RESUMEN

OBJECTIVES: The objectives of this study are to explore the potential benefits of combining AdGlipr1 (or AdGLIPR1) gene therapy with radiotherapy using subcutaneous prostate and bladder cancer models. MATERIALS AND METHODS: Combination adenoviral vector-mediated gene therapy and radiotherapy were applied to 178-2 BMA and TSU-Pr1 cells in vitro and colony formation and apoptosis were analyzed. In addition, combination therapies were administered to mice bearing subcutaneous 178-2 BMA and TSU-Pr1 tumors, and tumor growth suppression and survival extension were compared with the monotherapies (AdGlipr1/AdGLIPR1 and radiotherapy) or control vector Adv/CMV/ßgal, as well as single-cycle treatment with 2-cycle treatment. RESULTS: Combination treatment significantly suppressed colony formation and increased apoptosis in vitro. In vivo, combination therapy produced significant 178-2 BMA and TSU-Pr1 tumor growth suppression and survival extension compared with the monotherapies or the control. Further tumor growth suppression and survival extension were observed after 2 cycles of the combination treatment. CONCLUSIONS: Combining AdGlipr1 (AdGLIPR1) with radiotherapy may achieve additive or synergistic tumor control in selected prostate and bladder tumors, and additional therapeutic effects may result with repeated treatment cycles.

Asunto(s)

Terapia Genética/métodos , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata/terapia , Radioterapia/métodos , Neoplasias de la Vejiga Urinaria/terapia , Adenoviridae/genética , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Terapia Combinada , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/efectos de la radiación , Masculino , Proteínas de la Membrana , Ratones , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Resultado del Tratamiento , Carga Tumoral/genética , Carga Tumoral/efectos de la radiación , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Previously, we reported that caveolin-1 (cav-1) is overexpressed in metastatic prostate cancer and that virulent prostate cancer cells secrete biologically active cav-1. We also showed that cav-1 expression leads to prosurvival activities through maintenance of activated Akt and that cav-1 is taken up by other cav-1-negative tumor cells and/or endothelial cells, leading to stimulation of angiogenic activities through PI-3-K-Akt-eNOS signaling. To analyze the functional consequences of cav-1 overexpression on the development and progression of prostate cancer in vivo, we generated PBcav-1 transgenic mice. Adult male PBcav-1 mice showed significantly increased prostatic wet weight and higher incidence of epithelial hyperplasia compared with nontransgenic littermates. Increased immunostaining for cav-1, proliferative cell nuclear antigen, P-Akt, and reduced nuclear p27(Kip1) staining occurred in PBcav-1 hyperplastic prostatic lesions. PBcav-1 mice showed increased resistance to castration-induced prostatic regression and elevated serum cav-1 levels compared with nontransgenic littermates. Intraprostatic injection of androgen-sensitive, cav-1-secreting RM-9 mouse prostate cancer cells resulted in tumors that were larger in PBcav-1 mice than in nontransgenic littermates (P = 0.04). Tail vein inoculation of RM-9 cells produced significantly more experimental lung metastases in PBcav-1 males than in nontransgenic male littermates (P = 0.001), and in cav-1(+/+) mice than in cav-1(-/-) mice (P = 0.041). Combination treatment with surgical castration and systemic cav-1 antibody dramatically reduced the number of experimental metastases. These experimental data suggest a causal association of secreted cav-1 and prostate cancer growth and progression.

Asunto(s)

Caveolina 1/metabolismo , Neoplasias de la Próstata/metabolismo , Análisis de Varianza , Proteína de Unión a Andrógenos/genética , Andrógenos/metabolismo , Animales , Caveolina 1/sangre , Caveolina 1/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Orquiectomía , Regiones Promotoras Genéticas/genética , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/sangre , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Caveolin-1 (cav-1) is a major structural protein of caveolae, small invaginations of the plasma membrane that integrate and regulate signaling pathways involved in cell growth and differentiation. We previously generated a genetically engineered mice that are homozygous for a null mutation in exon 2 of cav-1 and documented increased incidence of urolithiasis in young male cav-1(-/-) mice. We attributed this, in part, to improper localization of plasma membrane calcium/calmodulin-dependent calcium ATPase in the distal convoluted tubules of the kidney. To document pathologies related to cav-1 function, we maintained cav-1(-/-) and control cav-1(+/+) mice for an extended time period. We report here that cav-1(-/-) mice demonstrate organ-specific growth-related disorders in stromal cells that normally have high levels of cav-1 expression. In many of these organs, epithelial cell growth/differentiation abnormalities were also observed, yet in most of these sites the epithelial cells normally express low to non-detectable levels of cav-1. We propose that loss of cav-1 function in stromal cells of various organs directly leads to a disorganized stromal compartment that, in turn, indirectly promotes abnormal growth and differentiation of adjacent epithelium.

Asunto(s)

Caveolina 1/genética , Células Epiteliales/citología , Regulación de la Expresión Génica/fisiología , Silenciador del Gen , Animales , Biomarcadores/metabolismo , Caveolina 1/metabolismo , Diferenciación Celular , Células Epiteliales/fisiología , Femenino , Genitales/metabolismo , Genitales/patología , Queratinas/metabolismo , Longevidad , Pulmón/metabolismo , Pulmón/patología , Masculino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/metabolismo , Páncreas/patología , Bazo/metabolismo , Bazo/patología , Células del Estroma/citología , Células del Estroma/fisiología , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología

RESUMEN

Glioma pathogenesis-related protein 1 (GLIPR1), a novel p53 target gene, is down-regulated by methylation in prostate cancer and has p53-dependent and -independent proapoptotic activities in tumor cells. These properties suggest an important tumor suppressor role for GLIPR1, yet direct genetic evidence of a tumor suppressor function for GLIPR1 is lacking and the molecular mechanism(s), through which GLIPR1 exerts its tumor suppressor functions, has not been shown. Here, we report that the expression of GLIPR1 is significantly reduced in human prostate tumor tissues compared with adjacent normal prostate tissues and in multiple human cancer cell lines. Overexpression of GLIPR1 in cancer cells leads to suppression of colony growth and induction of apoptosis. Mice with an inactivated Glipr1 gene had significantly shorter tumor-free survival times than either Glipr1(+/+) or Glipr1(+/-) mice in both p53(+/+) and p53(+/-) genetic backgrounds, owing to their development of a unique array of malignant tumors. Mechanistic analysis indicated that GLIPR1 up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through activation of the c-Jun-NH(2) kinase (JNK) signaling cascade. Thus, our results identify GLIPR1 as a proapoptotic tumor suppressor acting through the ROS-JNK pathway and support the therapeutic potential for this protein.

Asunto(s)

Apoptosis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas del Tejido Nervioso/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Transformación Celular Neoplásica/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/fisiología , Predisposición Genética a la Enfermedad , Células HCT116 , Humanos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Transducción de Señal/fisiología

RESUMEN

PURPOSE: To explore long-term immune responses after combined radio-gene-hormonal therapy. METHODS AND MATERIALS: Thirty-three patients with prostate specific antigen 10 or higher or Gleason score of 7 or higher or clinical stage T2b to T3 were treated with gene therapy that consisted of 3 separate intraprostatic injections of AdHSV-tk on Days 0, 56, and 70. Each injection was followed by 2 weeks of valacyclovir. Intensity-modulated radiation therapy was delivered 2 days after the second AdHSV-tk injection for 7 weeks. Hormonal therapy was initiated on Day 0 and continued for 4 months or 2.3 years. Blood samples were taken before, during, and after treatment. Lymphocytes were analyzed by fluorescent antibody cell sorting (FACS). RESULTS: Median follow-up was 26 months (range, 4-48 months). The mean percentages of DR+CD8+ T cells were increased at all timepoints up to 8 months. The mean percentages of DR+CD4+ T cells were increased later and sustained longer until 12 months. Long-term (2.3 years) use of hormonal therapy did not affect the percentage of any lymphocyte population. CONCLUSIONS: Sustained long-term (up to 8 to 12 months) systemic T-cell responses were noted after combined radio-gene-hormonal therapy for prostate cancer. Prolonged use of hormonal therapy does not suppress this response. These results suggest the potential for sustained activation of cell-mediated immune responses against cancer.

Asunto(s)

Terapia Genética/métodos , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Aciclovir/análogos & derivados , Aciclovir/uso terapéutico , Adenoviridae/genética , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/uso terapéutico , Antivirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Terapia Combinada/métodos , Humanos , Inmunidad Celular , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/radioterapia , Radioterapia de Intensidad Modulada , Timidina Quinasa/administración & dosificación , Timidina Quinasa/genética , Valaciclovir , Valina/análogos & derivados , Valina/uso terapéutico

RESUMEN

The advances in radiotherapy (3D-CRT, IMRT) have enabled high doses of radiation to be delivered with the least possible associated toxicity. However, the persistence of cancer (local recurrence after radiotherapy) despite these increased doses as well as distant failure suggesting the existence of micro-metastases, especially in the case of higher risk disease, have underscored the need for continued improvement in treatment strategies to manage local and micro-metastatic disease as definitively as possible. This has prompted the idea that an increase in the therapeutic index of radiotherapy might be achieved by combining it with in situ gene therapy. The goal of these combinatorial therapies is to maximize the selective pressure against cancer cell growth while minimizing treatment-associated toxicity. Major efforts utilizing different gene therapy strategies have been employed in conjunction with radiotherapy. We reviewed our and other published clinical trials utilizing this combined radio-genetherapy approach including their associated pre-clinical in vitro and in vivo models. The use of in situ gene therapy as an adjuvant to radiation therapy dramatically reduced cell viability in vitro and tumor growth in vivo. No significant worsening of the toxicities normally observed in single-modality approaches were identified in Phase I/II clinical studies. Enhancement of both local and systemic T-cell activation was noted with this combined approach suggesting anti-tumor immunity. Early clinical outcome including biochemical and biopsy data was very promising. These results demonstrate the increased therapeutic efficacy achieved by combining in situ gene therapy with radiotherapy in the management of local prostate cancer. The combined approach maximizes tumor control, both local-regional and systemic through radio-genetherapy induced cytotoxicity and anti-tumor immunity.

Asunto(s)

Terapia Genética , Neoplasias de la Próstata/terapia , Animales , Ensayos Clínicos como Asunto , Terapia Combinada , Humanos , Masculino , Neoplasias de la Próstata/radioterapia

RESUMEN

BACKGROUND: Over-expression of the oncogene c-Myc has been implicated in the development and progression of human prostate carcinoma. However, previous assessments of c-Myc expression have not revealed its potential for predicting prostate carcinoma progression. Caveolin-1 is associated with prostate carcinoma progression and is a downstream target gene of c-Myc. The observation that caveolin-1 can suppress c-Myc-induced apoptosis suggested the potential for cooperation between c-Myc and caveolin-1 in malignant progression. In this study, the authors evaluated the prognostic potential of combined c-Myc and caveolin-1 expression in human prostate carcinoma progression. METHODS: Immunostaining with c-Myc and caveolin-1-specific antibodies was performed on paraffin sections from 104 radical prostatectomy specimens from men with lymph node negative prostate carcinoma. Combined c-Myc and caveolin-1 immunostaining scores were related with the clinical and pathologic features and the probability of prostate-specific antigen recurrence after surgery. RESULTS: The combination of c-Myc and caveolin-1 immunopositivity correlated positively with Gleason score (rho = 0.219; P = 0.0253) and positive surgical margin (rho = 0.333; P = 0.0006). The combination of positive c-Myc and caveolin-1 in patients with clinically confined prostate carcinoma was a significant prognostic marker for the time to disease progression after surgery in both univariate analysis (P = 0.0039; hazard ratio, 3.035) and multivariate analysis (P = 0.0114; hazard ratio, 2.916). CONCLUSIONS: The coexpression of c-Myc and caveolin-1 showed potential as a useful prognostic marker for human prostate carcinoma. The current results suggest interactions between c-Myc and caveolin-1 in the progression of human prostate carcinoma.

Asunto(s)

Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Caveolinas/análisis , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Adenocarcinoma/cirugía , Distribución por Edad , Anciano , Biopsia con Aguja , Caveolina 1 , Estudios de Cohortes , Intervalos de Confianza , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Probabilidad , Pronóstico , Modelos de Riesgos Proporcionales , Prostatectomía/métodos , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/cirugía , Medición de Riesgo , Sensibilidad y Especificidad , Análisis de Supervivencia , Técnicas de Cultivo de Tejidos , Resultado del Tratamiento

RESUMEN

PURPOSE: In situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult metastatic disease. We are currently conducting three clinical trials using in situ adenoviral vector mediated herpes simplex virus-thymidine kinase (HSV-tk) gene delivery followed by the HSV-tk prodrug ganciclovir (GCV) or valacyclovir (VCV). This study evaluates the systemic T-cell response after gene therapy in each trial. METHODS AND MATERIALS: The study protocol included three separate clinical trials in the Baylor Prostate Cancer SPORE Program: Trial A gene therapy in prostate cancer patients failing radiotherapy (36 patients), Trial B neoadjuvant gene therapy in pre-radical prostatectomy patients (22 patients), and Trial C gene therapy in combination with radiotherapy for prostate cancer (27 patients). Heparinized blood was collected at the time of vector injection and at selected intervals afterward. A complete blood count was performed, and peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting after labeling with dual color-labeled antibody pairs. RESULTS: The pretreatment mean percentage of activated CD8+ T cells (DR+CD8+ T cells) was 12.23%, 16.72%, and 14.09% (Trials A, B, and C, respectively). Two weeks posttreatment, this increased to 22.87%, 26.15%, and 39.04% (Trials A, B, and C, respectively), and these increases were statistically significant (p = 0.0188, p = 0.0010, p < 0.0001, respectively). The increase of DR+CD8+ T cells was significantly larger in Trial C than in Trial A (p = 0.0044) or Trial B (p = 0.0288). Total CD8+ T cells significantly increased at 2 weeks posttreatment in Trial B and C (p = 0.0013, p = 0.0004, respectively). Interestingly, only in Trial C were significant increases in activated CD4+ T cells seen at 2 weeks (p = 0.0035). CONCLUSIONS: This is the first report of systemic T-cell responses after HSV-tk+GCV/VCV gene therapy under three clinical trial conditions. There was an increase in activated CD8+ T cells in the peripheral blood after vector injection, suggesting the potential for activation of components of cell-mediated immune response in all trial conditions. The addition of radiotherapy to in situ gene therapy seems to further increase the total CD8+ T cells and activated CD4+ T cells.

Asunto(s)

Aciclovir/análogos & derivados , Linfocitos T CD8-positivos/inmunología , Terapia Genética , Activación de Linfocitos/inmunología , Neoplasias de la Próstata/terapia , Timidina Quinasa/genética , Valina/análogos & derivados , Aciclovir/uso terapéutico , Adenoviridae/genética , Adulto , Anciano , Anciano de 80 o más Años , Antivirales/uso terapéutico , Virus del Sarcoma Aviar/genética , Linfocitos T CD4-Positivos/inmunología , Terapia Combinada , Ganciclovir/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Profármacos/uso terapéutico , Prostatectomía , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/radioterapia , Valaciclovir , Valina/uso terapéutico

RESUMEN

PURPOSE: There is an evolving role for combining radiotherapy (RT) with gene therapy in the management of prostate cancer. However, the clinical results of this combined approach are much needed. The preliminary results addressing the safety of this Phase I-II study combining RT and gene therapy (adenovirus/herpes simplex virus-thymidine kinase gene/valacyclovir with or without hormonal therapy) in the treatment of prostate cancer have been previously reported. We now report the prostate-specific antigen (PSA) response and biopsy data. METHODS AND MATERIALS: This trial was composed of three separate arms. Arm A consisted of low-risk patients (Stage T1-T2a, Gleason score <7, pretreatment PSA <10 ng/mL) treated with combined RT-gene therapy. A mean dose of 76 Gy was delivered to the prostate with intensity-modulated RT. They also received adenovirus/herpes simplex virus-thymidine kinase/valacyclovir gene therapy. Arm B consisted of high-risk patients (Stage T2b-T3, Gleason score >6, pretreatment PSA level >10 ng/mL) treated with combined RT-gene therapy and hormonal therapy (luteinizing hormone-releasing hormone agonist [30-mg Lupron, 4-month depot] and an antiandrogen [flutamide, 250 mg t.i.d. for 14 days]). Arm C consisted of patients with Stage D1 (positive pelvic lymph nodes) who received the same regimen as Arm B with the addition of 45 Gy to the pelvic lymphatics. PSA determination and biopsy were performed before, during, and after treatment. The American Society for Therapeutic Radiology and Oncology consensus definition (three consecutive rises in PSA level) was used to denote PSA failure. RESULTS: Fifty-nine patients (29 in Arm A, 26 in Arm B, and 4 in Arm C) completed the trial. The median age was 68 years (range, 39-85 years). The median follow-up for the entire group was 13.5 months (range, 1.4-27.8 months). Only Arm A patients were observed to have an increase in PSA on Day 14. The PSA then declined appropriately. All patients in Arm A (median follow-up, 13.4 months) and Arm B (median follow-up, 13.9 months) had biochemical control at last follow-up. Three patients in Arm C (with pretreatment PSA of 335, 19.6, and 2.5 ng/mL and a combined Gleason score of 8, 9, and 9 involving all biopsy cores) had biochemical failure at 3, 3, and 7.7 months. Two patients had distant failure in bone and 1 patient in the para-aortic lymph nodes outside the RT portal. Six to twelve prostate biopsies performed in these 3 patients revealed no evidence of residual carcinoma. In Arm A, biopsy showed no evidence of carcinoma in 66.7% (18 of 27), 92.3% (24 of 26), 91.7% (11 of 12), 100% (8 of 8), and 100% (6 of 6) at 6 weeks, 4 months, 12 months, 18 months, and 24 months after treatment, respectively. In Arm B, no evidence of carcinoma on biopsy was noted in 96% (24 of 25), 90.5% (19 of 21), 100% (14 of 14), 100% (7 of 7), and 100% (2 of 2), respectively, in the same interval after treatment. CONCLUSION: This is the first reported trial of its kind in the field of prostate cancer that aims to expand the therapeutic index of RT by combining it with in situ gene therapy. The initial transient PSA rise in the Arm A patients may have been a result of local immunologic response or inflammation elicited by in situ gene therapy. Additional investigation to elucidate the mechanisms is needed. Hormonal therapy may have obliterated this rise in Arm B and C patients. The biopsy data were encouraging and appeared to show no evidence of malignancy earlier than historical data. Combined RT, short-course hormonal therapy, and in situ therapy appeared to provide good locoregional control but inadequate systemic control in patients with positive pelvic lymph nodes. Longer term use of hormonal therapy in addition to gene therapy and RT has been adopted for this group of patients to maximize both locoregional and systemic control.

Asunto(s)

Aciclovir/análogos & derivados , Terapia Genética/métodos , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/terapia , Radioterapia Conformacional , Timidina Quinasa/uso terapéutico , Valina/análogos & derivados , Aciclovir/uso terapéutico , Adenoviridae/genética , Adulto , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/uso terapéutico , Antineoplásicos Hormonales/uso terapéutico , Antivirales/uso terapéutico , Biopsia , Terapia Combinada , Flutamida/uso terapéutico , Estudios de Seguimiento , Vectores Genéticos/uso terapéutico , Humanos , Leuprolida/uso terapéutico , Masculino , Persona de Mediana Edad , Profármacos/uso terapéutico , Próstata/patología , Neoplasias de la Próstata/patología , Dosificación Radioterapéutica , Timidina Quinasa/genética , Valaciclovir , Valina/uso terapéutico

RESUMEN

Gene-modified dendritic cells (DC) provide unique therapeutic strategies for prostate cancer; however, the comparative evaluation of specific delivery options using appropriate preclinical models has not been described. In this study, bone marrow-derived DC were genetically engineered to express high levels of interleukin-12 (IL-12) with or without the costimulatory molecule B7-1, by ex vivo infection with recombinant adenoviral vectors. We used an orthotopic metastatic mouse prostate cancer preclinical model (178-2 BMA) to compare two therapeutic protocols for DC delivery, in situ and subcutaneous. DC were generated from bone marrow of syngeneic 129/Sv mice by culturing in the presence of GM-CSF and IL-4. In vitro DC/IL-12 or DC/IL-12/B7 produced high levels of biologically active IL-12. In situ delivery of DC/IL-12 or DC/IL-12/B7 induced a significant suppression of primary tumor growth compared to DC/beta gal controls (P=.0328 and P=.0019, respectively), as well as reduced numbers of spontaneous lung metastatic nodules (P=.1404 and P=.0335, respectively). In survival experiments, in situ DC/IL-12 injection demonstrated a small but statistically significant advantage (P=.0041). Subcutaneous, tumor lysate pulsed DC/IL-12 significantly decreased tumor size (P=.0152) and increased survival (P=0.0433) compared to HBSS controls but the decrease in the number of spontaneous lung metastases did not achieve statistical significance. Both in situ and subcutaneous treatments enhanced cytolytic activities of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In this preclinical model, gene-modified DC-based intratumoral immunotherapy was shown to be an effective therapeutic strategy for locally advanced prostate cancer based on tumor growth suppression, inhibition of metastasis and survival improvement.

Asunto(s)

Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Inmunoterapia Adoptiva , Interleucina-12/biosíntesis , Neoplasias de la Próstata/terapia , Adenoviridae , Animales , Antígeno B7-1/biosíntesis , Antígeno B7-1/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , División Celular/efectos de los fármacos , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-12/genética , Interleucina-4/farmacología , Células Asesinas Naturales/inmunología , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Linfocitos T Citotóxicos/inmunología , Ingeniería de Tejidos , Transducción Genética

RESUMEN

We previously identified and characterized a novel p53-regulated gene in mouse prostate cancer cells that was homologous to a human gene that had been identified in brain cancers and termed RTVP-1 or GLIPR. In this report, we document that the human RTVP-1 gene is also regulated by p53 and induces apoptosis in human prostate cancer cell lines. We show that the expression of the human RTVP-1 gene is down-regulated in human prostate cancer specimens compared with normal human prostate tissue at the mRNA and protein levels. We further document epigenetic changes consistent with RTVP-1 being a tumor suppressor in human prostate cancer.

Asunto(s)

Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Neoplasias de la Próstata/genética , Apoptosis/genética , Sitios de Unión , División Celular/genética , Línea Celular Tumoral , Metilación de ADN , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Pérdida de Heterocigocidad , Masculino , Proteínas de la Membrana , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

We investigated the efficacy of intratumoral injection of macrophages transduced with murine IL-12 recombinant adenoviral vector (AdmIL-12) using the orthotopic 178-2 BMA mouse prostate cancer model. AdmIL-12-transduced macrophages secreted IL-12 in vitro and demonstrated increased surface expression of MHC classes I and II as well as F4/80 antigen compared with uninfected macrophages or those infected with an adenoviral vector containing beta-galactosidase (Adbetagal) in control macrophages. AdmIL-12-transduced macrophages injected into orthotopic 178-2 BMA tumors in vivo induced significant suppression of primary tumor growth and spontaneous lung metastases compared with controls. These antitumor and antimetastatic effects were comparable with those resulting from direct orthotopic delivery of the AdmIL-12 vector. Mice with orthotopic tumors treated with AdmIL-12-transduced macrophages survived significantly longer than controls. Analysis of tumors demonstrated significantly increased infiltration of CD4+ and CD8+ T cells in those injected with AdmIL-12-transduced macrophages compared with controls. Splenocyte-derived cytotoxic natural killer cell activity was enhanced on day 2 after AdmIL-12-transduced macrophage injection, and on day 14, tumor-specific T-lymphocyte activities were increased compared with control, Adbetagal-infected macrophages. Trafficking studies confirmed that intratumorally injected, AdmIL-12-transduced macrophages could migrate to draining lymph nodes. Overall, this novel approach to prostate cancer therapy demonstrates antitumor immune responses that provide effective antimetastatic activities in preclinical studies.

Asunto(s)

Inmunoterapia/métodos , Interleucina-12/inmunología , Macrófagos Peritoneales/inmunología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Adenoviridae/genética , Animales , División Celular/inmunología , Movimiento Celular/inmunología , Inmunohistoquímica , Interleucina-12/genética , Interleucina-12/metabolismo , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Linfocitos T Citotóxicos/inmunología , Transducción Genética

RESUMEN

PURPOSE: Caveolin-1 (cav-1), the major protein component of caveolae, plays an important role in multiple signaling pathways, molecular transport, and cellular proliferation and differentiation. The specific functions of cav-1/caveolae are highly cell and context dependent. We have previously shown that cav-1 expression is increased in metastatic human prostate cancer and that cav-1 cellular protein expression is predictive of recurrence of the disease after radical prostatectomy. Recently, we reported that cav-1 is secreted by androgen-insensitive prostate cancer cells, and we detected, by Western blotting, cav-1 in the high-density lipoprotein(3) fraction of serum specimens from patients with prostate cancer. EXPERIMENTAL DESIGN: Using rabbit polyclonal antibodies with specificity for cav-1, we developed a direct sandwich immunoassay for the determination of cav-1 in serum. A recombinant human cav-1 fusion protein was overexpressed and purified from 293 PE cells and used as a calibrator. RESULTS: The assay was highly specific and had a minimum detection limit of 0.017 ng/ml (mean + 3 SD of zero calibrator) and measuring range of up to 200 ng/ml. Intra-assay coefficient of variation was 2.29-6.74% and inter-assay coefficient of variation was 2.81-6.43% over the serum concentration tested 0.04-31.89 ng/ml. The recovery limit of cav-1 by the assay was 89.55-100.28%. The median serum cav-1 level in 102 prostate cancer patients with clinically localized disease (0.463 ng/ml) was significantly higher than 81 healthy control men (0.324 ng/ml; P = 0.0446, Mann-Whitney test) or 107 men with benign prostatic hyperplasia (0.172 ng/ml; P = 0.0317, Mann-Whitney test). CONCLUSIONS: Our results indicate that serum cav-1 has the power to differentiate between prostate cancer and benign prostatic hyperplasia patients and the potential to be an important biomarker for prostate cancer. Additional studies to test the potential of serum cav-1 as a diagnostic and/or prognostic marker in prostate cancer are warranted.

Asunto(s)

Biomarcadores de Tumor , Caveolinas/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoensayo/métodos , Neoplasias de la Próstata/sangre , Anciano , Animales , Anticuerpos/química , Western Blotting , Calibración , Caveolina 1 , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Persona de Mediana Edad , Hiperplasia Prostática/patología , Neoplasias de la Próstata/diagnóstico , Conejos , Proteínas Recombinantes/química , Sensibilidad y Especificidad

RESUMEN

We previously identified the mouse RTVP-1 (mRTVP-1; related to testes-specific, vespid, and pathogenesis proteins) gene as a direct target of p53 with proapoptotic activities in various cancer cell lines, including prostate cancer. To test the therapeutic potential of mRTVP-1 we constructed an adenoviral vector capable of efficient transduction and expression of mRTVP-1 (AdmRTVP-1) and used this vector in an orthotopic, metastatic mouse model of prostate cancer. A single intratumoral administration of AdmRTVP-1 gene therapy significantly reduced primary tumor wet weight compared with control Adbetagal-injected tumors at two time points after injection with two different vector doses (p < or = 0.01 at 7 and 14 days). Spontaneous metastasis to lung was also significantly reduced (p < or = 0.02). Evaluation of treated tumors revealed increased apoptosis and lower microvessel density counts. In a rat aortic ring sprouting assay, AdmRTVP-1 inhibited endothelial cell sprouting compared with Adbetagal, confirming its antiangiogenic activity. These therapeutic activities were associated with a significant increase in survival from 22.9 to 26.8 days (p = 0.003) in this aggressive model of prostate cancer. Interestingly, there were significant increases in the infiltration of tumor-associated macrophages, dendritic cells, and CD8+ T cells, which persisted at 14 days posttreatment in the AdmRTVP-1-treated tumors compared with Adbetagal control-treated tumors. In addition, significantly increased natural killer and cytotoxic T lymphocyte activities were demonstrated in the mice with AdmRTVP-1-treated tumors. The unique therapeutic properties of AdmRTVP-1 gene therapy demonstrated in this study provide new opportunities for gene and immunotherapy of prostate cancer and potentially other malignancies.

Asunto(s)

Adenoviridae , Terapia Genética , Vectores Genéticos , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso , Neoplasias de la Próstata/terapia , Animales , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Proteínas de la Membrana , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Ratas , Células Tumorales Cultivadas

RESUMEN

Prostate cancer is the most commonly diagnosed non-cutaneous cancer in adult males. Although prostate cancer that is confined to the gland can be cured in many patients using surgery or radiation, these treatments are only effective for localized tumors and the long-term failure rates for these treatments suggests that prostate cancer can metastasize relatively early in the course of the disease. Once prostate cancer has metastasized there are no curative therapies. The greatest challenge in the treatment of advanced prostate cancer is to access and eliminate metastatic cells. Therefore, effective prostate cancer therapy will require novel strategies to target cancer cells both at the site of the primary tumor and at distant metastatic sites. In this article we review several therapeutic targets and approaches that may provide new treatments for metastatic prostate cancer. We discuss the use of small molecules to target specific molecular events associated with metastatic prostate cancer, the use of specific antibodies that target unique metastasis associated molecules and the use of various gene therapy strategies to achieve anti-metastatic activities.

Asunto(s)

Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Animales , Anticuerpos/química , Anticuerpos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/inmunología , Vacunas contra el Cáncer/uso terapéutico , Terapia Genética/métodos , Humanos , Inmunoterapia , Masculino , Metástasis de la Neoplasia , Neoplasias de la Próstata/inmunología

RESUMEN

Using LoxP/Cre technology, we generated a knockout mouse homozygous for a null mutation in exon 2 of Cav1. In male Cav1-/- animals, we observed a dramatic increase in the incidence of urinary calcium stone formation. In 5-month-old male mice, the incidence of early urinary calculi was 67% in Cav1-/- mice compared to 19% in Cav1+/+ animals. Frank stone formation was observed in 13% of Cav1-/- males but was not seen in Cav1+/+ mice. Urine calcium concentration was significantly higher in Cav1-/- male mice compared to Cav1+/+ mice. In Cav1-/- mice, distal convoluted tubule cells were completely devoid of Cav1 and the localization of plasma membrane calcium ATPase was disrupted. Functional studies confirmed that active calcium absorption was significantly reduced in Cav1-/- compared to Cav1+/+ male mice. These results demonstrate that disruption of the Cav1 gene promotes the progressive steps required for urinary calcium stone formation and establish a new mouse model for urinary stone disease.

Asunto(s)

Trastornos del Metabolismo del Calcio/patología , Calcio/orina , Caveolinas/genética , Riñón/patología , Cálculos Urinarios/patología , Animales , Calcio/metabolismo , Trastornos del Metabolismo del Calcio/genética , Caveolina 1 , Caveolinas/deficiencia , Caveolinas/fisiología , Creatinina/orina , Cartilla de ADN , Modelos Animales de Enfermedad , Exones , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Cálculos Urinarios/genética

RESUMEN

BACKGROUND: The authors previously identified elevated caveolin-1 expression in human prostate carcinoma and determined that caveolin-1 levels as detected by immunohistochemistry of radical prostatectomy specimens offered novel prognostic information. A higher incidence of caveolin-1 expression also was reported in African-American men compared with white men in the U.S. To explore these ethnic/racial differences in caveolin-1 expression further, the authors evaluated caveolin-1 expression as a predictive marker in Japanese men with prostate carcinoma. METHODS: Immunohistochemical staining with a caveolin-1 specific antibody was performed on routinely processed paraffin sections from 152 consecutively collected radical prostatectomy specimens. The mean patient age was 64.3 years (range, 49-74 years; median, 64.5 years) and the mean follow-up period was 49.5 months (range, 1.3-103.3 months; median, 48.2 months). Caveolin-1 immunoreactivity was evaluated in association with patient's age; preoperative prostate specific antigen level; clinical stage; and pathologic features including Gleason score, extraprostatic extension, status of surgical margins, seminal vesicle involvement, lymph node involvement, and time to disease progression after surgery. RESULTS: Positive caveolin-1 immunostaining was detected in 46 of the 152 tumors (30.3%) and was found to be associated significantly with a positive surgical margin (P = 0.022). A higher incidence of caveolin-1 expression tended to be found in patients with poorly differentiated tumors (Gleason score > 7, 6-7, and < 6, 35.0% vs. 34.9% vs. 20.4%, respectively) or in patients with extraprostatic extension versus those without extraprostatic extension (35.4% vs. 24.7%) or patients with lymph node involvement compared with those without lymph node involvement (50% vs. 29.5%), although these differences did not reach statistical significance (P = 0.100, P = 0.150, and P = 0.178, respectively, by the Spearman correlation test). Kaplan-Meier analysis revealed that increased caveolin-1 expression was associated with an increased risk of disease progression at 5 years (P = 0.0122 by the log-rank test). In patients with organ-confined (pT2N0) disease, univariate Cox proportional hazards regression analysis revealed that positive caveolin-1 expression was the only significant predictor of disease recurrence after radical prostatectomy (P = 0.011; hazards ratio = 4.75; and 95% confidence interval, 1.43-15.76). CONCLUSIONS: The results of the current study confirm that positive caveolin-1 expression is associated with clinical markers of disease progression and is predictive of poor clinical outcome after surgery in Japanese patients with pT2N0 prostate carcinoma.

Asunto(s)

Caveolinas/metabolismo , Recurrencia Local de Neoplasia , Neoplasias de la Próstata/metabolismo , Anciano , Biomarcadores de Tumor , Caveolina 1 , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Etnicidad , Humanos , Inmunohistoquímica , Incidencia , Japón/epidemiología , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Análisis de Supervivencia

RESUMEN

Prostate cancer is the most common noncutaneous cancer in man. When confined to the prostate it can be cured by radical prostatectomy or irradiation therapy. However, there are no curative therapies for locally advanced, recurrent or metastatic disease. Prostate cancer gene therapy has recently transition from preclinical studies to clinical trials with the goal of developing novel treatments for prostate cancer. The greatest challenge in treating advanced prostate cancer is therapeutic access to and the elimination of metastases. This review details two aspects of prostate cancer gene therapy, the types of delivery systems under development and specific categories of therapeutic genes available with an emphasis on the mechanism of action of specific gene therapy strategies.

Asunto(s)

Adyuvantes Inmunológicos/uso terapéutico , Genes Supresores de Tumor , Terapia Genética/métodos , Neoplasias de la Próstata/terapia , Adyuvantes Inmunológicos/genética , Apoptosis/genética , Bacterias/enzimología , Bacterias/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/uso terapéutico , Citosina Desaminasa , Toxina Diftérica/genética , Toxina Diftérica/uso terapéutico , Vectores Genéticos , Humanos , Masculino , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Nucleósido Desaminasas/genética , Nucleósido Desaminasas/uso terapéutico , Profármacos/metabolismo , Profármacos/uso terapéutico , Neoplasias de la Próstata/genética , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genética , Timidina Quinasa/uso terapéutico

RESUMEN

We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.

Asunto(s)

Apoptosis/genética , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Apoptosis/fisiología , Tamaño de la Célula , Doxorrubicina/farmacología , Rayos gamma , Genes , Genes Reporteros , Humanos , Proteínas de la Membrana , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , Señales de Clasificación de Proteína , Alineación de Secuencia , Células Tumorales Cultivadas , Regulación hacia Arriba