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229 INHIBITION OF THE GROWTH AND TUMOR ANGIOGENESIS OF RENAL CELL CARCINOMA BY THE STAT3 INHIBITOR WP1066
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
cyclical hypoxia/reoxygenation using 2% oxygen for 30 minutes followed by reoxygenation with 21% oxygen for one hour; cycling in this manner for 48 hours. After 48 hours, samples were collected and processed for fluorometric and enzyme immunoassay of cell injury and transmission electron microscopy. RESULTS: Lipid peroxidation was found in cells exposed to hypoxia and oxidative stress while protein oxidation was evident in cellular oxidative stress only. The levels of oxidatively modified products increased after cell exposure to oxidative stress while increased nitrosative products were evident in both hypoxic and oxidative stress conditions. Cellular antioxidant capacity decreased in oxidative stress but remained unchanged in hypoxia. Forty-eight hours of hypoxia and oxidative stress had no significant effect on cell senescence. Electron microscopy of cells showed thickened deformed cell membrane, swollen mitochondria and enlarged endoplasmic reticulum (ER) in cells exposed to hypoxia. Distorted partially lost cell membrane with increased caveolae, enlarged mitochondria with degraded or lost cristae, splintered ER, and increased cytoplasmic lysosomes and multivesicular bodies were evident in oxidative stress. CONCLUSIONS: Human bladder smooth muscle cells are highly sensitive to hypoxic and oxidative stress conditions exhibiting specific biological and ultrastructural reactions. Bladder smooth muscle cell reactions to hypoxia are consistent with cell survival signaling to cope with lack of oxygen. Changes in oxidative stress correspond to extensive damage, cellular ultrastructural disintegration and deterioration of the subcellular elements. Source of Funding: A Merit Review grant from the Department of Veterans Affairs
228 ALTERATION IN CAVEOLAR ELEMENTS AFFECTS BLADDER FUNCTIONAL RESPONSES IN ANIMAL MODELS OF DETRUSOR OVERACTIVITY Vivian Cristofaro*, Samar K. Lowalekar, Subbarao V. Yalla, Maryrose P. Sullivan, Boston, MA INTRODUCTION AND OBJECTIVES: Loss of bladder smooth muscle (BSM) caveolae, membrane invaginations that modulate agonist-induced responses, may contribute to detrusor dysfunction. Reduction in caveolae was described in bladder outlet obstruction (BOO) and spontaneously hypertensive rats (SHR), two animal models with detrusor overactivity (DO). We investigated whether alteration in caveolar elements in BSM tissue from BOO and SHR results in abnormal regulation of contractile responses to physiologic agonists thus contributing to DO. METHODS: BOO was created by placing a ligature around the proximal urethra of male Sprague Dawley (SD) rats for 2 weeks. Adult male SHR were used as a model of non-obstructive DO. Sham operated animals and Wistar Kyoto (WKY) rats served as controls respectively. Bladder tissue from each animal was stretched in organ bath at 37°C and the contractile responses induced by phenylephrine (PE), bradykinin (BK), and carbachol (CCh) were measured before and after caveolar depletion achieved by methyl--cyclodextrin (mCD). The density of BSM caveolae and the distribution of caveolin (Cav) protein were compared among animals by electron microscopy and immunofluorescence respectively. RESULTS: BSM caveolar density was significantly lower in BOO and SHR compared with sham and WKY. Cav-1, and Cav-3 protein expression decreased in BOO and SHR compared to their respective controls. mCD significantly increased the contractile response to PE and BK in sham and WKY bladders, consistent with the negative regulation imparted by caveolae of these agonist responses. In BOO and SHR, the amplitudes of PE and BK contractions under baseline conditions were significantly higher compared to their respective controls, consistent with a loss of negative regulation due to the reduced caveolae. In addition, the effect of caveolar depletion on PE and BK responses in BOO and SHR was less effective. CCh-induced contractions were unaffected by mCD in bladders from both DO models as well as in controls.
THE JOURNAL OF UROLOGY姞
e93
CONCLUSIONS: In normal bladders, several agonist-induced responses are negatively regulated by caveolae. In overactive bladders from BOO and SHR, a loss of caveolar elements is associated with significant alteration of contractile responses, suggesting an impaired negative regulation of these receptor-mediated responses. A decrease in caveolar elements seems to occur with DO independently from its etiology (secondary to obstruction or idiopathic) and may be potentially related to the development of this dysfunction Source of Funding: Department of Veterans Affairs, Washington DC
Kidney Cancer: Basic Research Moderated Poster 10 Sunday, May 15, 2011
10:30 AM-12:30 PM
229 INHIBITION OF THE GROWTH AND TUMOR ANGIOGENESIS OF RENAL CELL CARCINOMA BY THE STAT3 INHIBITOR WP1066 Akio Horiguchi*, Takako Asano, Kenji Kuroda, Akinori Sato, Junichi Asakuma, Keiichi Ito, Masamichi Hayakawa, Makoto Sumitomo, Tomohiko Asano, Tokorozawa, Japan INTRODUCTION AND OBJECTIVES: Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes that mediate cell survival, proliferation, and angiogenesis and is aberrantly activated in various types of malignancies, including renal cell carcinoma. We used the STAT3 inhibitor WP1066 to see if STAT3 could be a therapeutic target for renal cell carcinoma. METHODS: Effects of WP1066 on the viability and proliferation of Caki-1 and 786-O human renal cancer cells were examined by cell counting and MTS assay. Apoptotic cells were assessed by double staining with FITC-conjugated annexin-V and propidium iodide. The effects of WP1066 on the intracellular signaling pathways were analyzed by western blotting. In vitro angiogenesis was assessed according to the formation of capillary-like structures when human umbilical vascular endothelial cells were co-cultured with human diploid fibroblasts. The antitumor efficacy of WP1066 was examined using Caki-1 cell xenografts. RESULTS: In Caki-1 and 786-O renal cancer cells, 5 M WP1066 prevented the phosphorylation of STAT3 and 2.5 M WP1066 inhibited cell survival and proliferation significantly (P ⬍ 0.01). WP1066 suppressed the expression of Bcl-2, induced apoptosis, and inhibited the basal and hypoxia-induced expressions of HIF1␣ and HIF2␣ as well as VEGF secretion into the cell culture medium. Human umbilical vascular endothelial cells co-cultured with media from WP1066-treated cells showed significantly reduced tubulogenesis (P ⬍ 0.05). Systemic oral administration of WP1066 to mice for 19 days inhibited the growth of Caki-1 xenograft tumors significantly (P ⬍ 0.05), and pathological analysis of xenografts of WP1066-treated mice showed decreased immunostaining of phosphorylated STAT3 and shorter CD34-positive vessels (P ⬍ 0.05). CONCLUSIONS: Our results suggest that using WP1066 to inhibit the STAT3 signaling pathway could be a novel therapeutic strategy against renal cell carcinoma. Source of Funding: None
cyclical hypoxia/reoxygenation using 2% oxygen for 30 minutes followed by reoxygenation with 21% oxygen for one hour; cycling in this manner for 48 hours. After 48 hours, samples were collected and processed for fluorometric and enzyme immunoassay of cell injury and transmission electron microscopy. RESULTS: Lipid peroxidation was found in cells exposed to hypoxia and oxidative stress while protein oxidation was evident in cellular oxidative stress only. The levels of oxidatively modified products increased after cell exposure to oxidative stress while increased nitrosative products were evident in both hypoxic and oxidative stress conditions. Cellular antioxidant capacity decreased in oxidative stress but remained unchanged in hypoxia. Forty-eight hours of hypoxia and oxidative stress had no significant effect on cell senescence. Electron microscopy of cells showed thickened deformed cell membrane, swollen mitochondria and enlarged endoplasmic reticulum (ER) in cells exposed to hypoxia. Distorted partially lost cell membrane with increased caveolae, enlarged mitochondria with degraded or lost cristae, splintered ER, and increased cytoplasmic lysosomes and multivesicular bodies were evident in oxidative stress. CONCLUSIONS: Human bladder smooth muscle cells are highly sensitive to hypoxic and oxidative stress conditions exhibiting specific biological and ultrastructural reactions. Bladder smooth muscle cell reactions to hypoxia are consistent with cell survival signaling to cope with lack of oxygen. Changes in oxidative stress correspond to extensive damage, cellular ultrastructural disintegration and deterioration of the subcellular elements. Source of Funding: A Merit Review grant from the Department of Veterans Affairs
228 ALTERATION IN CAVEOLAR ELEMENTS AFFECTS BLADDER FUNCTIONAL RESPONSES IN ANIMAL MODELS OF DETRUSOR OVERACTIVITY Vivian Cristofaro*, Samar K. Lowalekar, Subbarao V. Yalla, Maryrose P. Sullivan, Boston, MA INTRODUCTION AND OBJECTIVES: Loss of bladder smooth muscle (BSM) caveolae, membrane invaginations that modulate agonist-induced responses, may contribute to detrusor dysfunction. Reduction in caveolae was described in bladder outlet obstruction (BOO) and spontaneously hypertensive rats (SHR), two animal models with detrusor overactivity (DO). We investigated whether alteration in caveolar elements in BSM tissue from BOO and SHR results in abnormal regulation of contractile responses to physiologic agonists thus contributing to DO. METHODS: BOO was created by placing a ligature around the proximal urethra of male Sprague Dawley (SD) rats for 2 weeks. Adult male SHR were used as a model of non-obstructive DO. Sham operated animals and Wistar Kyoto (WKY) rats served as controls respectively. Bladder tissue from each animal was stretched in organ bath at 37°C and the contractile responses induced by phenylephrine (PE), bradykinin (BK), and carbachol (CCh) were measured before and after caveolar depletion achieved by methyl--cyclodextrin (mCD). The density of BSM caveolae and the distribution of caveolin (Cav) protein were compared among animals by electron microscopy and immunofluorescence respectively. RESULTS: BSM caveolar density was significantly lower in BOO and SHR compared with sham and WKY. Cav-1, and Cav-3 protein expression decreased in BOO and SHR compared to their respective controls. mCD significantly increased the contractile response to PE and BK in sham and WKY bladders, consistent with the negative regulation imparted by caveolae of these agonist responses. In BOO and SHR, the amplitudes of PE and BK contractions under baseline conditions were significantly higher compared to their respective controls, consistent with a loss of negative regulation due to the reduced caveolae. In addition, the effect of caveolar depletion on PE and BK responses in BOO and SHR was less effective. CCh-induced contractions were unaffected by mCD in bladders from both DO models as well as in controls.
THE JOURNAL OF UROLOGY姞
e93
CONCLUSIONS: In normal bladders, several agonist-induced responses are negatively regulated by caveolae. In overactive bladders from BOO and SHR, a loss of caveolar elements is associated with significant alteration of contractile responses, suggesting an impaired negative regulation of these receptor-mediated responses. A decrease in caveolar elements seems to occur with DO independently from its etiology (secondary to obstruction or idiopathic) and may be potentially related to the development of this dysfunction Source of Funding: Department of Veterans Affairs, Washington DC
Kidney Cancer: Basic Research Moderated Poster 10 Sunday, May 15, 2011
10:30 AM-12:30 PM
229 INHIBITION OF THE GROWTH AND TUMOR ANGIOGENESIS OF RENAL CELL CARCINOMA BY THE STAT3 INHIBITOR WP1066 Akio Horiguchi*, Takako Asano, Kenji Kuroda, Akinori Sato, Junichi Asakuma, Keiichi Ito, Masamichi Hayakawa, Makoto Sumitomo, Tomohiko Asano, Tokorozawa, Japan INTRODUCTION AND OBJECTIVES: Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes that mediate cell survival, proliferation, and angiogenesis and is aberrantly activated in various types of malignancies, including renal cell carcinoma. We used the STAT3 inhibitor WP1066 to see if STAT3 could be a therapeutic target for renal cell carcinoma. METHODS: Effects of WP1066 on the viability and proliferation of Caki-1 and 786-O human renal cancer cells were examined by cell counting and MTS assay. Apoptotic cells were assessed by double staining with FITC-conjugated annexin-V and propidium iodide. The effects of WP1066 on the intracellular signaling pathways were analyzed by western blotting. In vitro angiogenesis was assessed according to the formation of capillary-like structures when human umbilical vascular endothelial cells were co-cultured with human diploid fibroblasts. The antitumor efficacy of WP1066 was examined using Caki-1 cell xenografts. RESULTS: In Caki-1 and 786-O renal cancer cells, 5 M WP1066 prevented the phosphorylation of STAT3 and 2.5 M WP1066 inhibited cell survival and proliferation significantly (P ⬍ 0.01). WP1066 suppressed the expression of Bcl-2, induced apoptosis, and inhibited the basal and hypoxia-induced expressions of HIF1␣ and HIF2␣ as well as VEGF secretion into the cell culture medium. Human umbilical vascular endothelial cells co-cultured with media from WP1066-treated cells showed significantly reduced tubulogenesis (P ⬍ 0.05). Systemic oral administration of WP1066 to mice for 19 days inhibited the growth of Caki-1 xenograft tumors significantly (P ⬍ 0.05), and pathological analysis of xenografts of WP1066-treated mice showed decreased immunostaining of phosphorylated STAT3 and shorter CD34-positive vessels (P ⬍ 0.05). CONCLUSIONS: Our results suggest that using WP1066 to inhibit the STAT3 signaling pathway could be a novel therapeutic strategy against renal cell carcinoma. Source of Funding: None