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EFFECTS OF HYPOXIA ON MNSOD EXPRESSION AND SUBSEQUENT EPIGALLOCATECHIN GALLATE INDUCED PROSTATE CANCER CELL APOPTOSIS
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EFFECTS OF HYPOXIA ON MNSOD EXPRESSION AND SUBSEQUENT EPIGALLOCATECHIN GALLATE INDUCED PROSTATE CANCER CELL APOPTOSIS O’Neill A.1 , Morrissey C.1 , Cunningham K.1 , Taylor C.1 , Fitzpatrick J.M.2 , Watson R.W.G.1 1 Conway Institute, University College Dublin, School of Medicine & Medical Science, Dublin, Ireland; 2 Conway Institute, UCD & Mater Misericordiae Hospital, School of Medicine & Medical Science, Dublin, Ireland
INTRODUCTION & OBJECTIVES: Antioxidants may reduce tumorigenesis in prostate cancer due to their chemopreventative properties, however they have also been studied from a chemotherapeutic standpoint. Epigallocatechin gallate (EGCG) a potent antioxidant has been shown to induce apoptosis in prostate cancer cells in vitro via a contradictory oxidative pathway. The aims of this study were to determine if a hypoxic environment alters the effects of EGCG on metastatic prostate cell number and apoptosis, and investigate the mechanism involved specifically at the level of the mitochondria. MATERIAL & METHODS: Two androgen independent prostate cancer cell lines, PC3 and DU-145 were cultured to confluence and treated with different concentrations of EGCG in hypoxic and normoxic conditions. Cell number and apoptosis was assessed using crystal violet assays and flow cytometric analysis. The effect of EGCG on mitochondrial activity was assessed using the MTT assay and MnSOD expression by western analysis. The influence of hypoxia and EGCG on the oxidative environment and superoxide production in PC-3 and DU-145 cells was measured by flow cytometry and luminometery using DCFH-DA. RESULTS: Despite EGCG anti-oxidant potential, it was shown to increase reactive oxygen species generation with a decrease in MnSOD expression under normoxic conditions in the PC-3 and DU-145 cells resulting in the induction of apoptosis. Hypoxia increased MnSOD protein expression, which was not decreased on incubation with EGCG and may represent an important protective mechanism in hypoxic conditions. Further studies have confirmed that PC-3 cells over-expressing MnSOD are more resistant to TRAIL and Fas induced cell death compared to the parental PC-3 cell line. CONCLUSIONS: Increased expression of MnSOD under hypoxic conditions may represent an important mechanism by which cancer cells protect themselves against subsequent treatment with apoptotic inducing agents. EGCG differential effects maybe explained by altered tumour environments associated with different stages of prostate cancer progression.
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THE ROLE OF NITRIC OXIDE NON STEROIDAL DRUGS IN THE HYPOXIC RESPONSE OF PROSTATE CELLS Brown D.1 , Habib F.K.2 1 Queen Margaret Hospital, Urology, Dunfermline, United Kingdom; 2 University of Edinburgh, Prostate Research Group, Edinburgh, United Kingdom
INTRODUCTION & OBJECTIVES: Tumour hypoxia is associated with resistance to treatment and possible early metastatic spread. This has been shown for prostate cancer and is secondary to the stabilisation of hypoxia inducible factor 1 alpha h.i.f.1.alpha. Inhibition of this could potentially sensitise prostate cancer to treatment. Nitric oxide non steroidals n.o.-.n.s.a.i.ds have been shown to inhibit the growth of, and induce apoptosis in cultured prostate cancer cells. This work aimed to determine if there was an associated inhibition of the hypoxic response and a subsequent sensitisation to radiation. MATERIAL & METHODS: Using tissue culture techniques, PC3, PNT1 and primary stromal cells were used. Proliferation studies were performed and flow cytometric analysis of apoptosis. The cells were treated with n.o.-.n.s.a.i.d or control and exposed to a hypoxic or normoxic environment. The cells were then used fo western blot analysis to detect changes in h.i.f.1.alpha and the medium used for ELISA analysis of vascular endothelial growth factor v.e.g.f. Primary stroma was then cultured and treated with control or n.o.-.n.s.a.i.d and exposed to normoxia or hypoxia. They were then irradiated (10Gy) and differential cell count performed at 16 hours to detect cell survival. RESULTS: Flow cytometry identified an increase in apoptosis in PC3 and Stroma cells with time of treatment. Apoptosis was inhibited in PNT1 cells. Western blot analysis identified a reduction in h.i.f in PC3 cells and stromal cells under hypoxia in n.o.-.n.s.a.i.d treated cells. There was an up-regulation of h.i.f in PNT1 cells. v.e.g.f levels were reduced in the hypoxic PC3 cells (P = 0.023)but raised in PNT1 cells and Stroma cells (P = 0.199 and 0.042). Treatment of stromal cells caused massive rises in radiation induced cell death in both normoxic and hypoxic cells (P = 0.003 and < 0.001) CONCLUSIONS: n.o.-.n.s.a.i.d inhibits the hypoxic response in cultured PC3 cells. They inhibit HIF expression in stroma and promote hypoxic response in PNT1 cells. There is a massive rise in radiation cell death with them which is irrespective of oxygen tensions. This could identify a potential treatment adjunct for patients undergoing radiotherapy. Further work is being performed to identify effects on PC3 cells and chemosensitivity.
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GALIELLALACTONE INHIBITS GROWTH OF PROSTATE CANCER CELL XENOGRAFTS AND INDUCES APOPTOSIS OF HUMAN PROSTATE CANCER CELLS EXPRESSING ACTIVE STAT3 Hellsten R.1 , Johansson M.2 , Sterner O.2 , Bjartell A.3 1 Division of Urological Research, Department of Clinical Sciences Malm¨o, Lund University, Malm¨o, Sweden; 2 Organic Chemistry, Department of Chemistry, Lund University, Lund, Sweden; 3 Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, USA
DEFINING AGGRESSIVE PROSTATE CANCER USING A 12 GENE MODEL Demichelis F.1 , Chinnaiyan A.M.2 , Febo P.3 , Hofer M.4 , Kuefer R.5 , Tang J.1 , Aster J.1 , Kutok J.1 , He L.1 , Varambally S.2 , Kim R.1 , Riva A.3 , Bismar T.A.3 , Rubin M.1 1 Brigham and Womens Hospital, Dept. of Pathology, Boston, USA; 2 University of Michigan, Dept. of Pathology and Urology, Boston, USA; 3 Harvard Medical School, Boston, USA; 4 Brigham and Womens Hospital, Boston, USA; 5 University Hospital of Ulm, Ulm, Germany
INTRODUCTION & OBJECTIVES: Signal transducers and activators of transcription 3 (Stat3) are transcription factors involved in cell growth, differentiation and immune defence. Normaly Stat3 signaling is highly regulated and transient. However, constitutive activation of Stat3 is observed in prostate cancer (PCa) and many other cancers. Tumor cells expressing active Stat3 become dependent on this signaling pathway for survival and blocking induces apoptosis. The fungal metabolite galiellalactone is shown to inhibit interleukin-6 (IL-6) mediated Stat3 signaling but has not yet been studied on human cancer cells. This study aims to investigate the effect of galiellalactone on human PCa cells both in vivo and in vitro with focus on the Stat3 signaling pathway. MATERIAL & METHODS: The human PCa cell lines DU145, PC-3 and LNCaP were used. Nude mice with subcutaneous DU145 and PC-3 cell xenografts were subjected to daily i p injections of galiellalactone for three weeks. Tumor size was measured every 3-4 days. Induction of apoptosis by galiellalactone of cultured PCa cells was investigated by Western blot analysis and immunocytochemistry for detection of apoptotic proteins and by annexin V staining followed by flow cytometry. The effect of galiellalactone on Stat3 signaling was studied by a luciferase reporter gene assay. Expression of p-Stat3 and galiellalactones effect on Stat3 associated proteins was investigated by Western blot analysis. RESULTS: p-Stat3 was constitutively expressed in PC-3 and DU145 cells and induced by IL-6 (50 ng/ml) in LNCaP cells. Treatment with galiellalactone (3 mg/kg) significantly reduced tumor growth of DU145 xenografts by 42% (p = 0.0016) and reduced PC-3 xenograft growth by 25%, however not significantly (p = 0.56). Galiellalactone (25 mM, 72 h) induced apoptosis of DU145 (30% apoptotic cells) and PC-3 cells (17%), but not of LNCaP cells. The expression of cleaved-PARP was increased in PC-3 and DU145 cells but not in LNCaP cells after galiellalactone stimulation (10–25 mM, 72 h). Galiellalactone down-regulated the Stat3 regulated anti-apoptotic proteins BCl-xL and BCl-2 and the cell cycle regulatory proteins c-myc and cyclin D1 in DU145 cells. Stat3-mediated luciferase activity was inhibited by galiellalactone in a dose dependent manner (IC50 ≈ 5 mM). CONCLUSIONS: Galiellalactone inhibited growth of PCa cell xenografts and induced apoptosis of p-Stat3 expressing PCa cells. Galiellalactone may act as a potential therapeutic agent for hormone refractory PCa tumors expressing constitutive Stat3.
INTRODUCTION & OBJECTIVES: The critical clinical question in prostate cancer research is to develop means of distinguishing aggressive from indolent disease. Expression array technology has lead to the development of discrete molecular signatures but has thus far failed to identify a robust signature to characterize aggressive prostate. We describe the development of a refined model for prostate cancer progression. MATERIAL & METHODS: Previous work from our group analyzed the expression of 1383 distinct proteins or post-translational modifications expression in benign prostate tissue, localized prostate cancer, and metastatic prostate cancer. Integration of these proteomic alterations with transcriptomic data derived from 8 prostate cancer profiling studies, revealed a set of genes that demonstrated concordance between protein and transcript levels. 41 of these genes were evaluated on a prostate cancer progression tissue micorarray. The protein products of these genes were tested using quantitative analysis of immunohistochemistry. The best model was validated using prostate cancer expression array data with associated clinical outcomes data. RESULTS: Linear discriminant analysis determined that the optimal model consisted of 12 proteins. This 12 protein model accurately distinguishes stages of prostate cancer progression. Intriguingly, the transcriptional levels of the 12 genes encoding for these proteins predict PSA-failure in 80 men following surgery for clinically localized prostate cancer (p = 0.0015). CONCLUSIONS: This study demonstrates that cross platform models can lead to predictive models with the possible advantage of being more robust through this selection process. The approach led to a 12 gene model that was validated on a separate patient cohort using PSA-failure following surgery for clinically localized prostate cancer as the endpoint. We are currently validating and further refining the model on a population based clinical cohort. Importantly, this model can be feasibly used in a clinical setting.
Eur Urol Suppl 2006;5:797
EFFECTS OF HYPOXIA ON MNSOD EXPRESSION AND SUBSEQUENT EPIGALLOCATECHIN GALLATE INDUCED PROSTATE CANCER CELL APOPTOSIS O’Neill A.1 , Morrissey C.1 , Cunningham K.1 , Taylor C.1 , Fitzpatrick J.M.2 , Watson R.W.G.1 1 Conway Institute, University College Dublin, School of Medicine & Medical Science, Dublin, Ireland; 2 Conway Institute, UCD & Mater Misericordiae Hospital, School of Medicine & Medical Science, Dublin, Ireland
INTRODUCTION & OBJECTIVES: Antioxidants may reduce tumorigenesis in prostate cancer due to their chemopreventative properties, however they have also been studied from a chemotherapeutic standpoint. Epigallocatechin gallate (EGCG) a potent antioxidant has been shown to induce apoptosis in prostate cancer cells in vitro via a contradictory oxidative pathway. The aims of this study were to determine if a hypoxic environment alters the effects of EGCG on metastatic prostate cell number and apoptosis, and investigate the mechanism involved specifically at the level of the mitochondria. MATERIAL & METHODS: Two androgen independent prostate cancer cell lines, PC3 and DU-145 were cultured to confluence and treated with different concentrations of EGCG in hypoxic and normoxic conditions. Cell number and apoptosis was assessed using crystal violet assays and flow cytometric analysis. The effect of EGCG on mitochondrial activity was assessed using the MTT assay and MnSOD expression by western analysis. The influence of hypoxia and EGCG on the oxidative environment and superoxide production in PC-3 and DU-145 cells was measured by flow cytometry and luminometery using DCFH-DA. RESULTS: Despite EGCG anti-oxidant potential, it was shown to increase reactive oxygen species generation with a decrease in MnSOD expression under normoxic conditions in the PC-3 and DU-145 cells resulting in the induction of apoptosis. Hypoxia increased MnSOD protein expression, which was not decreased on incubation with EGCG and may represent an important protective mechanism in hypoxic conditions. Further studies have confirmed that PC-3 cells over-expressing MnSOD are more resistant to TRAIL and Fas induced cell death compared to the parental PC-3 cell line. CONCLUSIONS: Increased expression of MnSOD under hypoxic conditions may represent an important mechanism by which cancer cells protect themselves against subsequent treatment with apoptotic inducing agents. EGCG differential effects maybe explained by altered tumour environments associated with different stages of prostate cancer progression.
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THE ROLE OF NITRIC OXIDE NON STEROIDAL DRUGS IN THE HYPOXIC RESPONSE OF PROSTATE CELLS Brown D.1 , Habib F.K.2 1 Queen Margaret Hospital, Urology, Dunfermline, United Kingdom; 2 University of Edinburgh, Prostate Research Group, Edinburgh, United Kingdom
INTRODUCTION & OBJECTIVES: Tumour hypoxia is associated with resistance to treatment and possible early metastatic spread. This has been shown for prostate cancer and is secondary to the stabilisation of hypoxia inducible factor 1 alpha h.i.f.1.alpha. Inhibition of this could potentially sensitise prostate cancer to treatment. Nitric oxide non steroidals n.o.-.n.s.a.i.ds have been shown to inhibit the growth of, and induce apoptosis in cultured prostate cancer cells. This work aimed to determine if there was an associated inhibition of the hypoxic response and a subsequent sensitisation to radiation. MATERIAL & METHODS: Using tissue culture techniques, PC3, PNT1 and primary stromal cells were used. Proliferation studies were performed and flow cytometric analysis of apoptosis. The cells were treated with n.o.-.n.s.a.i.d or control and exposed to a hypoxic or normoxic environment. The cells were then used fo western blot analysis to detect changes in h.i.f.1.alpha and the medium used for ELISA analysis of vascular endothelial growth factor v.e.g.f. Primary stroma was then cultured and treated with control or n.o.-.n.s.a.i.d and exposed to normoxia or hypoxia. They were then irradiated (10Gy) and differential cell count performed at 16 hours to detect cell survival. RESULTS: Flow cytometry identified an increase in apoptosis in PC3 and Stroma cells with time of treatment. Apoptosis was inhibited in PNT1 cells. Western blot analysis identified a reduction in h.i.f in PC3 cells and stromal cells under hypoxia in n.o.-.n.s.a.i.d treated cells. There was an up-regulation of h.i.f in PNT1 cells. v.e.g.f levels were reduced in the hypoxic PC3 cells (P = 0.023)but raised in PNT1 cells and Stroma cells (P = 0.199 and 0.042). Treatment of stromal cells caused massive rises in radiation induced cell death in both normoxic and hypoxic cells (P = 0.003 and < 0.001) CONCLUSIONS: n.o.-.n.s.a.i.d inhibits the hypoxic response in cultured PC3 cells. They inhibit HIF expression in stroma and promote hypoxic response in PNT1 cells. There is a massive rise in radiation cell death with them which is irrespective of oxygen tensions. This could identify a potential treatment adjunct for patients undergoing radiotherapy. Further work is being performed to identify effects on PC3 cells and chemosensitivity.
44
GALIELLALACTONE INHIBITS GROWTH OF PROSTATE CANCER CELL XENOGRAFTS AND INDUCES APOPTOSIS OF HUMAN PROSTATE CANCER CELLS EXPRESSING ACTIVE STAT3 Hellsten R.1 , Johansson M.2 , Sterner O.2 , Bjartell A.3 1 Division of Urological Research, Department of Clinical Sciences Malm¨o, Lund University, Malm¨o, Sweden; 2 Organic Chemistry, Department of Chemistry, Lund University, Lund, Sweden; 3 Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, USA
DEFINING AGGRESSIVE PROSTATE CANCER USING A 12 GENE MODEL Demichelis F.1 , Chinnaiyan A.M.2 , Febo P.3 , Hofer M.4 , Kuefer R.5 , Tang J.1 , Aster J.1 , Kutok J.1 , He L.1 , Varambally S.2 , Kim R.1 , Riva A.3 , Bismar T.A.3 , Rubin M.1 1 Brigham and Womens Hospital, Dept. of Pathology, Boston, USA; 2 University of Michigan, Dept. of Pathology and Urology, Boston, USA; 3 Harvard Medical School, Boston, USA; 4 Brigham and Womens Hospital, Boston, USA; 5 University Hospital of Ulm, Ulm, Germany
INTRODUCTION & OBJECTIVES: Signal transducers and activators of transcription 3 (Stat3) are transcription factors involved in cell growth, differentiation and immune defence. Normaly Stat3 signaling is highly regulated and transient. However, constitutive activation of Stat3 is observed in prostate cancer (PCa) and many other cancers. Tumor cells expressing active Stat3 become dependent on this signaling pathway for survival and blocking induces apoptosis. The fungal metabolite galiellalactone is shown to inhibit interleukin-6 (IL-6) mediated Stat3 signaling but has not yet been studied on human cancer cells. This study aims to investigate the effect of galiellalactone on human PCa cells both in vivo and in vitro with focus on the Stat3 signaling pathway. MATERIAL & METHODS: The human PCa cell lines DU145, PC-3 and LNCaP were used. Nude mice with subcutaneous DU145 and PC-3 cell xenografts were subjected to daily i p injections of galiellalactone for three weeks. Tumor size was measured every 3-4 days. Induction of apoptosis by galiellalactone of cultured PCa cells was investigated by Western blot analysis and immunocytochemistry for detection of apoptotic proteins and by annexin V staining followed by flow cytometry. The effect of galiellalactone on Stat3 signaling was studied by a luciferase reporter gene assay. Expression of p-Stat3 and galiellalactones effect on Stat3 associated proteins was investigated by Western blot analysis. RESULTS: p-Stat3 was constitutively expressed in PC-3 and DU145 cells and induced by IL-6 (50 ng/ml) in LNCaP cells. Treatment with galiellalactone (3 mg/kg) significantly reduced tumor growth of DU145 xenografts by 42% (p = 0.0016) and reduced PC-3 xenograft growth by 25%, however not significantly (p = 0.56). Galiellalactone (25 mM, 72 h) induced apoptosis of DU145 (30% apoptotic cells) and PC-3 cells (17%), but not of LNCaP cells. The expression of cleaved-PARP was increased in PC-3 and DU145 cells but not in LNCaP cells after galiellalactone stimulation (10–25 mM, 72 h). Galiellalactone down-regulated the Stat3 regulated anti-apoptotic proteins BCl-xL and BCl-2 and the cell cycle regulatory proteins c-myc and cyclin D1 in DU145 cells. Stat3-mediated luciferase activity was inhibited by galiellalactone in a dose dependent manner (IC50 ≈ 5 mM). CONCLUSIONS: Galiellalactone inhibited growth of PCa cell xenografts and induced apoptosis of p-Stat3 expressing PCa cells. Galiellalactone may act as a potential therapeutic agent for hormone refractory PCa tumors expressing constitutive Stat3.
INTRODUCTION & OBJECTIVES: The critical clinical question in prostate cancer research is to develop means of distinguishing aggressive from indolent disease. Expression array technology has lead to the development of discrete molecular signatures but has thus far failed to identify a robust signature to characterize aggressive prostate. We describe the development of a refined model for prostate cancer progression. MATERIAL & METHODS: Previous work from our group analyzed the expression of 1383 distinct proteins or post-translational modifications expression in benign prostate tissue, localized prostate cancer, and metastatic prostate cancer. Integration of these proteomic alterations with transcriptomic data derived from 8 prostate cancer profiling studies, revealed a set of genes that demonstrated concordance between protein and transcript levels. 41 of these genes were evaluated on a prostate cancer progression tissue micorarray. The protein products of these genes were tested using quantitative analysis of immunohistochemistry. The best model was validated using prostate cancer expression array data with associated clinical outcomes data. RESULTS: Linear discriminant analysis determined that the optimal model consisted of 12 proteins. This 12 protein model accurately distinguishes stages of prostate cancer progression. Intriguingly, the transcriptional levels of the 12 genes encoding for these proteins predict PSA-failure in 80 men following surgery for clinically localized prostate cancer (p = 0.0015). CONCLUSIONS: This study demonstrates that cross platform models can lead to predictive models with the possible advantage of being more robust through this selection process. The approach led to a 12 gene model that was validated on a separate patient cohort using PSA-failure following surgery for clinically localized prostate cancer as the endpoint. We are currently validating and further refining the model on a population based clinical cohort. Importantly, this model can be feasibly used in a clinical setting.
Eur Urol Suppl 2006;5:797