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MP88-06 ANTICANCER AND CHEMOSENSITIZING EFFECTS OF 2-DEOXYGLUCOSE TARGETING GLYCOLYSIS IN HUMAN BLADDER CANCER CELLS
THE JOURNAL OF UROLOGYâ
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induced cell cycle changes. Further, compared to mock transfected UMUC3 cells, G1P3 knockdown resulted in decreased cell viability, and it prevented HTB9 colony growth. CONCLUSIONS: G1P3 may play a role in cisplatin-resistance and proliferation in bladder cancer cells through suppression of apoptotic pathways. Source of Funding: The Leo and Anne Albert Institute for Bladder Cancer Research
MP88-04 ANTI-TUMOR EFFECT OF NEW TYROSIN KINASE INHIBITORS (BOSUTINIB, NINTEDANIB, VATALANIB) IN HUMAN BLADDER CANCER CELLS Kwangmo Kim*, Beyong do Song, Jin-Nyoung Ho, Jong Jin Oh, Sung Kyu Hong, Sang Eun Lee, Seok-Soo Byun, Sang Chul Lee, Seong-Jin Jeong, Seongnam, Korea, Republic of INTRODUCTION AND OBJECTIVES: Angiogenesis is known to play a major role in the pathogenesis of bladder cancer. Bosutinib is an orally bioavailable dual Src/Abl tyrosin kinase inhibitor (TKI). Nintedanib and vatalanib are TKI that inhibits the vascular endothelial growth factor receptor (VEGFR) and the platelet-derived growth factor receptor (PDGFR), has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. We examined anti-tumor effects of bosutinib, nintedanib, and vatalanib in human bladder cancer cells (T24, cisplatinresistant-T24R2). METHODS: Bladder cancer cells were treated with bosutinib, nintedanib, and vatalanib and cancer cell proliferation was determined using the cell counting kit-8 (CCK-8) assay and clonogenic assay. Cell cycle and apoptosis rate were measured by flow cytometry. Apoptosisand cell cycle-related protein expression was analyzed by Western blot assays. RESULTS: Bladder cancer cell proliferation and colony formation was significantly reduced by bosutinib and vatalanib treatment in a dose-dependent manner. Bosutinib, nintedanib, and vatalanib induced apoptosis and cell cycle arrest at G1 phase. Expression of apoptosis related proteins (caspase-3, -8, -9, PARP, cytochrome c, bad, and bax) was elevated by bosutinib, nintedanib, and vatalanib treatment. CONCLUSIONS: In conclusion, new TKI (bosutinib, nintedanib, and vatalanib) showed anticancer effect in bladder cancer cells through induce apoptosis and cell cycle arrest. These data suggest that bosutinib, nintedanib, and vatalanib may be a novel therapeutic approach in the bladder cancer.
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
INTRODUCTION AND OBJECTIVES: Osteopontin (OPN) is a secreted extracellular matrix protein known to play a key role in tissue remodeling, inflammation and tumor development. In a laser capture microdissection-coupled microarray gene expression analysis of chemoresistant vs. non-chemoresistant bladder tumor samples, OPN was found be the most highly differentially (24-fold) expressed gene. We sought to explore the role of OPN in bladder cancer chemoresistance. METHODS: Bladder cancer cell lines (HTB4, HTB5, HTB9, UMUC3) were cultured in ATCC defined media. Total RNA was extracted and qPCR was performed. OPN protein expression was analyzed in cell lysates by Western Blotting and in culture medium by ELISA. Transient transfections with OPN gene-specific siRNAs were performed. Apoptosis and cell cycle analysis were evaluated by flow cytometry. Clonogenic survival and XTT-based cell viability assays were also performed. RESULTS: OPN transcript and protein expression in cell lysates and culture medium were observed only in HTB9 cell line. Treatment of HTB9 cells with cisplatin increased OPN gene expression by 29%. siRNA knockdown of OPN expression in HTB9 cells increased the cisplatin (3 mM) treated apoptotic cells by 28% compared to mocktransfected cells at 24h. Similarly, a dose dependent decrease in cell viability was observed in HTB9 cells with cisplatin treatment (0.1 - 3 mM) with OPN knockdown. Correspondingly, cell cycle analyses in OPN knockdown cells showed a 12% decrease in S-phase and a 39% increase in G0-G1 phase after 48h incubation with 3 mM cisplatin. In a clonogenic assay, cisplatin treatment decreased the number of colonies in a dose-dependent fashion and silencing of OPN expression completely inhibited the colony-forming ability. In silico analysis of OPN expression in the Oncomine database showed 3-8 fold upregulated expression in infiltrating bladder urothelial carcinoma vs. superficial bladder cancer. CONCLUSIONS: These findings suggest that inhibiting OPN expression sensitizes bladder cancer cells to cisplatin by conferring a greater propensity for G1 arrest and inhibiting colony formation. Therefore, upregulated OPN expression may contribute to the development of chemoresistance in bladder cancer. Source of Funding: The Leo & Anne Albert Institute for Bladder Cancer Research
MP88-06 ANTICANCER AND CHEMOSENSITIZING EFFECTS OF 2-DEOXYGLUCOSE TARGETING GLYCOLYSIS IN HUMAN BLADDER CANCER CELLS Vladimir Valera*, Derek Prabharasuth, Jonathan Bloom, John Phillips, Muhammad Choudhury, Sensuke Konno, Valhalla, NY
Source of Funding: none
MP88-05 OSTEOPONTIN e A POTENTIAL MARKER FOR CHEMOTHERAPY RESISTANCE IN BLADDER CANCER Nora Gibson*, Dharamainder Choudhary, Carol Pilbeam, John Taylor III, Farmington, CT
INTRODUCTION AND OBJECTIVES: Intravesical administration of BCG is currently the most effective immunotherapy for superficial bladder cancer. However, due to its severe side effects, an alternative intervention with few side effects needs to be established. The glycolytic pathway is considered a strategic target and has been often investigated in various cancers. Particularly, 2-deoxyglucose (2DG), a glycolysis inhibitor, has been intensely studied and shown to be well tolerated by patients. Accordingly, we investigated anticancer effect of 2DG on bladder cancer cells in vitro, implying its potential intravesical application. METHODS: The human bladder cancer 5637 cells were cultured with varying concentrations of 2DG (0-5 mM) and cell viability was assessed by the MTT assay in 72 h. The anticancer mechanism of 2DG was explored examining activity of key glycolytic enzyme (glyceraldehyde-3-phosphate dehydrogenase, G3PDH), ATP synthesis, specific signaling pathways, and induction of apoptosis. Additionally, whether 2DG could improve anticancer effect of doxorubicin (DOX) was also assessed for its possible chemosensitizing effect.
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
e1131
RESULTS: 2DG treatment for 72 h led to a 50-80% reduction in cell viability in a dose-dependent manner. G3PDH activity and the cellular ATP level were significantly decreased by ~22% and ~47% with 24-h 2DG treatment, respectively. Coupled with such a reduced ATP level, AMP-activated protein kinase (AMPK) was activated while protein kinase B (Akt) opposed to AMPK was inactivated and concomitantly mammalian target of rapamycin (mTOR) was inhibited. These results may account for the significantly reduced cell viability by 2DG. Glucose-regulated protein 78 (GRP78) was also downregulated, indicating cells under endoplasmic reticulum (ER) stress. Additionally, enzymatic activities of caspases-3 and 9 increased by ~2.7- and ~3.5-fold by 2DG, respectively, indicating induction of apoptosis. Moreover, 2DG was capable of sensitizing or enhancing anticancer activity of DOX when combined, resulting in a >70% cell viability reduction. CONCLUSIONS: This study demonstrates that 2DG has anticancer activity on bladder cancer cells and its anticancer mechanism involves the complex cellular events, including glycolysis inhibition, modulation of several signaling pathways, ER stress, and induction of apoptosis. Thus, 2DG appears to be an anticancer agent by itself as well as an adjuvant agent sensitizing certain chemotherapeutic drugs, providing us an alternative option for intravesical therapy for superficial bladder cancer. Source of Funding: Seize the Ribbon Source of Funding: none
MP88-07 RSPH9 METHYLATION PATTERN AS A PROGNOSTIC INDICATOR IN PATIENTS WITH NON-MUSCLE INVASIVE BLADDER CANCER Yong-June Kim*, Ho-Won Kang, Sung Phil Seo, Cheonju, Korea, Republic of; Hoon Jang, Daejeon, Korea, Republic of; Tongwook Kim, Cheongju, Korea, Republic of; Won Tae Kim, Seok Joong Yun, Sang-Cheol Lee, Wun-Jae Kim, Cheonju, Korea, Republic of INTRODUCTION AND OBJECTIVES: DNA methylation is a frequent and early epigenetic event with potential application as a biomarker for cancer detection and an indicator of disease evolution. The aim of the present study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation in samples from long-term follow-up patients with nonmuscle invasive bladder cancer (NMIBC). METHODS: Candidate methylation markers were selected from our previous published genome-wide methylation profiles. The clinical relevance of candidate methylation markers was determined by quantitative pyrosequencing analysis of 136 human bladder specimens [8 normal controls (NC) and 128 NMIBCs]. RESULTS: The methylation patterns of candidate markers were significantly associated with aggressive clinicopathological features. In multivariate regression analysis, hypermethylation of radial spoke head 9 homolog (RSPH9) was an independent predictor of disease recurrence (hazard ratio, 3.02; p ¼ 0.001) and progression (hazard ratio, 8.25; p ¼ 0.028). CONCLUSIONS: RSPH9 methylation is an independent prognostic indicator in NMIBC patients, and could be of value for the assessment of disease recurrence and progression and for clinical decision making regarding treatment.
MP88-08 GPX2 HAS THERAPEUTIC POTENTIAL THROUGH THE REGULATION OF OXIDATIVE STRESS IN BLADDER CANCER Taku Naiki*, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Kenjiro Kohri, Satoru Takahashi, Takahiro Yasui, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against the damage caused by ROS in the human body. In this study, we investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: (i) Glutathione peroxidase 2 (GPX2) and antigen Ki67 (KI67) protein expression was analyzed by immunohistochemistry in human radical cystectomy specimens. (ii) F344 rats were given either 0.05% BBN in drinking water or 0.001 to 0.1% PEITC in their diet for 36 weeks. Bladder tissue samples were collected from each animal for histopathological and protein expression analyses. (iii) The rat bladder and human cancer cell lines were used. GPX2 small interfering RNA (siRNA) and negative control siRNA (NC) were used to transfect T24 and BC31 cells. After transfection, we investigated the proliferation rates and ROS levels by employing cell counts, DCFH assay, western blotting, and flow cytometry. (iv) siRNA or NC transfected BC31 cells diluted in a buffered solution were subcutaneously implanted into nude mice. Three weeks after implantation, the mice were sacrificed, and the subcutaneous tumors were isolated. RESULTS: (i) GPX2 was strongly expressed in low grade and low MIB1 index cancer on analyses of the radical cystectomy specimen. (ii) In the analyses using invasive bladder cancer animal model, Gpx2 expression in the normal epithelium of the control group was significantly lower than in that of the treated group with dysplasia and urothelial carcinoma. (iii) BC31 and RT4 had strong expression of GPX2 compared with other cell lines. Silencing of GPX2 caused significant growth inhibition and the DCFH assay revealed that the intracellular ROS levels were significantly decreased in the siRNA treated groups. The death mechanism was revealed to be caspase dependent apoptosis. (iv) On BC31 implantation in nude mice using the siRNA, tumor growth of BC31 was significantly inhibited.
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induced cell cycle changes. Further, compared to mock transfected UMUC3 cells, G1P3 knockdown resulted in decreased cell viability, and it prevented HTB9 colony growth. CONCLUSIONS: G1P3 may play a role in cisplatin-resistance and proliferation in bladder cancer cells through suppression of apoptotic pathways. Source of Funding: The Leo and Anne Albert Institute for Bladder Cancer Research
MP88-04 ANTI-TUMOR EFFECT OF NEW TYROSIN KINASE INHIBITORS (BOSUTINIB, NINTEDANIB, VATALANIB) IN HUMAN BLADDER CANCER CELLS Kwangmo Kim*, Beyong do Song, Jin-Nyoung Ho, Jong Jin Oh, Sung Kyu Hong, Sang Eun Lee, Seok-Soo Byun, Sang Chul Lee, Seong-Jin Jeong, Seongnam, Korea, Republic of INTRODUCTION AND OBJECTIVES: Angiogenesis is known to play a major role in the pathogenesis of bladder cancer. Bosutinib is an orally bioavailable dual Src/Abl tyrosin kinase inhibitor (TKI). Nintedanib and vatalanib are TKI that inhibits the vascular endothelial growth factor receptor (VEGFR) and the platelet-derived growth factor receptor (PDGFR), has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. We examined anti-tumor effects of bosutinib, nintedanib, and vatalanib in human bladder cancer cells (T24, cisplatinresistant-T24R2). METHODS: Bladder cancer cells were treated with bosutinib, nintedanib, and vatalanib and cancer cell proliferation was determined using the cell counting kit-8 (CCK-8) assay and clonogenic assay. Cell cycle and apoptosis rate were measured by flow cytometry. Apoptosisand cell cycle-related protein expression was analyzed by Western blot assays. RESULTS: Bladder cancer cell proliferation and colony formation was significantly reduced by bosutinib and vatalanib treatment in a dose-dependent manner. Bosutinib, nintedanib, and vatalanib induced apoptosis and cell cycle arrest at G1 phase. Expression of apoptosis related proteins (caspase-3, -8, -9, PARP, cytochrome c, bad, and bax) was elevated by bosutinib, nintedanib, and vatalanib treatment. CONCLUSIONS: In conclusion, new TKI (bosutinib, nintedanib, and vatalanib) showed anticancer effect in bladder cancer cells through induce apoptosis and cell cycle arrest. These data suggest that bosutinib, nintedanib, and vatalanib may be a novel therapeutic approach in the bladder cancer.
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
INTRODUCTION AND OBJECTIVES: Osteopontin (OPN) is a secreted extracellular matrix protein known to play a key role in tissue remodeling, inflammation and tumor development. In a laser capture microdissection-coupled microarray gene expression analysis of chemoresistant vs. non-chemoresistant bladder tumor samples, OPN was found be the most highly differentially (24-fold) expressed gene. We sought to explore the role of OPN in bladder cancer chemoresistance. METHODS: Bladder cancer cell lines (HTB4, HTB5, HTB9, UMUC3) were cultured in ATCC defined media. Total RNA was extracted and qPCR was performed. OPN protein expression was analyzed in cell lysates by Western Blotting and in culture medium by ELISA. Transient transfections with OPN gene-specific siRNAs were performed. Apoptosis and cell cycle analysis were evaluated by flow cytometry. Clonogenic survival and XTT-based cell viability assays were also performed. RESULTS: OPN transcript and protein expression in cell lysates and culture medium were observed only in HTB9 cell line. Treatment of HTB9 cells with cisplatin increased OPN gene expression by 29%. siRNA knockdown of OPN expression in HTB9 cells increased the cisplatin (3 mM) treated apoptotic cells by 28% compared to mocktransfected cells at 24h. Similarly, a dose dependent decrease in cell viability was observed in HTB9 cells with cisplatin treatment (0.1 - 3 mM) with OPN knockdown. Correspondingly, cell cycle analyses in OPN knockdown cells showed a 12% decrease in S-phase and a 39% increase in G0-G1 phase after 48h incubation with 3 mM cisplatin. In a clonogenic assay, cisplatin treatment decreased the number of colonies in a dose-dependent fashion and silencing of OPN expression completely inhibited the colony-forming ability. In silico analysis of OPN expression in the Oncomine database showed 3-8 fold upregulated expression in infiltrating bladder urothelial carcinoma vs. superficial bladder cancer. CONCLUSIONS: These findings suggest that inhibiting OPN expression sensitizes bladder cancer cells to cisplatin by conferring a greater propensity for G1 arrest and inhibiting colony formation. Therefore, upregulated OPN expression may contribute to the development of chemoresistance in bladder cancer. Source of Funding: The Leo & Anne Albert Institute for Bladder Cancer Research
MP88-06 ANTICANCER AND CHEMOSENSITIZING EFFECTS OF 2-DEOXYGLUCOSE TARGETING GLYCOLYSIS IN HUMAN BLADDER CANCER CELLS Vladimir Valera*, Derek Prabharasuth, Jonathan Bloom, John Phillips, Muhammad Choudhury, Sensuke Konno, Valhalla, NY
Source of Funding: none
MP88-05 OSTEOPONTIN e A POTENTIAL MARKER FOR CHEMOTHERAPY RESISTANCE IN BLADDER CANCER Nora Gibson*, Dharamainder Choudhary, Carol Pilbeam, John Taylor III, Farmington, CT
INTRODUCTION AND OBJECTIVES: Intravesical administration of BCG is currently the most effective immunotherapy for superficial bladder cancer. However, due to its severe side effects, an alternative intervention with few side effects needs to be established. The glycolytic pathway is considered a strategic target and has been often investigated in various cancers. Particularly, 2-deoxyglucose (2DG), a glycolysis inhibitor, has been intensely studied and shown to be well tolerated by patients. Accordingly, we investigated anticancer effect of 2DG on bladder cancer cells in vitro, implying its potential intravesical application. METHODS: The human bladder cancer 5637 cells were cultured with varying concentrations of 2DG (0-5 mM) and cell viability was assessed by the MTT assay in 72 h. The anticancer mechanism of 2DG was explored examining activity of key glycolytic enzyme (glyceraldehyde-3-phosphate dehydrogenase, G3PDH), ATP synthesis, specific signaling pathways, and induction of apoptosis. Additionally, whether 2DG could improve anticancer effect of doxorubicin (DOX) was also assessed for its possible chemosensitizing effect.
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
e1131
RESULTS: 2DG treatment for 72 h led to a 50-80% reduction in cell viability in a dose-dependent manner. G3PDH activity and the cellular ATP level were significantly decreased by ~22% and ~47% with 24-h 2DG treatment, respectively. Coupled with such a reduced ATP level, AMP-activated protein kinase (AMPK) was activated while protein kinase B (Akt) opposed to AMPK was inactivated and concomitantly mammalian target of rapamycin (mTOR) was inhibited. These results may account for the significantly reduced cell viability by 2DG. Glucose-regulated protein 78 (GRP78) was also downregulated, indicating cells under endoplasmic reticulum (ER) stress. Additionally, enzymatic activities of caspases-3 and 9 increased by ~2.7- and ~3.5-fold by 2DG, respectively, indicating induction of apoptosis. Moreover, 2DG was capable of sensitizing or enhancing anticancer activity of DOX when combined, resulting in a >70% cell viability reduction. CONCLUSIONS: This study demonstrates that 2DG has anticancer activity on bladder cancer cells and its anticancer mechanism involves the complex cellular events, including glycolysis inhibition, modulation of several signaling pathways, ER stress, and induction of apoptosis. Thus, 2DG appears to be an anticancer agent by itself as well as an adjuvant agent sensitizing certain chemotherapeutic drugs, providing us an alternative option for intravesical therapy for superficial bladder cancer. Source of Funding: Seize the Ribbon Source of Funding: none
MP88-07 RSPH9 METHYLATION PATTERN AS A PROGNOSTIC INDICATOR IN PATIENTS WITH NON-MUSCLE INVASIVE BLADDER CANCER Yong-June Kim*, Ho-Won Kang, Sung Phil Seo, Cheonju, Korea, Republic of; Hoon Jang, Daejeon, Korea, Republic of; Tongwook Kim, Cheongju, Korea, Republic of; Won Tae Kim, Seok Joong Yun, Sang-Cheol Lee, Wun-Jae Kim, Cheonju, Korea, Republic of INTRODUCTION AND OBJECTIVES: DNA methylation is a frequent and early epigenetic event with potential application as a biomarker for cancer detection and an indicator of disease evolution. The aim of the present study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation in samples from long-term follow-up patients with nonmuscle invasive bladder cancer (NMIBC). METHODS: Candidate methylation markers were selected from our previous published genome-wide methylation profiles. The clinical relevance of candidate methylation markers was determined by quantitative pyrosequencing analysis of 136 human bladder specimens [8 normal controls (NC) and 128 NMIBCs]. RESULTS: The methylation patterns of candidate markers were significantly associated with aggressive clinicopathological features. In multivariate regression analysis, hypermethylation of radial spoke head 9 homolog (RSPH9) was an independent predictor of disease recurrence (hazard ratio, 3.02; p ¼ 0.001) and progression (hazard ratio, 8.25; p ¼ 0.028). CONCLUSIONS: RSPH9 methylation is an independent prognostic indicator in NMIBC patients, and could be of value for the assessment of disease recurrence and progression and for clinical decision making regarding treatment.
MP88-08 GPX2 HAS THERAPEUTIC POTENTIAL THROUGH THE REGULATION OF OXIDATIVE STRESS IN BLADDER CANCER Taku Naiki*, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Kenjiro Kohri, Satoru Takahashi, Takahiro Yasui, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against the damage caused by ROS in the human body. In this study, we investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: (i) Glutathione peroxidase 2 (GPX2) and antigen Ki67 (KI67) protein expression was analyzed by immunohistochemistry in human radical cystectomy specimens. (ii) F344 rats were given either 0.05% BBN in drinking water or 0.001 to 0.1% PEITC in their diet for 36 weeks. Bladder tissue samples were collected from each animal for histopathological and protein expression analyses. (iii) The rat bladder and human cancer cell lines were used. GPX2 small interfering RNA (siRNA) and negative control siRNA (NC) were used to transfect T24 and BC31 cells. After transfection, we investigated the proliferation rates and ROS levels by employing cell counts, DCFH assay, western blotting, and flow cytometry. (iv) siRNA or NC transfected BC31 cells diluted in a buffered solution were subcutaneously implanted into nude mice. Three weeks after implantation, the mice were sacrificed, and the subcutaneous tumors were isolated. RESULTS: (i) GPX2 was strongly expressed in low grade and low MIB1 index cancer on analyses of the radical cystectomy specimen. (ii) In the analyses using invasive bladder cancer animal model, Gpx2 expression in the normal epithelium of the control group was significantly lower than in that of the treated group with dysplasia and urothelial carcinoma. (iii) BC31 and RT4 had strong expression of GPX2 compared with other cell lines. Silencing of GPX2 caused significant growth inhibition and the DCFH assay revealed that the intracellular ROS levels were significantly decreased in the siRNA treated groups. The death mechanism was revealed to be caspase dependent apoptosis. (iv) On BC31 implantation in nude mice using the siRNA, tumor growth of BC31 was significantly inhibited.