOS4a/b) gene polymorphism and clinical features of superficial bladder cancer (SBC). METHODS: The present study includes healthy controls (n⫽ 143, mean age⫽ 63.1 years) and patients with a diagnosis of histopathologically confirmed SBC (n⫽ 111, mean age⫽ 64.5 years). Genotypes (aa, bb, ab) for eNOS4a/b gene polymorphisms were identified by polymerase chain reaction (PCR) analysis. Meanwhile, plasma nitrate and nitrite levels (NOx) were used to estimate the amounts of endogeneous NO formation for both groups of patients. Chi-square and Student’s t tests, and Mann-Whitney U tests were used as statistical analyses where appropriate.