RESUMO
BACKGROUND: Neuroendocrine (NE) cells promote the progression of prostate cancer to a castration-resistant state through the production of paracrine growth factors. We have demonstrated this principle using in vitro and in vivo proliferative endpoints; however, the contributions of NE-derived pro-survival factors and anti-apoptosis to this phenomenon have not been thoroughly investigated. METHODS: Here, we utilized conditioned-medium (CM) from LNCaP cells, engineered to undergo NE differentiation, and examined its effects on PC3 and LNCaP cell survival. RESULTS: Statistically significant changes in clonogenic survival, Annexin V staining, PARP cleavage and trypan blue positivity of approximately twofold were observed in the presence of NE-derived CM relative to control-CM for both LNCaP and PC3 cells. These changes were partially abrogated by antagonists of the neuropeptides neurotensin, bombesin, and PTHrP. Selective inhibitors of IGF-1R, EGFR or Src caused significant and nearly complete blockade of prostate cancer cell survival due to NE secretions. Similar increases in cell survival were observed for LNCaP or PC3 cells treated with NE-derived medium in the presence of docetaxel. Increased phosphorylation of IGF-1R, following treatment with NE-derived medium, was accompanied by decreased protein tyrosine phosphatase, receptor type F (PTPRF) mRNA, and protein levels. Overexpression of PTPRF decreased cell survival, the amplitude and duration of IGF-1R phosphorylation, and enhanced PARP cleavage in the presence of NE-derived medium. CONCLUSIONS: These data support the hypothesis that NE-derived factors act upon prostate cancer cells to stimulate pro-survival signaling and describe a novel mechanism of cross-talk between NE-derived factors and IGF-1R, mediated in part by PTPRF.
Assuntos
Neoplasias Hormônio-Dependentes/metabolismo , Sistemas Neurossecretores/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/genética , Proteína Relacionada ao Hormônio Paratireóideo/antagonistas & inibidores , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Transdução de SinaisRESUMO
2-Methoxyestradiol (2ME2) is an endogenous estradiol metabolite that inhibits microtubule polymerization, tumor growth, and angiogenesis. Because prostate cancer is often treated with radiotherapy, and 2ME2 has shown efficacy as a single agent against human prostate carcinoma, we evaluated 2ME2 as a potential radiosensitizer in prostate cancer models. A dose-dependent decrease in mitogen-activated protein kinase phosphorylation was observed in human PC3 prostate cancer cells treated with 2ME2 for 18 h. This decrease correlated with in vitro radiosensitization measured by clonogenic assays, and these effects were blocked by the expression of constitutively active MEK. Male nude mice with subcutaneous PC3 xenografts in the hind leg were treated with 2ME2 (75 mg/kg) p.o. for 5 days, and 2 Gy radiation fractions were delivered each day at 4 h after drug treatment. A statistically significant super-additive effect between radiation and 2ME2 was observed in this subcutaneous model, using analysis of within-animal slopes. A PC-3M orthotopic model was also used, with bioluminescence imaging as an end point. PC-3M cells stably expressing the luciferase gene were surgically implanted into the prostates of male nude mice. Mice were given oral doses of 2ME2 (75 mg/kg), with radiation fractions (3 Gy) delivered 4 h later. Mice were then imaged weekly for 4 to 5 weeks with a Xenogen system. A significant super-additive effect was also observed in the orthotopic model. These data show that 2ME2 is an effective radiosensitizing agent against human prostate cancer xenografts, and that the mechanism may involve a decrease in mitogen-activated protein kinase phosphorylation by 2ME2.
Assuntos
Carcinoma/metabolismo , Carcinoma/radioterapia , Estradiol/análogos & derivados , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/radioterapia , Radiossensibilizantes/farmacologia , 2-Metoxiestradiol , Animais , Carcinoma/enzimologia , Estradiol/farmacologia , Estradiol/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Neoplasias da Próstata/enzimologia , Tela Subcutânea , Transplante Heterotópico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The neuroendocrine status of prostatic adenocarcinomas is considered a prognostic indicator for development of aggressive, androgen-independent disease. Neuroendocrine-like cells are thought to function by providing growth and survival signals to surrounding tumor cells, particularly following androgen ablation therapy. To test this hypothesis directly, LNCaP cells were engineered to inducibly express a constitutively activated form of the cyclic AMP-dependent protein kinase A catalytic subunit (caPKA), which was previously found upon transient transfection to be sufficient for acquisition of neuroendocrine-like characteristics and loss of mitotic activity. Clonal cells that inducibly expressed caPKA enhanced the growth of prostate tumor cells in anchorage-dependent and anchorage-independent in vitro assays as well as the growth of prostate tumor xenografts in vivo, with the greatest effects seen under conditions of androgen deprivation. These results suggest that neuroendocrine-like cells of prostatic tumors have the potential to enhance androgen-independent tumor growth in a paracrine manner, thereby contributing to progression of the disease.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Tumores Neuroendócrinos/patologia , Neoplasias da Próstata/patologia , Animais , Domínio Catalítico , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitose/fisiologia , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/genética , Tumores Neuroendócrinos/enzimologia , Oligopeptídeos , Peptídeos/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genéticaRESUMO
Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca(2+)) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca(2+) channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca(2+) channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer.
Assuntos
Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Carboplatina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Mibefradil/farmacologia , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Survivina , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose- and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell-attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy.