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103 HIGH DENSITY LIPOPROTEIN INDUCES PROLIFERATION AND MIGRATION OF HUMAN PROSTATE ANDROGEN INDEPENDENT CANCER CELLS VIA ATP-BINDING CASSETTE TRANSPORTER A1 BY A CHOLESTEROL-INDEPENDENT MECHANISM
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Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
Addition of other metabolites, such as farnesol, ubiquinone (Co-enzyme Q10), and squalene (precursor for synthesis of cholesterol) along with atorvastatin in culture medium did not affect atorvastatin-induced expression of LC3-II in PC3 cells (lanes 4-6). Furthermore, addition of geranylgeraniol, along with atorvastatin into culture medium reversed atorvastatin-caused PC3 cell death, suggesting a connection of atorvastatin-induced autophagy to rapid cell death. CONCLUSIONS: Atorvastatin mediated inhibition of biosynthesis of geranylgeranyl, not farnesyl, ubiquinone or cholesterol, is the cause for the effects of atorvastatin on autophagy and autophagyassociated cell death in PC3 cells.
Source of Funding: None
103 Source of Funding: Department of Defense Prostate Cancer Research Program under award number W81XWH-07-1-0146
102 STATINS INDUCE AUTOPHAGY BY INHIBITION OF GERANYLGERANYL BIOSYNTHESIS IN PROSTATE CANCER PC3 CELLS Ankur Parikh*, Chandra Childress, Qiong Lin, Daniel Rukstalis, Wannian Yang, Danville, PA INTRODUCTION AND OBJECTIVES: We have previously demonstrated that statins induce autophagy and cause rapid cell death in PC-3 cells. Autophagy, or “self-eating”, is a process responsible for intracellular material degradation, in which cytoplasmic components are engulfed by autophagosomes and delivered to lysosomes for degradation. Statins, a class of inhibitors of 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are commonly prescribed medications for treatment of hypercholesterolemia. Inhibition of HMG-CoA reductase halts biosynthesis of metabolites in mevalonate pathway that are required for production of cholesterol, ubiquinone (Coenzyme Q10), geranylgeranyl pyrophosphate and farnesyl pyrophosphate. To define which metabolite biosynthesis is involved in atorvastatin-induced autophagy, we performed rescue experiments by supplementation of various metabolites to cell culture medium. METHODS: PC-3 cells were plated and treated with atorvastatin. Rescue experiments were performed with the addition of geranylgeraniol, farnesol, coenzyme Q 10, and squalene. Autophagic response was evaluated with Western Blot assays for expression of LC3-II, a widely accepted marker for autophagy. RESULTS: As shown in Fig. 1, addition of geranylgeraniol in culture medium along with atorvastatin completely reversed atorvastatin-induced LC3-II expression in PC3 cells (lane 3), while geranylgeraniol alone did not have any effect on LC3-II expression (lane 7).
HIGH DENSITY LIPOPROTEIN INDUCES PROLIFERATION AND MIGRATION OF HUMAN PROSTATE ANDROGEN INDEPENDENT CANCER CELLS VIA ATP-BINDING CASSETTE TRANSPORTER A1 BY A CHOLESTEROL-INDEPENDENT MECHANISM Yoshitaka Sekine*, Bethesda, MD; Yosuke Furuya, Hidekazu Koike, Kazuhiro Suzuki, Maebashi, Japan; Alan Remaley, Bethesda, MD INTRODUCTION AND OBJECTIVES: Androgen deprivation therapy in men with prostate cancer (PC) leads to a significant increase of HDL. The increase is generally viewed as beneficial, particularly for cardiovascular disease, but the effects of HDL on prostate cancer are unknown. ATP-binding Cassette Transporter A1 (ABCA1), ATP-binding Cassette Transporter G1 and scavenger receptor class B member 1 are receptors for HDL and have important roles in HDL-induced signal transductions. In this study, we investigated the effect of HDL on prostate cancer cell proliferation, migration, cholesterol levels, and signal transductions, and if they are mediated by one of the receptors for HDL. METHODS: HDL was isolated from health volunteer men. mRNA expressions of PC cells and human prostate biopsy samples were evaluated by quantitative real-time PCR. Cell viability was determined by MTS assay. Cell migration was shown by would healing assay. The activation of MAPK and Akt were detected by Western blot analysis. The functional role of ABCA1 in any of the observed phenomena was examined after transfection with a siRNA against ABCA1. RESULTS: HDL could induce PC-3 and DU145 cell proliferations, migrations and both MAPK and Akt signal transductions, but not in LNCaP cells. Treatment with HDL did not significantly alter cellular cholesterol levels in PC cells. In cells grown in 10% FCS, the expression of ABCA1 was much lower in LNCaP than PC-3 and DU145. After culturing in androgen-free condition, ABCA1 expression increased 10 times in LNCaP cells. In human prostate biopsy samples, ABCA1 expression was significantly higher in androgen deprivation therapy group than BPH and pre-treatment prostate cancer group. Knockdown of ABCA1 by siRNA inhibited HDL-induced cell proliferation, migration
Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
and both MAPK and Akt signal transductions in PC-3. Moreover, after treatment of androgen free condition for 96 hour, HDL could induce MAPK activation, and knockdown of ABCA1 expression by siRNA inhibited HDL-induced MAPK activation in LNCaP cells. Simvastatin, which inhibited ABCA1 expression in PC-3 and DU145 cells, inhibited HDL-induced PC-3 and DU145 cell proliferation, migration and both MAPK and Akt signal transduction. CONCLUSIONS: HDL can induce androgen independent prostate cancer cell proliferation and migration via ABCA1, which can be reversed by simvastatin. These results suggest that therapies that modulate HDL and lipid metabolism can potentially affect prostate cancer growth. Source of Funding: Intramural research funds from the National Heart, Lung and Blood Institute.
104 DIRECT EFFECTS OF ZOLEDRONIC ACID ON HORMONE RESPONSIVE AND HORMONE REFRACTORY PROSTATE CANCER CELLS Sweaty Koul, Binod Kumar, Paul Maroni, Randall Meacham, Hari Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Bone metastasis is a common site of metastasis in patients with advanced prostate cancer develop bone metastasis. Interleukin 6 (IL-6), plays an important role in bone remodeling associated with progression of prostate cancer, evolution of androgen independence and finally for resistance to chemotherapeutics. Previous studies have shown that IL-6 is not only a paracrine factor essential for promoting survival of neighboring cells, but also may act in an autocrine fashion, promoting survival of the tumor cells. Moreover, NF-KB is known to promote survival of cancer cells. The bisphosphonate zoledronic acid has been shown to decrease bone resorption due to tumor metastasis probably due to its effect on the growth and viability of osteoclasts present at the site of metastasis. Presently, zoledronic acid is also the only bisphosphonate approved for the treatment of bone metastases from prostate cancer in patients who have progressed after treatment with at least one hormonal therapy. METHODS: We investigated the effect of zoledronic acid on IL-6 production and the associated transcription factor NF-kB in PC3 (hormone independent) and LNCaP (hormone sensitive) prostate cancer cells. Gene expression was monitored by relative quantitative PCR. NFKB was monitored by Immunofluorescence microscopy. Cell growth and viability were measured by crystal violet and MTT assays. RESULTS: Treatment with zoledronic acid resulted in a drastic reduction in the levels of IL-6 mRNA in a concentration dependent manner in PC3 cells. Treatment with zoledronic acid also reduced nuclear localization of the transcription factor NF-kB which is responsible for IL-6 synthesis. We also observed that LNCaP cells synthesize IL-6 in response to TNF-a and zoledronic acid inhibited TNF-a induced ˆ B nuclear localization in LNCaP cells as well. These results show NF-fU that zoledronic acid inhibits both constitutive and TNF-stimulated IL-6 and NF-KB production in both hormone independent and hormone sensitive prostate cancer cell lines. In addition treatments with zoledronic acid resulted in dose and time dependent decrease in survival of prostate cancer cells. CONCLUSIONS: The results presented here suggest that zoledronic acid may not only have effects on bone resorption, but may also directly impact cancer cells. Zoledronic acid could potentially serve as a therapeutic agent in early stages of prostate cancer and perhaps other cancers targeting bone. Source of Funding: Supported in part by Investigator initiated Grant from Novartis (CZOL446EUS105) and by The Dept. of Surgery-School of Medicine, CU Denver School of Medicine Academic Enrichment Funds to H. K. Koul
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105 NOGGIN CONTRIBUTES TO THE OSTEOLYTIC RESPONSE IN BONE METASTASIS OF PROSTATE CANCER Chiara Secondini, Antoinette Wetterwald, Ruth Schwaninger, Marco G. Cecchini, George N. Thalmann*, Bern, Switzerland INTRODUCTION AND OBJECTIVES: Members of the bone morphogenetic protein (BMP) and the wingless (Wnt) protein family play a relevant role in the osteoblast response to prostate cancer (CaP) bone metastasis and their activity is modulated by extracellular antagonists. Lack of expression of the BMP antagonist noggin by CaP cells determines their osteoinductive activity in vivo. Osteolytic cells express noggin constitutively. The Wnt-antagonist dickkopf-1 (DKK-1) represses bone formation and noggin may act in analogy on BMP activity. We utilized a RNA silencing strategy to investigate whether constitutive noggin expression by an osteolytic CaP cell line determines the osteolytic phenotype. METHODS: Luciferase-expressing PC-3 cell clones were transfected with shRNA vectors to produce stable noggin-knock down (Nog-KD) clones. A vector encoding a non-targeting shRNA (mock) was used as negative control. Silencing efficiency was monitored by real time PCR and immunoblotting, respectively. Nog-KD and mock clones were xenografted into the tibia of nude mice and intra-osseous growth and osteolytic effect were monitored by bioluminescent imaging (BLI) and radiography, respectively. 3-D images of the xenografts were generated by micro-tomography (£gCT). Bone structural parameters were analyzed by peripheral quantitative computerized tomography (pQCT) and histomorphometry. RESULTS: Significant noggin mRNA and protein expression decrease in several Nog-KD clones with unchanged growth rates, osteolytic cytokine expression (PTHrP and IL-8), and of DKK-1 In vitro were observed. Tumor growth in bone xenografts of different mock and Nog-KD clones was only marginally or not affected. Radiographic and fY´CT analysis showed that the bones xenografted with the Nog-KD clones have structural modifications indicative of bone formation/repair activity but not parental PC-3 cells and mock clones. Bone histomorphometry and pQCT further corroborated these findings. CONCLUSIONS: Noggin expression by prostate cancer cells contribute to the induction of an osteolytic lesion. Conversely, shRNAmediated suppression of noggin expression in the osteolytic cell line PC-3 restored bone formation and may represent a new therapeutic option for the treatment of osteolytic bone metastases. Source of Funding: None
106 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-2 (IGFBP-2) ALTERS PTEN ACTIVITY AND REDUCES THE EFFICACY OF DOCETAXEL IN THE TREATMENT OF PROSTATE CANCER (PCA) Christopher Uzoh*, Jeff Holly, Raj Persad, Amit Bahl, Claire Perks, Bristol, United Kingdom INTRODUCTION AND OBJECTIVES: PCa is 5th most diagnosed cancer, worldwide. Aggressive disease, androgen-independence and treatment failure are predominant reasons for high mortality. We aim to identify new molecular targets that can be exploited to slow progression. Inactivation of tumor suppressor genes, changes in androgen regulation and activation of the insulin-like growth factor axis are common events in PCa pathogenesis and progression. Tumor suppressor gene PTEN is most frequently mutated in metastatic PCa and it dictates progression. IGFBP-2 can modulate IGF actions but also has intrinsic, IGF-independent actions, promoting growth of PCa cells. Its levels increase 2-3-fold in serum of PCa patients and correlate with serum PSA and Gleason score. It plays a key role in promoting androgen-independent and metastatic phenotype and is a biomarker of PTEN status. However, its mechanism of action and relationship to PTEN remain unknown. METHODS: DU145 and PC3 PCa cell lines were treated with either IGFBP-2 (0-1000ng/ml)(GroPep, Australia) or IGFBP-2 siRNA
THE JOURNAL OF UROLOGY姞
Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
Addition of other metabolites, such as farnesol, ubiquinone (Co-enzyme Q10), and squalene (precursor for synthesis of cholesterol) along with atorvastatin in culture medium did not affect atorvastatin-induced expression of LC3-II in PC3 cells (lanes 4-6). Furthermore, addition of geranylgeraniol, along with atorvastatin into culture medium reversed atorvastatin-caused PC3 cell death, suggesting a connection of atorvastatin-induced autophagy to rapid cell death. CONCLUSIONS: Atorvastatin mediated inhibition of biosynthesis of geranylgeranyl, not farnesyl, ubiquinone or cholesterol, is the cause for the effects of atorvastatin on autophagy and autophagyassociated cell death in PC3 cells.
Source of Funding: None
103 Source of Funding: Department of Defense Prostate Cancer Research Program under award number W81XWH-07-1-0146
102 STATINS INDUCE AUTOPHAGY BY INHIBITION OF GERANYLGERANYL BIOSYNTHESIS IN PROSTATE CANCER PC3 CELLS Ankur Parikh*, Chandra Childress, Qiong Lin, Daniel Rukstalis, Wannian Yang, Danville, PA INTRODUCTION AND OBJECTIVES: We have previously demonstrated that statins induce autophagy and cause rapid cell death in PC-3 cells. Autophagy, or “self-eating”, is a process responsible for intracellular material degradation, in which cytoplasmic components are engulfed by autophagosomes and delivered to lysosomes for degradation. Statins, a class of inhibitors of 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are commonly prescribed medications for treatment of hypercholesterolemia. Inhibition of HMG-CoA reductase halts biosynthesis of metabolites in mevalonate pathway that are required for production of cholesterol, ubiquinone (Coenzyme Q10), geranylgeranyl pyrophosphate and farnesyl pyrophosphate. To define which metabolite biosynthesis is involved in atorvastatin-induced autophagy, we performed rescue experiments by supplementation of various metabolites to cell culture medium. METHODS: PC-3 cells were plated and treated with atorvastatin. Rescue experiments were performed with the addition of geranylgeraniol, farnesol, coenzyme Q 10, and squalene. Autophagic response was evaluated with Western Blot assays for expression of LC3-II, a widely accepted marker for autophagy. RESULTS: As shown in Fig. 1, addition of geranylgeraniol in culture medium along with atorvastatin completely reversed atorvastatin-induced LC3-II expression in PC3 cells (lane 3), while geranylgeraniol alone did not have any effect on LC3-II expression (lane 7).
HIGH DENSITY LIPOPROTEIN INDUCES PROLIFERATION AND MIGRATION OF HUMAN PROSTATE ANDROGEN INDEPENDENT CANCER CELLS VIA ATP-BINDING CASSETTE TRANSPORTER A1 BY A CHOLESTEROL-INDEPENDENT MECHANISM Yoshitaka Sekine*, Bethesda, MD; Yosuke Furuya, Hidekazu Koike, Kazuhiro Suzuki, Maebashi, Japan; Alan Remaley, Bethesda, MD INTRODUCTION AND OBJECTIVES: Androgen deprivation therapy in men with prostate cancer (PC) leads to a significant increase of HDL. The increase is generally viewed as beneficial, particularly for cardiovascular disease, but the effects of HDL on prostate cancer are unknown. ATP-binding Cassette Transporter A1 (ABCA1), ATP-binding Cassette Transporter G1 and scavenger receptor class B member 1 are receptors for HDL and have important roles in HDL-induced signal transductions. In this study, we investigated the effect of HDL on prostate cancer cell proliferation, migration, cholesterol levels, and signal transductions, and if they are mediated by one of the receptors for HDL. METHODS: HDL was isolated from health volunteer men. mRNA expressions of PC cells and human prostate biopsy samples were evaluated by quantitative real-time PCR. Cell viability was determined by MTS assay. Cell migration was shown by would healing assay. The activation of MAPK and Akt were detected by Western blot analysis. The functional role of ABCA1 in any of the observed phenomena was examined after transfection with a siRNA against ABCA1. RESULTS: HDL could induce PC-3 and DU145 cell proliferations, migrations and both MAPK and Akt signal transductions, but not in LNCaP cells. Treatment with HDL did not significantly alter cellular cholesterol levels in PC cells. In cells grown in 10% FCS, the expression of ABCA1 was much lower in LNCaP than PC-3 and DU145. After culturing in androgen-free condition, ABCA1 expression increased 10 times in LNCaP cells. In human prostate biopsy samples, ABCA1 expression was significantly higher in androgen deprivation therapy group than BPH and pre-treatment prostate cancer group. Knockdown of ABCA1 by siRNA inhibited HDL-induced cell proliferation, migration
Vol. 183, No. 4, Supplement, Sunday, May 30, 2010
and both MAPK and Akt signal transductions in PC-3. Moreover, after treatment of androgen free condition for 96 hour, HDL could induce MAPK activation, and knockdown of ABCA1 expression by siRNA inhibited HDL-induced MAPK activation in LNCaP cells. Simvastatin, which inhibited ABCA1 expression in PC-3 and DU145 cells, inhibited HDL-induced PC-3 and DU145 cell proliferation, migration and both MAPK and Akt signal transduction. CONCLUSIONS: HDL can induce androgen independent prostate cancer cell proliferation and migration via ABCA1, which can be reversed by simvastatin. These results suggest that therapies that modulate HDL and lipid metabolism can potentially affect prostate cancer growth. Source of Funding: Intramural research funds from the National Heart, Lung and Blood Institute.
104 DIRECT EFFECTS OF ZOLEDRONIC ACID ON HORMONE RESPONSIVE AND HORMONE REFRACTORY PROSTATE CANCER CELLS Sweaty Koul, Binod Kumar, Paul Maroni, Randall Meacham, Hari Koul*, Aurora, CO INTRODUCTION AND OBJECTIVES: Bone metastasis is a common site of metastasis in patients with advanced prostate cancer develop bone metastasis. Interleukin 6 (IL-6), plays an important role in bone remodeling associated with progression of prostate cancer, evolution of androgen independence and finally for resistance to chemotherapeutics. Previous studies have shown that IL-6 is not only a paracrine factor essential for promoting survival of neighboring cells, but also may act in an autocrine fashion, promoting survival of the tumor cells. Moreover, NF-KB is known to promote survival of cancer cells. The bisphosphonate zoledronic acid has been shown to decrease bone resorption due to tumor metastasis probably due to its effect on the growth and viability of osteoclasts present at the site of metastasis. Presently, zoledronic acid is also the only bisphosphonate approved for the treatment of bone metastases from prostate cancer in patients who have progressed after treatment with at least one hormonal therapy. METHODS: We investigated the effect of zoledronic acid on IL-6 production and the associated transcription factor NF-kB in PC3 (hormone independent) and LNCaP (hormone sensitive) prostate cancer cells. Gene expression was monitored by relative quantitative PCR. NFKB was monitored by Immunofluorescence microscopy. Cell growth and viability were measured by crystal violet and MTT assays. RESULTS: Treatment with zoledronic acid resulted in a drastic reduction in the levels of IL-6 mRNA in a concentration dependent manner in PC3 cells. Treatment with zoledronic acid also reduced nuclear localization of the transcription factor NF-kB which is responsible for IL-6 synthesis. We also observed that LNCaP cells synthesize IL-6 in response to TNF-a and zoledronic acid inhibited TNF-a induced ˆ B nuclear localization in LNCaP cells as well. These results show NF-fU that zoledronic acid inhibits both constitutive and TNF-stimulated IL-6 and NF-KB production in both hormone independent and hormone sensitive prostate cancer cell lines. In addition treatments with zoledronic acid resulted in dose and time dependent decrease in survival of prostate cancer cells. CONCLUSIONS: The results presented here suggest that zoledronic acid may not only have effects on bone resorption, but may also directly impact cancer cells. Zoledronic acid could potentially serve as a therapeutic agent in early stages of prostate cancer and perhaps other cancers targeting bone. Source of Funding: Supported in part by Investigator initiated Grant from Novartis (CZOL446EUS105) and by The Dept. of Surgery-School of Medicine, CU Denver School of Medicine Academic Enrichment Funds to H. K. Koul
THE JOURNAL OF UROLOGY姞
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105 NOGGIN CONTRIBUTES TO THE OSTEOLYTIC RESPONSE IN BONE METASTASIS OF PROSTATE CANCER Chiara Secondini, Antoinette Wetterwald, Ruth Schwaninger, Marco G. Cecchini, George N. Thalmann*, Bern, Switzerland INTRODUCTION AND OBJECTIVES: Members of the bone morphogenetic protein (BMP) and the wingless (Wnt) protein family play a relevant role in the osteoblast response to prostate cancer (CaP) bone metastasis and their activity is modulated by extracellular antagonists. Lack of expression of the BMP antagonist noggin by CaP cells determines their osteoinductive activity in vivo. Osteolytic cells express noggin constitutively. The Wnt-antagonist dickkopf-1 (DKK-1) represses bone formation and noggin may act in analogy on BMP activity. We utilized a RNA silencing strategy to investigate whether constitutive noggin expression by an osteolytic CaP cell line determines the osteolytic phenotype. METHODS: Luciferase-expressing PC-3 cell clones were transfected with shRNA vectors to produce stable noggin-knock down (Nog-KD) clones. A vector encoding a non-targeting shRNA (mock) was used as negative control. Silencing efficiency was monitored by real time PCR and immunoblotting, respectively. Nog-KD and mock clones were xenografted into the tibia of nude mice and intra-osseous growth and osteolytic effect were monitored by bioluminescent imaging (BLI) and radiography, respectively. 3-D images of the xenografts were generated by micro-tomography (£gCT). Bone structural parameters were analyzed by peripheral quantitative computerized tomography (pQCT) and histomorphometry. RESULTS: Significant noggin mRNA and protein expression decrease in several Nog-KD clones with unchanged growth rates, osteolytic cytokine expression (PTHrP and IL-8), and of DKK-1 In vitro were observed. Tumor growth in bone xenografts of different mock and Nog-KD clones was only marginally or not affected. Radiographic and fY´CT analysis showed that the bones xenografted with the Nog-KD clones have structural modifications indicative of bone formation/repair activity but not parental PC-3 cells and mock clones. Bone histomorphometry and pQCT further corroborated these findings. CONCLUSIONS: Noggin expression by prostate cancer cells contribute to the induction of an osteolytic lesion. Conversely, shRNAmediated suppression of noggin expression in the osteolytic cell line PC-3 restored bone formation and may represent a new therapeutic option for the treatment of osteolytic bone metastases. Source of Funding: None
106 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-2 (IGFBP-2) ALTERS PTEN ACTIVITY AND REDUCES THE EFFICACY OF DOCETAXEL IN THE TREATMENT OF PROSTATE CANCER (PCA) Christopher Uzoh*, Jeff Holly, Raj Persad, Amit Bahl, Claire Perks, Bristol, United Kingdom INTRODUCTION AND OBJECTIVES: PCa is 5th most diagnosed cancer, worldwide. Aggressive disease, androgen-independence and treatment failure are predominant reasons for high mortality. We aim to identify new molecular targets that can be exploited to slow progression. Inactivation of tumor suppressor genes, changes in androgen regulation and activation of the insulin-like growth factor axis are common events in PCa pathogenesis and progression. Tumor suppressor gene PTEN is most frequently mutated in metastatic PCa and it dictates progression. IGFBP-2 can modulate IGF actions but also has intrinsic, IGF-independent actions, promoting growth of PCa cells. Its levels increase 2-3-fold in serum of PCa patients and correlate with serum PSA and Gleason score. It plays a key role in promoting androgen-independent and metastatic phenotype and is a biomarker of PTEN status. However, its mechanism of action and relationship to PTEN remain unknown. METHODS: DU145 and PC3 PCa cell lines were treated with either IGFBP-2 (0-1000ng/ml)(GroPep, Australia) or IGFBP-2 siRNA