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INHIBITION OF ACID CERAMIDASE SENSITIZES PROSTATE CANCER TO RADIATION THERAPY BOTH IN VITRO AND IN VIVO
42
THE JOURNAL OF UROLOGY®
VHQVLWL]HGWRWXPRUVDQGDOVRWUDI¿FWKURXJKWLVVXHDQGFDQDFWGLUHFWO\ as cytolytic effector cells. METHODS: We previously presented the spontaneous prostate cancer model by knocking out PTEN gene and cultured PTEN-CaP8 cells (CaP8) ex-vivo. RCR spreading among the tumor ZDVGHWHFWHGZLWKJUHHQÀXRUHVFHQWSURWHLQ *)3 E\ÀRZF\WRPHWU\ Therapeutic effect of RCR suicide gene vector carrying the yeast cytosine GHDPLQDVH &' JHQHDQGWXPRUVSHFL¿F&7/VZHUHH[DPLQHGZLWK076 cytotoxicity assay. In vivoWXPRUWUDI¿FNLQJHIIHFWRI&7/VDQGF\WRWR[LFLW\ of locally admnistered CTLs were shown by live imaging of luciferase. To determine the treatment effect, we followed the CaP8 subcutaneous WXPRU VL]H DQG OXFLIHUDVH H[SUHVVLRQ DIWHU LQMHFWLRQ RI WXPRUVSHFL¿F CTLs to check the cytotoxicity. RESULTS: First, RCR vectors were tested for their ability to spread among the CaP8 cells, and GFP was detected more than 90% E\GD\VDIWHUWUDQVGXFWLRQZLWK5&5*)3DW02,:HFRQ¿UPHG CaP8 cells transduced with RCR-CD at various ratios could be killed dose- and time-dependently. CTLs also killed the prostate cancer cells dose-dependently in vitro. CTLs’ effects were tracked by in vivo image ¿J 7XPRUVL]HVKRZHGVLJQL¿FDQWUHGXFWLRQRIWKHWXPRUDIWHUWUHDWHG with CTLs. &21&/86,216 7KHVH UHVXOWV FRQ¿UP WKDW 5&5 YHFWRUV spread in prostate cancer and impart them with susceptibility to suicide gene therapy in the novel PTEN knock-out spontaneous prostate cancer PRGHO&7/VDOVRHI¿FLHQWO\NLOOWKHWXPRUGHPRQVWUDWLQJWKHIHDVLELOLW\ of combining adoptive immunotherapy with viroreplicative gene therapy for prostate cancer.
Source of Funding: None
119 INHIBITION OF ACID CERAMIDASE SENSITIZES PROSTATE CANCER TO RADIATION THERAPY BOTH IN VITRO AND IN VIVO Ayman Mahdy*, Xiang Liu, Jun Li, Saeed Elojeimy, Joe Cheng, Lorianne Turner, Aiping Bai, Ahmed M El-Zawahry, Alicja Bielawska, Yusuf A Hannun, Thomas E Keane, Mohammed I Taha, Hisham M Hammouda, James S Norris. Weston, FL, Charleston, SC, Charletsion, SC, and Assiut, Egypt. INTRODUCTION AND OBJECTIVE: Approximately one half of prostate cancer (PCa) is treated with radiotherapy. For intermediate and unfavorable risk groups, radiation resistance, local recurrence and impotence is an issue. We have data that the tumor suppressor lipid ceramide is an important factor for modulation of radiation-induced UHVLVWDQFH&HUDPLGHLVPHWDEROL]HGE\DFLGFHUDPLGDVH $& SUHYLRXVO\ reported as over-expressed in >60% of PCa tissue samples (Norris et al., CGT, June 2006). This promotes a shift in the ceramide/sphingosine1-phosphate (S1P) rheostat toward anti-apoptotic S1P. We now report radiation up regulates AC expression in a time dependent manner. In view of these data our objective is to investigate AC inhibition as a PHFKDQLVPRIVHQVLWL]DWLRQRI 3&D WRUDGLDWLRQWKHUDS\ METHODS: AC Protein level was measured by Western blotting. Trypan blue (TB) exclusion and MTT assays were used to measure cell viability and Western blotting to detect caspase 3 activation.
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
Small interfering RNA (siRNA) transfection was used for AC knock down in PPC1 PCa cells. Cell survival was determined by clonogenic assay DQGTXDQWL¿HGE\GHQVLWRPHWULFDQDO\VLV3&D[HQRJUDIWPRGHOVZHUH treated with different treatment combinations including radiation, alone or with the AC inhibitor LCL 385 and tumor growth was followed. RESULTS: In this study we show that PPC1 cells are radiation resistant and radio-resistance is related to increased levels of AC. With SiRNA, AC knock down increased cell killing by the MTS assay by 40% when compared with the non SiRNA transfected cells. Using the AC inhibitor LCL385 at a subtoxic dose we prevented both AC upregulation and activity in response to radiation. Combining LCL 385 with radiation also resulted in a 50% increased cell killing and caspase 3 activation when compared with either treatment alone. In the animal models and 4 weeks after treatment, combining radiation with LCL385 resulted in DQGGHFUHDVHLQWXPRUVL]HFRPSDUHGZLWKUDGLDWLRQRU/&/ 385 alone respectively. CONCLUSIONS: AC upregulation in PCa in response to UDGLDWLRQWKHUDS\LVK\SRWKHVL]HGWREHDPHFKDQLVPRIUDGLRUHVLVWDQFH $&LQKLELWLRQVHQVLWL]HV3&DFHOOVWRUDGLDWLRQWUHDWPHQW:HVXJJHVW /&/LVDUDGLRVHQVLWL]HUZRUWKLQYHVWLJDWLQJIRUFOLQLFDOUHOHYDQFH Source of Funding: PPG.
120 INTER-RELATED EFFECTS OF ANDROGENS, FATTY ACIDS AND OXIDATIVE STRESS IN PROSTATE CANCER – A MECHANISTIC SUPPORT FOR PREVENTION STRATEGIES Jehonathan H Pinthus*, Helen Lin, Inna Bryskin, Nir Kleinmann, Brian Wilson, Gurmit Singh. Hamilton, ON, Canada, and Toronto, ON, Canada. INTRODUCTION AND OBJECTIVE: Epidemiological evidences suggest that dietary fat may play a role in the etiology of prostate cancer (PC). Oxidation of fatty acids (FA) results in the generation of reactive oxygen species (ROS) which have been postulated to play a key role in the initiation and progression of PC. The purpose of this study was to determine whether androgens, which stimulate the growth of PC, also play a role in fatty acid (FA) uptake and degradation and consequently increase mitochondrial ROS production. METHODS: The model used compared the effect of a synthetic androgen (R1881) and an androgen receptor (AR) blocker (bicalutamide) on androgen-sensitive LNCaP and androgen responsive 22rv1 cells versus that on the androgen-independent CL1 subline that was derived from the parental androgen-sensitive LNCaP cells. Methods 8VHG ZHUH LPPXQRÀXRUHVFHQFH FRQIRFDO PLFURVFRS\ ZHVWHUQ EORW ÀRZF\WRPHWU\+ROHDWHXSWDNHDQG&UDGLRODEHOHGORQJFKDLQ)$ degradation studies. RESULTS: Androgen supplementation increased the cellular and surface expression of the plasma membrane fatty acid binding SURWHLQ )$%3SP OHDGLQJWRLQFUHDVHXSWDNHRIÀXRUHVFHQWO\ODEHOHG FA and of 3H-oleate only in PC cells that express the AR. Bicalutamide inhibited this phenomenon. Furthermore, androgen supplementation VLJQL¿FDQWO\ LQFUHDVHG WKH R[LGDWLRQ RI )$ E\ LQFUHDVLQJ WKH OHYHOV RI &37 WKH UDWH OLPLWLQJ HQ]\PH LQ WKLV SDWKZD\ 6XEVHTXHQWO\ ZH demonstrated that blockage of mitochondrial ROS generation by 2 GLIIHUHQWLQKLELWRUVURWHQRQHDQGWKHQR\OWULÀXRURDFHWRQHFRXOGHOLPLQDWH the androgen induced generation of cellular ROS which is coupled to FA oxidation, bringing it to the same levels of PC cells that were deprived of androgens or treated with bicalutamide. CONCLUSIONS: We propose that the uptake of FA into PC cells is androgen regulated through the increased expression of FABPpm. More FA are therefore available for mitochondrial oxidation, a process that is also positively regulated by androgens, leading to the increased production of ROS that are associated with cancer cell proliferation and mutagenesis. These results may support the rationale for PC prevention using 5-alpha reductase inhibitors, dietary restrictions or anti-oxidants, all of which each have different inhibitory but complementary effect on the proposed mechanism. Source of Funding: None
THE JOURNAL OF UROLOGY®
VHQVLWL]HGWRWXPRUVDQGDOVRWUDI¿FWKURXJKWLVVXHDQGFDQDFWGLUHFWO\ as cytolytic effector cells. METHODS: We previously presented the spontaneous prostate cancer model by knocking out PTEN gene and cultured PTEN-CaP8 cells (CaP8) ex-vivo. RCR spreading among the tumor ZDVGHWHFWHGZLWKJUHHQÀXRUHVFHQWSURWHLQ *)3 E\ÀRZF\WRPHWU\ Therapeutic effect of RCR suicide gene vector carrying the yeast cytosine GHDPLQDVH &' JHQHDQGWXPRUVSHFL¿F&7/VZHUHH[DPLQHGZLWK076 cytotoxicity assay. In vivoWXPRUWUDI¿FNLQJHIIHFWRI&7/VDQGF\WRWR[LFLW\ of locally admnistered CTLs were shown by live imaging of luciferase. To determine the treatment effect, we followed the CaP8 subcutaneous WXPRU VL]H DQG OXFLIHUDVH H[SUHVVLRQ DIWHU LQMHFWLRQ RI WXPRUVSHFL¿F CTLs to check the cytotoxicity. RESULTS: First, RCR vectors were tested for their ability to spread among the CaP8 cells, and GFP was detected more than 90% E\GD\VDIWHUWUDQVGXFWLRQZLWK5&5*)3DW02,:HFRQ¿UPHG CaP8 cells transduced with RCR-CD at various ratios could be killed dose- and time-dependently. CTLs also killed the prostate cancer cells dose-dependently in vitro. CTLs’ effects were tracked by in vivo image ¿J 7XPRUVL]HVKRZHGVLJQL¿FDQWUHGXFWLRQRIWKHWXPRUDIWHUWUHDWHG with CTLs. &21&/86,216 7KHVH UHVXOWV FRQ¿UP WKDW 5&5 YHFWRUV spread in prostate cancer and impart them with susceptibility to suicide gene therapy in the novel PTEN knock-out spontaneous prostate cancer PRGHO&7/VDOVRHI¿FLHQWO\NLOOWKHWXPRUGHPRQVWUDWLQJWKHIHDVLELOLW\ of combining adoptive immunotherapy with viroreplicative gene therapy for prostate cancer.
Source of Funding: None
119 INHIBITION OF ACID CERAMIDASE SENSITIZES PROSTATE CANCER TO RADIATION THERAPY BOTH IN VITRO AND IN VIVO Ayman Mahdy*, Xiang Liu, Jun Li, Saeed Elojeimy, Joe Cheng, Lorianne Turner, Aiping Bai, Ahmed M El-Zawahry, Alicja Bielawska, Yusuf A Hannun, Thomas E Keane, Mohammed I Taha, Hisham M Hammouda, James S Norris. Weston, FL, Charleston, SC, Charletsion, SC, and Assiut, Egypt. INTRODUCTION AND OBJECTIVE: Approximately one half of prostate cancer (PCa) is treated with radiotherapy. For intermediate and unfavorable risk groups, radiation resistance, local recurrence and impotence is an issue. We have data that the tumor suppressor lipid ceramide is an important factor for modulation of radiation-induced UHVLVWDQFH&HUDPLGHLVPHWDEROL]HGE\DFLGFHUDPLGDVH $& SUHYLRXVO\ reported as over-expressed in >60% of PCa tissue samples (Norris et al., CGT, June 2006). This promotes a shift in the ceramide/sphingosine1-phosphate (S1P) rheostat toward anti-apoptotic S1P. We now report radiation up regulates AC expression in a time dependent manner. In view of these data our objective is to investigate AC inhibition as a PHFKDQLVPRIVHQVLWL]DWLRQRI 3&D WRUDGLDWLRQWKHUDS\ METHODS: AC Protein level was measured by Western blotting. Trypan blue (TB) exclusion and MTT assays were used to measure cell viability and Western blotting to detect caspase 3 activation.
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
Small interfering RNA (siRNA) transfection was used for AC knock down in PPC1 PCa cells. Cell survival was determined by clonogenic assay DQGTXDQWL¿HGE\GHQVLWRPHWULFDQDO\VLV3&D[HQRJUDIWPRGHOVZHUH treated with different treatment combinations including radiation, alone or with the AC inhibitor LCL 385 and tumor growth was followed. RESULTS: In this study we show that PPC1 cells are radiation resistant and radio-resistance is related to increased levels of AC. With SiRNA, AC knock down increased cell killing by the MTS assay by 40% when compared with the non SiRNA transfected cells. Using the AC inhibitor LCL385 at a subtoxic dose we prevented both AC upregulation and activity in response to radiation. Combining LCL 385 with radiation also resulted in a 50% increased cell killing and caspase 3 activation when compared with either treatment alone. In the animal models and 4 weeks after treatment, combining radiation with LCL385 resulted in DQGGHFUHDVHLQWXPRUVL]HFRPSDUHGZLWKUDGLDWLRQRU/&/ 385 alone respectively. CONCLUSIONS: AC upregulation in PCa in response to UDGLDWLRQWKHUDS\LVK\SRWKHVL]HGWREHDPHFKDQLVPRIUDGLRUHVLVWDQFH $&LQKLELWLRQVHQVLWL]HV3&DFHOOVWRUDGLDWLRQWUHDWPHQW:HVXJJHVW /&/LVDUDGLRVHQVLWL]HUZRUWKLQYHVWLJDWLQJIRUFOLQLFDOUHOHYDQFH Source of Funding: PPG.
120 INTER-RELATED EFFECTS OF ANDROGENS, FATTY ACIDS AND OXIDATIVE STRESS IN PROSTATE CANCER – A MECHANISTIC SUPPORT FOR PREVENTION STRATEGIES Jehonathan H Pinthus*, Helen Lin, Inna Bryskin, Nir Kleinmann, Brian Wilson, Gurmit Singh. Hamilton, ON, Canada, and Toronto, ON, Canada. INTRODUCTION AND OBJECTIVE: Epidemiological evidences suggest that dietary fat may play a role in the etiology of prostate cancer (PC). Oxidation of fatty acids (FA) results in the generation of reactive oxygen species (ROS) which have been postulated to play a key role in the initiation and progression of PC. The purpose of this study was to determine whether androgens, which stimulate the growth of PC, also play a role in fatty acid (FA) uptake and degradation and consequently increase mitochondrial ROS production. METHODS: The model used compared the effect of a synthetic androgen (R1881) and an androgen receptor (AR) blocker (bicalutamide) on androgen-sensitive LNCaP and androgen responsive 22rv1 cells versus that on the androgen-independent CL1 subline that was derived from the parental androgen-sensitive LNCaP cells. Methods 8VHG ZHUH LPPXQRÀXRUHVFHQFH FRQIRFDO PLFURVFRS\ ZHVWHUQ EORW ÀRZF\WRPHWU\+ROHDWHXSWDNHDQG&UDGLRODEHOHGORQJFKDLQ)$ degradation studies. RESULTS: Androgen supplementation increased the cellular and surface expression of the plasma membrane fatty acid binding SURWHLQ )$%3SP OHDGLQJWRLQFUHDVHXSWDNHRIÀXRUHVFHQWO\ODEHOHG FA and of 3H-oleate only in PC cells that express the AR. Bicalutamide inhibited this phenomenon. Furthermore, androgen supplementation VLJQL¿FDQWO\ LQFUHDVHG WKH R[LGDWLRQ RI )$ E\ LQFUHDVLQJ WKH OHYHOV RI &37 WKH UDWH OLPLWLQJ HQ]\PH LQ WKLV SDWKZD\ 6XEVHTXHQWO\ ZH demonstrated that blockage of mitochondrial ROS generation by 2 GLIIHUHQWLQKLELWRUVURWHQRQHDQGWKHQR\OWULÀXRURDFHWRQHFRXOGHOLPLQDWH the androgen induced generation of cellular ROS which is coupled to FA oxidation, bringing it to the same levels of PC cells that were deprived of androgens or treated with bicalutamide. CONCLUSIONS: We propose that the uptake of FA into PC cells is androgen regulated through the increased expression of FABPpm. More FA are therefore available for mitochondrial oxidation, a process that is also positively regulated by androgens, leading to the increased production of ROS that are associated with cancer cell proliferation and mutagenesis. These results may support the rationale for PC prevention using 5-alpha reductase inhibitors, dietary restrictions or anti-oxidants, all of which each have different inhibitory but complementary effect on the proposed mechanism. Source of Funding: None