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PHELLODENDRON AMURENSE BARK EXTRACT ENHANCES RADIOSENSITIVITY BY INHIBITION OF NF-KAPPA B IN TRANSGENIC ADENOCARCINOMA OF MOUSE PROSTATE MODEL AND HUMAN PROSTATE CANCER CELLS
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Tuesday, April 28, 2009
RESULTS: IL-18Bpa was expressed and secreted by the prostate cancer cell lines DU145 and PC3, but not by LNCaP and CWR22, upon interferon-G (IFN-G) stimulation. IFN-G-induced secretion of IL-18Bpa was enhanced by added TNF-A, IFN-A and IFN-B. The IL-18Bpa secreted from DU145 and PC3 functionally inhibited IL-18. Conditioned medium from IL-18Bpa-overexpressed PC3 cells suppressed CD8+ IFN-G+ cells and CD4+ cells and appeared to decrease TH1 cells in human peripheral blood cultures. Immunohistochemical analyses showed positive IL18Bpa staining in prostate cancer cells as well as in macrophages in radical prostatectomy specimens. Significant differences in post-DRE urinary IL-18Bpa levels (normalized by total protein) were found between cases with and without cancer on biopsy (P = 0.02) and serum IL-18Bpa levels correlated with Gleason score (P = 0.03). CONCLUSIONS: Our finding of elevated IL-18Bpa secretion from prostate cancer cells suggests an attempt by cancer to escape immune surveillance. IL-18Bpa merits further study as a marker of aggressive prostate cancer and as a therapeutic target. Source of Funding: NIH/NIDDK grant 1K23DK071262, Department of Defense grant PC041214 and NIH/NCI grant U24 CA115102
1342 PHELLODENDRON AMURENSE BARK EXTRACT ENHANCES RADIOSENSITIVITY BY INHIBITION OF NF-KAPPA B IN TRANSGENIC ADENOCARCINOMA OF MOUSE PROSTATE MODEL AND HUMAN PROSTATE CANCER CELLS Craig H Robson*, Manonmani Ganapathy, Gregory P Swanson, Mohan Natarajan, Niko Papanikolaou, Martha A Hanes, I-Tien Yeh, Rita Ghosh, Addanki P Kumar, San Antonio, TX INTRODUCTION AND OBJECTIVES: Evidence supports that the activation of nuclear factor-kappa B (NF-kB) by ionizing radiation in prostate cancer results in adaptive resistance to radiation therapy. Therefore, altering expression of NF-kB may enhance radiation-induced apoptosis. Phellodendron amurense bark extract has been shown to compromise transcriptional activity of NF-kB and several dependent genes such as Cyclin D1 and COX-2 resulting in inhibition of prostate cancer cell proliferation by inducing apoptosis. We investigated if intervention with a complex botanical in vivo would potentiate cytotoxic effects of radiotherapy. METHODS: Twenty-week-old transgenic adenocarcinoma of mouse prostate (TRAMP) mice (at which time they display invasive carcinoma) were fed with pelleted diet containing 600mg/ kg Phellodendron Amurense bark extract 6 weeks prior to undergoing radiation therapy. These animals (n=6) were compared to a group that received radiation alone. Efficacy was evaluated by histological analysis of prostate tissue at the termination of the experiment at 34 weeks. Mechanistic studies were conducted in tumor tissue as well as in human prostate cancer cells. RESULTS: Preliminary data demonstrate that majority of animals that received Phellodendron amurense bark extract for 6 weeks prior to radiation therapy had no overt cancer but exhibited features consistent with high grade prostatic intraepithelial neoplasia. In contrast animals that received radiation alone exhibited features consistent with well to poorly differentiated adenocarcinoma. Western blot analysis using whole cell extracts prepared from prostate tumors and PC3 cells showed decreased expression of p65. CONCLUSIONS: The current study demonstrates that Phellodendron amurense bark extract potentiates radiation-induced apoptosis and may work by inhibition of NF-kB. Supported by pilot funds through Cancer Therapy and Research Center (APK). Source of Funding: Pilot Funds through Cancer Therapy and Research Center (CTRC) University of Texas Health Science Center, San Antonio - Dr. Addanki P. Kumar - Lead Investigator
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1343 METHYLATION OF THE TRANSCRIPTIONAL REPRESSOR SLUG/ SNAI2 IS ASSOCIATED WITH GENE SILENCING AND CAN BE REACTIVATED BY TREATMENT WITH 5-AZA-2’-DEOXYCITIDINE IN HUMAN PROSTATE CANCER CELLS. Takumi Shiraishi*, Hang Wang, Mark W Ball, Mark L Gonzalgo, Baltimore, MD INTRODUCTION AND OBJECTIVES: Expression of the transcriptional repressor Slug/Snai2 has been associated with tumor progression in a variety of human cancers. The significance of this gene and mechanisms of transcriptional regulation in prostate cancer have not been investigated. We examined the expression level of Slug/Snai2 in prostate cancer cell lines and whether methylation of the CpG island associated with Slug/Snai2 could regulate expression. METHODS: We initially obtained expression data of Slug/ Snai2 in normal prostate and prostate cancer using the ONCOMINE database. The mRNA expression level of Slug/Snai2 was determined by quantitative real-time PCR in 4 prostate cancer cell lines (LNCaP, CWR22Rv1, PC3, and DU145) and one normal prostate epithelial cell line (PrEC). The methylation level of the CpG island associated with Slug/Snai2 was measured by quantitative real-time methylation specific PCR. Cell lines were treated with 5-aza-2’-deoxycytidine to determine the effects on Slug/Snai2 methylation and expression. RESULTS: The expression of Slug/Snai2 was significantly lower in prostate cancer than in normal prostate according to the ONCOMINE database. Slug/Snai2 mRNA expression was downregulated in all 4 prostate cancer cell lines examined compared to human PrEC cells. The CpG island associated with the Slug/Snai2 promoter was found to be hypermethylated in DU145 prostate cancer cells. Treatment of DU145 cells with the demethylating agent 5-Aza-2’-deoxycytidine reactivated Slug/Snai2 expression. CONCLUSIONS: Slug/Snai2 expression is decreased in prostate cancer cells. Methylation of Slug/Snai2 was observed in DU145 prostate cancer cells and was associated with gene silencing. Treatment of DU145 cancer cells with 5-Aza-2’-deoxycytidine restored Slug/Snai2 expression. This is the first study which demonstrates that hypermethylation of Slug/ Snai2 is associated with transcriptional downregulation of this gene in prostate cancer cells. Source of Funding: Prostate Cancer Foundation Career Development Award, Patrick C. Walsh Cancer Research Fund.
1344 BEYOND MATRIGEL: EXTRACELLULAR MATRIX DERIVED FROM HUMAN PLEOMORPHIC ADENOMAS Gustavo E Ayala*, Yi Ding, Michael Ittmann, David R Rowley, Houston, TX INTRODUCTION AND OBJECTIVES: Currently Matrigel and immunodeficient mice are the “gold” standards for in culture and in vivo growth respectively. Matrigel has inherent problems. First, it is a mouse derived chondorsarcoma matrix and therefore its components are different from human extracelullar matrices. The most common epithelial tumor that produces extracellular matrix is the benign pleomorphic adenoma, a salivary gland tumor composed of myoepithelial cells and that is known to produce large amounts of matrix. The histologic features of this tumor are very similar to those of EHS METHODS: Fragments of pleomorphic adenoma tumors were placed in 6-well plates with Bfs medium [DMEMhigh glucose with 5% Nu-serum (BD-Biosciences), 5% FBS, 5 μg/ ml insulin (Sigma) and antibiotics]. Cell growing in the periphery of these fragments were passaged 5-8 times. Cells were pelleted to produce a cellblock and immunohistochemistry performed to determine lineage using antibodies against cytokeratin, Smooth muscle actin, GFAP and Ki67. Cell were also grown into 3 dimensional BD collagen scaffold. The scaffolds were fixed in formalin, sectioned and embedded in paraffin. Antibodies against laminin, collagen IV and V were used to determine the components off the secreted matrix. Supernatants were run on 12% gels . Later western blots against collagen V (CHEMICON cat# MAB3302)
Vol. 181, No. 4, Supplement, Tuesday, April 28, 2009
RESULTS: IL-18Bpa was expressed and secreted by the prostate cancer cell lines DU145 and PC3, but not by LNCaP and CWR22, upon interferon-G (IFN-G) stimulation. IFN-G-induced secretion of IL-18Bpa was enhanced by added TNF-A, IFN-A and IFN-B. The IL-18Bpa secreted from DU145 and PC3 functionally inhibited IL-18. Conditioned medium from IL-18Bpa-overexpressed PC3 cells suppressed CD8+ IFN-G+ cells and CD4+ cells and appeared to decrease TH1 cells in human peripheral blood cultures. Immunohistochemical analyses showed positive IL18Bpa staining in prostate cancer cells as well as in macrophages in radical prostatectomy specimens. Significant differences in post-DRE urinary IL-18Bpa levels (normalized by total protein) were found between cases with and without cancer on biopsy (P = 0.02) and serum IL-18Bpa levels correlated with Gleason score (P = 0.03). CONCLUSIONS: Our finding of elevated IL-18Bpa secretion from prostate cancer cells suggests an attempt by cancer to escape immune surveillance. IL-18Bpa merits further study as a marker of aggressive prostate cancer and as a therapeutic target. Source of Funding: NIH/NIDDK grant 1K23DK071262, Department of Defense grant PC041214 and NIH/NCI grant U24 CA115102
1342 PHELLODENDRON AMURENSE BARK EXTRACT ENHANCES RADIOSENSITIVITY BY INHIBITION OF NF-KAPPA B IN TRANSGENIC ADENOCARCINOMA OF MOUSE PROSTATE MODEL AND HUMAN PROSTATE CANCER CELLS Craig H Robson*, Manonmani Ganapathy, Gregory P Swanson, Mohan Natarajan, Niko Papanikolaou, Martha A Hanes, I-Tien Yeh, Rita Ghosh, Addanki P Kumar, San Antonio, TX INTRODUCTION AND OBJECTIVES: Evidence supports that the activation of nuclear factor-kappa B (NF-kB) by ionizing radiation in prostate cancer results in adaptive resistance to radiation therapy. Therefore, altering expression of NF-kB may enhance radiation-induced apoptosis. Phellodendron amurense bark extract has been shown to compromise transcriptional activity of NF-kB and several dependent genes such as Cyclin D1 and COX-2 resulting in inhibition of prostate cancer cell proliferation by inducing apoptosis. We investigated if intervention with a complex botanical in vivo would potentiate cytotoxic effects of radiotherapy. METHODS: Twenty-week-old transgenic adenocarcinoma of mouse prostate (TRAMP) mice (at which time they display invasive carcinoma) were fed with pelleted diet containing 600mg/ kg Phellodendron Amurense bark extract 6 weeks prior to undergoing radiation therapy. These animals (n=6) were compared to a group that received radiation alone. Efficacy was evaluated by histological analysis of prostate tissue at the termination of the experiment at 34 weeks. Mechanistic studies were conducted in tumor tissue as well as in human prostate cancer cells. RESULTS: Preliminary data demonstrate that majority of animals that received Phellodendron amurense bark extract for 6 weeks prior to radiation therapy had no overt cancer but exhibited features consistent with high grade prostatic intraepithelial neoplasia. In contrast animals that received radiation alone exhibited features consistent with well to poorly differentiated adenocarcinoma. Western blot analysis using whole cell extracts prepared from prostate tumors and PC3 cells showed decreased expression of p65. CONCLUSIONS: The current study demonstrates that Phellodendron amurense bark extract potentiates radiation-induced apoptosis and may work by inhibition of NF-kB. Supported by pilot funds through Cancer Therapy and Research Center (APK). Source of Funding: Pilot Funds through Cancer Therapy and Research Center (CTRC) University of Texas Health Science Center, San Antonio - Dr. Addanki P. Kumar - Lead Investigator
479
1343 METHYLATION OF THE TRANSCRIPTIONAL REPRESSOR SLUG/ SNAI2 IS ASSOCIATED WITH GENE SILENCING AND CAN BE REACTIVATED BY TREATMENT WITH 5-AZA-2’-DEOXYCITIDINE IN HUMAN PROSTATE CANCER CELLS. Takumi Shiraishi*, Hang Wang, Mark W Ball, Mark L Gonzalgo, Baltimore, MD INTRODUCTION AND OBJECTIVES: Expression of the transcriptional repressor Slug/Snai2 has been associated with tumor progression in a variety of human cancers. The significance of this gene and mechanisms of transcriptional regulation in prostate cancer have not been investigated. We examined the expression level of Slug/Snai2 in prostate cancer cell lines and whether methylation of the CpG island associated with Slug/Snai2 could regulate expression. METHODS: We initially obtained expression data of Slug/ Snai2 in normal prostate and prostate cancer using the ONCOMINE database. The mRNA expression level of Slug/Snai2 was determined by quantitative real-time PCR in 4 prostate cancer cell lines (LNCaP, CWR22Rv1, PC3, and DU145) and one normal prostate epithelial cell line (PrEC). The methylation level of the CpG island associated with Slug/Snai2 was measured by quantitative real-time methylation specific PCR. Cell lines were treated with 5-aza-2’-deoxycytidine to determine the effects on Slug/Snai2 methylation and expression. RESULTS: The expression of Slug/Snai2 was significantly lower in prostate cancer than in normal prostate according to the ONCOMINE database. Slug/Snai2 mRNA expression was downregulated in all 4 prostate cancer cell lines examined compared to human PrEC cells. The CpG island associated with the Slug/Snai2 promoter was found to be hypermethylated in DU145 prostate cancer cells. Treatment of DU145 cells with the demethylating agent 5-Aza-2’-deoxycytidine reactivated Slug/Snai2 expression. CONCLUSIONS: Slug/Snai2 expression is decreased in prostate cancer cells. Methylation of Slug/Snai2 was observed in DU145 prostate cancer cells and was associated with gene silencing. Treatment of DU145 cancer cells with 5-Aza-2’-deoxycytidine restored Slug/Snai2 expression. This is the first study which demonstrates that hypermethylation of Slug/ Snai2 is associated with transcriptional downregulation of this gene in prostate cancer cells. Source of Funding: Prostate Cancer Foundation Career Development Award, Patrick C. Walsh Cancer Research Fund.
1344 BEYOND MATRIGEL: EXTRACELLULAR MATRIX DERIVED FROM HUMAN PLEOMORPHIC ADENOMAS Gustavo E Ayala*, Yi Ding, Michael Ittmann, David R Rowley, Houston, TX INTRODUCTION AND OBJECTIVES: Currently Matrigel and immunodeficient mice are the “gold” standards for in culture and in vivo growth respectively. Matrigel has inherent problems. First, it is a mouse derived chondorsarcoma matrix and therefore its components are different from human extracelullar matrices. The most common epithelial tumor that produces extracellular matrix is the benign pleomorphic adenoma, a salivary gland tumor composed of myoepithelial cells and that is known to produce large amounts of matrix. The histologic features of this tumor are very similar to those of EHS METHODS: Fragments of pleomorphic adenoma tumors were placed in 6-well plates with Bfs medium [DMEMhigh glucose with 5% Nu-serum (BD-Biosciences), 5% FBS, 5 μg/ ml insulin (Sigma) and antibiotics]. Cell growing in the periphery of these fragments were passaged 5-8 times. Cells were pelleted to produce a cellblock and immunohistochemistry performed to determine lineage using antibodies against cytokeratin, Smooth muscle actin, GFAP and Ki67. Cell were also grown into 3 dimensional BD collagen scaffold. The scaffolds were fixed in formalin, sectioned and embedded in paraffin. Antibodies against laminin, collagen IV and V were used to determine the components off the secreted matrix. Supernatants were run on 12% gels . Later western blots against collagen V (CHEMICON cat# MAB3302)