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415 ANDROGRAPHOLIDE TARGETS AR PATHWAY IN CASTRATION-RESISTANT PROSTATE CANCER
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
414 VACCINATION WITH RAD5 CARRYING A FUSION CONSTRUCT OF PROSTATE-SPECIFIC ANTIGENS GENERATES EFFECTOR T CELLS Felipe Rosso*, Jeffrey Holzbeierlein, Peter Van Veldhuizen, Dev Karan, Kansas City, KS INTRODUCTION AND OBJECTIVES: Immunotherapy has emerged as a promising approach in the development of novel therapies for prostate cancer. While most currently approved vaccines are targeted toward a single antigen, the heterogeneity of prostate cancer may create a limitation toward achieving a maximal effector response with this approach. Our goal is to expand the range of anti-tumor responses by creating a multi-antigenic T cell response utilizing a single immunization with a recombinant viral construct containing a fusion of two known prostate cancer antigens. METHODS: A fusion construct (PSPA) containing prostatespecific antigen (PSA) and prostate stem cell antigen (PSCA) was created and combined with a recombinant adenovirus type 5 (rAd5) vector and injected into mice. Following immunization, spleen cell suspensions were processed to analyze the production of IFN-␥ in the vaccinated mice compared to control. In vivo cytotoxic T cell assays and tumor growth challenge studies were compared in the vaccinated group compared to control mice. Student’s t-test was used to determine levels of significance among groups studied. RESULTS: Average immune responses of anti-PSA (p⫽0.037) and anti-PSCA (p⫽0.026) CD8 T cells following Ad5PSPA immunization were significantly higher as compared to controls. A reduced level of anti-PSA IFN-␥ was noted in comparison to anti-PSCA following immunization (p⫽0.056). Immunized mice showed a strong cytotoxic response, being able to lyse 80% and 100% of PSA or PSCA coated target cells, respectively (p⫽0.034 and 0.005, respectively). Immunized mice demonstrated a significant inhibition of tumor outgrowth derived from PSA-expressing murine tumor cell lines with a total survival rate of 60%. CONCLUSIONS: Immunization of mice with rAd5 vector carrying a fusion construct of PSA and PSCA simultaneously induces the expansion of anti-PSA and anti-PSCA CD8 T cells. The developed antigen-specific T cell responses were efficient in eliminating target cells expressing cognate antigens, and strong anti-tumor immunity was elicited when challenged with prostate tumor cell lines. The effector response caused by the use of multiple antigens may have a synergistic effect to significantly reduce tumor burden and have important implications for human clinical trials targeted at prostate cancer. Source of Funding: Hearst Foundation and the Veterans Affairs Medical Center
415 ANDROGRAPHOLIDE TARGETS AR PATHWAY IN CASTRATION-RESISTANT PROSTATE CANCER Chengfei Liu*, Meng Sun, Wei Lou, Jaeyeon Chun, Nagalakshmi Nadiminty, Christopher Evans, Allen Gao, Sacramento, CA INTRODUCTION AND OBJECTIVES: The Androgen Receptor pathway plays a central role in growth and survival of castrationresistant prostate cancer (CRPC). The first line of treatment of androgen-dependent prostate cancer is the use of androgen-deprivation therapy. However, most of the patients will eventually relapse due to development of CRPC. Bicalutamide is used as a second line treatment option for patients who fail androgen&endash;deprivation therapy. Unfortunately, patients will also develop a resistance to bicalutamide treatment. Thus develop a strategy to treat bicalutamide resistant prostate cancer is urgently needed. METHODS: We have previously identified andrographolide can suppress tumor growth of prostate cancer cells by screening compounds from the Natural compound library. LNCaP and C4-2 cells were treated with either bicalutamide or andrographolide in FBS or charcoalstripped FBS conditions. Cell growth was determined and the levels of
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AR expression were measured by qRT-PCR and Western blots. The effects on PSA and NKX3.1 were also examined by ELISA and luciferase reporter assays. AR nuclear translocation was examined by immunofluorescence assays. The effects of andrographolide on the recruitment of AR to the androgen responsive genes were examined by Chromatin Immunoprecipitation (ChIP) Assays. RESULTS: We identified andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata which exhibit a wide spectrum of biological activities of therapeutic importance, can significantly inhibit prostate cancer cell growth and induce apoptosis. Andrographolide not only inhibits AR activity, such as inhibition of the expression of androgen regulated genes (PSA and NKX3.1), but is also able to down regulate AR expression at both mRNA and protein levels and prevents its nuclear translocation. Andrographolide prevents the binding of Hsp90 to AR, resulting in proteasome-mediated AR degradation. Furthermore, andrographolide inhibits bicalutamide resistant C4-2 cell growth by reducing AR expression and activity. Combination of andrographolide with bicalutamide achieved synergistic effect in inhibition of C4-2 cell growth and AR expression. CONCLUSIONS: We have characterized and identified andrographolide as a potential therapeutic agent for prostate cancer by inhibiting androgen receptor signaling. Andrographolide inhibits the growth of castration resistant prostate cancer after failure of bicalutamide treatment. Andrographolide could be developed as a potential anti-androgen against castration-resistant prostate cancer. Source of Funding: R01CA140468
416 IMPACT OF COMBINED HDAC AND MTOR INHIBITION ON THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS Steffen Wedel*, Lucasz Hudak, Jasmina Makarevic, Jens-Michael Seibel, Eva Juengel, Axel Haferkamp, Roman Blaheta, Frankfurt am Main, Germany INTRODUCTION AND OBJECTIVES: The concept of molecular tumor targeting has recently become standard in the therapy of renal cell carcinoma and might also provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. METHODS: RAD001 or VPA were applied to PC-3, DU-145 and LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin ␣ and  subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. RESULTS: Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase deactivation. Integrin subtype expression was altered significantly by both compounds (VPA ⬎ RAD001). Particularly,3 coding mRNA was diminished drastically with the combination being superior then single drug treatment. Furthermore, when both drugs were used in concert additive effects were observed on the migratory and invasive behavior of the prostate cancer cells. CONCLUSIONS: Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA application alone with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility. Source of Funding: supported by the ”Jung-Stiftung”
414 VACCINATION WITH RAD5 CARRYING A FUSION CONSTRUCT OF PROSTATE-SPECIFIC ANTIGENS GENERATES EFFECTOR T CELLS Felipe Rosso*, Jeffrey Holzbeierlein, Peter Van Veldhuizen, Dev Karan, Kansas City, KS INTRODUCTION AND OBJECTIVES: Immunotherapy has emerged as a promising approach in the development of novel therapies for prostate cancer. While most currently approved vaccines are targeted toward a single antigen, the heterogeneity of prostate cancer may create a limitation toward achieving a maximal effector response with this approach. Our goal is to expand the range of anti-tumor responses by creating a multi-antigenic T cell response utilizing a single immunization with a recombinant viral construct containing a fusion of two known prostate cancer antigens. METHODS: A fusion construct (PSPA) containing prostatespecific antigen (PSA) and prostate stem cell antigen (PSCA) was created and combined with a recombinant adenovirus type 5 (rAd5) vector and injected into mice. Following immunization, spleen cell suspensions were processed to analyze the production of IFN-␥ in the vaccinated mice compared to control. In vivo cytotoxic T cell assays and tumor growth challenge studies were compared in the vaccinated group compared to control mice. Student’s t-test was used to determine levels of significance among groups studied. RESULTS: Average immune responses of anti-PSA (p⫽0.037) and anti-PSCA (p⫽0.026) CD8 T cells following Ad5PSPA immunization were significantly higher as compared to controls. A reduced level of anti-PSA IFN-␥ was noted in comparison to anti-PSCA following immunization (p⫽0.056). Immunized mice showed a strong cytotoxic response, being able to lyse 80% and 100% of PSA or PSCA coated target cells, respectively (p⫽0.034 and 0.005, respectively). Immunized mice demonstrated a significant inhibition of tumor outgrowth derived from PSA-expressing murine tumor cell lines with a total survival rate of 60%. CONCLUSIONS: Immunization of mice with rAd5 vector carrying a fusion construct of PSA and PSCA simultaneously induces the expansion of anti-PSA and anti-PSCA CD8 T cells. The developed antigen-specific T cell responses were efficient in eliminating target cells expressing cognate antigens, and strong anti-tumor immunity was elicited when challenged with prostate tumor cell lines. The effector response caused by the use of multiple antigens may have a synergistic effect to significantly reduce tumor burden and have important implications for human clinical trials targeted at prostate cancer. Source of Funding: Hearst Foundation and the Veterans Affairs Medical Center
415 ANDROGRAPHOLIDE TARGETS AR PATHWAY IN CASTRATION-RESISTANT PROSTATE CANCER Chengfei Liu*, Meng Sun, Wei Lou, Jaeyeon Chun, Nagalakshmi Nadiminty, Christopher Evans, Allen Gao, Sacramento, CA INTRODUCTION AND OBJECTIVES: The Androgen Receptor pathway plays a central role in growth and survival of castrationresistant prostate cancer (CRPC). The first line of treatment of androgen-dependent prostate cancer is the use of androgen-deprivation therapy. However, most of the patients will eventually relapse due to development of CRPC. Bicalutamide is used as a second line treatment option for patients who fail androgen&endash;deprivation therapy. Unfortunately, patients will also develop a resistance to bicalutamide treatment. Thus develop a strategy to treat bicalutamide resistant prostate cancer is urgently needed. METHODS: We have previously identified andrographolide can suppress tumor growth of prostate cancer cells by screening compounds from the Natural compound library. LNCaP and C4-2 cells were treated with either bicalutamide or andrographolide in FBS or charcoalstripped FBS conditions. Cell growth was determined and the levels of
THE JOURNAL OF UROLOGY姞
e167
AR expression were measured by qRT-PCR and Western blots. The effects on PSA and NKX3.1 were also examined by ELISA and luciferase reporter assays. AR nuclear translocation was examined by immunofluorescence assays. The effects of andrographolide on the recruitment of AR to the androgen responsive genes were examined by Chromatin Immunoprecipitation (ChIP) Assays. RESULTS: We identified andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata which exhibit a wide spectrum of biological activities of therapeutic importance, can significantly inhibit prostate cancer cell growth and induce apoptosis. Andrographolide not only inhibits AR activity, such as inhibition of the expression of androgen regulated genes (PSA and NKX3.1), but is also able to down regulate AR expression at both mRNA and protein levels and prevents its nuclear translocation. Andrographolide prevents the binding of Hsp90 to AR, resulting in proteasome-mediated AR degradation. Furthermore, andrographolide inhibits bicalutamide resistant C4-2 cell growth by reducing AR expression and activity. Combination of andrographolide with bicalutamide achieved synergistic effect in inhibition of C4-2 cell growth and AR expression. CONCLUSIONS: We have characterized and identified andrographolide as a potential therapeutic agent for prostate cancer by inhibiting androgen receptor signaling. Andrographolide inhibits the growth of castration resistant prostate cancer after failure of bicalutamide treatment. Andrographolide could be developed as a potential anti-androgen against castration-resistant prostate cancer. Source of Funding: R01CA140468
416 IMPACT OF COMBINED HDAC AND MTOR INHIBITION ON THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS Steffen Wedel*, Lucasz Hudak, Jasmina Makarevic, Jens-Michael Seibel, Eva Juengel, Axel Haferkamp, Roman Blaheta, Frankfurt am Main, Germany INTRODUCTION AND OBJECTIVES: The concept of molecular tumor targeting has recently become standard in the therapy of renal cell carcinoma and might also provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. METHODS: RAD001 or VPA were applied to PC-3, DU-145 and LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin ␣ and  subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. RESULTS: Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase deactivation. Integrin subtype expression was altered significantly by both compounds (VPA ⬎ RAD001). Particularly,3 coding mRNA was diminished drastically with the combination being superior then single drug treatment. Furthermore, when both drugs were used in concert additive effects were observed on the migratory and invasive behavior of the prostate cancer cells. CONCLUSIONS: Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA application alone with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility. Source of Funding: supported by the ”Jung-Stiftung”