RESUMO
Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.
Assuntos
Apoptose , Citocromos b/metabolismo , Mitocôndrias/metabolismo , Receptor fas/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Galinhas , Citocromos c/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Supressão Genética , Receptor fas/genéticaRESUMO
Recent reports have shown that the AR is the key determinant of the molecular changes required for driving prostate cancer cells from an androgen-dependent to an androgen-independent or androgen depletion-independent (ADI) state. Several recent publications suggest that down-regulation of AR expression should therefore be considered the principal strategy for the treatment of ADI prostate cancer. However, no valid data is available about how androgen-dependent prostate cancer cells respond to apoptosis-inducing drugs after knocking down AR expression and whether prostate cancer cells escape apoptosis after inhibition of AR expression. This review will focus on mechanisms of prostate cancer cell survival after inhibition of AR activity mediated either by androgen depletion or by targeting the expression of AR by siRNA. We have shown that knocking down AR expression by siRNA induced PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We also showed that the expression of CaMKII genes is under AR control: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in an elevated level of kinase activity and in enhanced expression of CaMKII genes. This in turn activates the anti-apoptotic PI3K/Akt pathways. CaMKII also express anti-apoptotic activity that is independent from the Akt pathway. This may therefore be an important mechanism by which prostate cancer cells escape apoptosis after androgen depletion or knocking down AR expression. In addition, we have found that there is another way to escape cell death after AR inhibition: DNA damaging agents cannot fully activate p53 in the absence of AR and as a result p53 down stream targets, for example, microRNA-34, cannot be activated and induce apoptosis. This implies that there may be a need for re-evaluation of the therapeutic approaches to human prostate cancer.
Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/deficiência , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Ativação Enzimática/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Luciferases , Masculino , Camundongos , Regiões Promotoras Genéticas , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
It has been suggested in many studies that combined treatment with chemotherapeutic agents and apoptosis-inducing ligands belonging to TNFR family is a more effective strategy for cancer treatment. However, the role of androgen regulation of TNFR family-induced apoptosis in prostate cancer is poorly understood. In this study, we investigated the dose-dependent effects of androgen on TNF-alpha and TRAIL-mediated apoptosis in LNCaP. To investigate the interaction between the androgen receptor (AR) and the caspase-2 gene, chromatin immunoprecipitation analysis was used, and we are the first to identify that AR interacts in vivo with an androgen-responsive elements in intron 8 of caspase-2 gene. We have found that DHT inhibited apoptosis in dose-dependent manner. There is a direct, androgen-dependent correlation between the levels of activated Akt and caspase activation after treatment with TNF-alpha and TRAIL. We have also found that there are at least two different regulatory mechanisms of p53 expression by androgen: at the gene and protein levels. At the same time, the level of AR was found to be higher in LNCaP-si-p53 compared to LNCaP-mock cells. These data indicate that there is a mutual regulation of expression between p53 and AR. Our study suggests that androgen-dependent outcome of apoptotic treatment can occur, at least in part, via the caspase-2, Akt and p53-mediated pathways.
Assuntos
Androgênios/fisiologia , Apoptose/fisiologia , Transdução de Sinais/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Sequência de Bases , Caspase 2 , Inibidores de Caspase , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Primers do DNA , Ativação Enzimática , Humanos , Imunoprecipitação , Masculino , Glicoproteínas de Membrana/metabolismo , Receptores Androgênicos/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.
Assuntos
Proteínas de Transporte/metabolismo , Caspase 2/metabolismo , Ácidos Hidroxâmicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteína Adaptadora de Sinalização CRADD/metabolismo , Proteínas de Transporte/genética , Caspase 2/genética , Caspase 8/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Ativação Enzimática/efeitos dos fármacos , Inibidores de Histona Desacetilases , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor kappaB (NFkappaB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFkappaB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFkappaB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFkappaB, plays the role of a general "buffer" of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFkappaB in tumors.
Assuntos
Inflamação/prevenção & controle , NF-kappa B/antagonistas & inibidores , Proteína Supressora de Tumor p53/fisiologia , Animais , Ceco/cirurgia , Quimiocinas/genética , Citocinas/genética , DNA/metabolismo , Humanos , Inflamação/induzido quimicamente , Ligadura , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/análise , NF-kappa B/fisiologia , Neutrófilos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Peritonite/etiologia , Peroxidase/sangue , Fagocitose , Regiões Promotoras Genéticas/genética , Punções , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/mortalidade , Tioglicolatos , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/fisiologia , Transfecção , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Both inactivation of p53 function and loss of sensitivity to Fas contribute to a malignant phenotype and frequently occur during tumor progression. Although in the majority of cases only one of the p53 alleles is mutated, some tumors acquire mutations in both alleles of the p53 gene. To determine the biological significance of this phenomenon, we analyzed p53 mutants, p53(223Leu) and p53(274Phe), from Fas-resistant prostate carcinoma cell line DU145. Both mutants differed from wild-type p53 in their conformation, transactivation ability, and effect on the growth of p53-deficient cells, with p53(223Leu) being more similar to wild-type p53 than was p53(274Phe). Interestingly, the biological effect of coexpression of the DU145-derived mutants was dramatically different from that of each mutant expressed alone. Whereas neither of the two mutants was found to be dominant-negative against wild-type p53, each neutralized the other's growth-suppressive effects and, in combination, were capable of down-regulating Fas expression and converting Fas-sensitive prostate carcinoma cells PC3 into Fas-resistant ones. These results indicate that two different p53 mutants that are separately rather weak can cooperate to generate p53 protein with anti-Fas function that is likely to provide additional selective advantages to the tumor.
Assuntos
Alelos , Genes p53/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína Supressora de Tumor p53/fisiologia , Receptor fas/fisiologia , Animais , Apoptose/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Humanos , Masculino , Camundongos , Mutação , Conformação Proteica , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-alpha and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-alpha and anti-Fas Ab resulted in 55%, 70% and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: (1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and (2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.
Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Glicoproteínas de Membrana/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligantes , Masculino , Modelos Biológicos , Neoplasias da Próstata/metabolismo , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
It has been suggested that some nuclear transcription factors may participate in the regulation of mitochondrial functions through transcriptional control of mitochondrial DNA. Very little is known about the response of transcription factors within mitochondria to the activation of death receptors. Recent publications indicate that nuclear factor-kappaB (NF-kappaB) is localized in mitochondria of mammalian cells. Because of the critical role of mitochondria in the execution of many apoptotic pathways, we suggest that NF-kappaB-dependent mechanisms operating at the level of mitochondria contribute to its role in regulating death receptor signaling. We have found NF-kappaB p65 and p50 subunits with DNA binding activity in the mitochondria of prostatic carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) affects DNA binding activity of mitochondria-associated NF-kappaB but does not change the amount of p65 in mitochondria, which suggests activation of mitochondrial NF-kappaB without additional translocation of NF-kappaB subunits to mitochondria. We have also shown that TRAIL decreases mitochondrial genome encoded mRNA levels and inhibition of NF-kappaB prevents this decrease. TRAIL effects on mitochondrial NF-kappaB-DNA binding and mitochondrial genome encoded mRNA levels also depend on Bcl-2 overexpression. In addition, transcription factor activator protein-1 with DNA binding activity is also found in mitochondria of prostatic carcinoma cells and TRAIL treatment affects this binding. In summary, NF-kappaB is found in mitochondria of prostatic carcinoma cells, where it is thought to regulate mitochondria genome encoded mRNA levels in response to TRAIL treatment.
Assuntos
DNA Mitocondrial/metabolismo , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Neoplasias da Próstata , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Glicoproteínas de Membrana/genética , Mitocôndrias/fisiologia , Subunidade p50 de NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/genéticaRESUMO
Here we show that LNCaP, which is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, becomes sensitive to TRAIL after overexpression of full-length, wild-type BAD (BAD WT). TRAIL induces caspase-dependent cleavage of BAD WT that results in generation of a M(r) 15,000 protein. LNCaP stably expressing truncated BAD (tBAD) and cells expressing mutated BAD at the caspase cleavage site were less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Cytochrome c and Smac/DIABLO release from mitochondria into cytosol was found after TRAIL treatment only in cells overexpressing BAD WT. Furthermore, differences in phosphorylation of serine residues for BAD WT and tBAD were identified. BAD WT was phosphorylated at positions S136 and S155, whereas tBAD was phosphorylated at positions S112, S136, and S155. LNCaP stably expressing BAD mutated at serine 112 to alanine was less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Lastly, recombinant BAD cleaved by caspase-3 is a more potent inducer of cytochrome c and Smac/DIABLO release than BAD WT. In summary, BAD-mediated sensitivity of LNCaP to TRAIL depends on the phosphorylation status of BAD WT and tBAD.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/genética , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular , Citosol/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Fosforilação , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Proteína de Morte Celular Associada a bclRESUMO
We have previously shown that Bisindolylmaleimide (Bis) IX is localized in mitochondria but also acts as an inhibitor of transcription and facilitates tumor necrosis factor receptor family-mediated apoptosis. In this study, we found that Bis IX is freely distributed both within cells and extracellular medium and acts as a reversible apoptosis-inducing agent. Bis IX was found to induce time-dependent apoptosis in combination with TNF-a, TRAIL, and anti-Fas Ab. Using human prostatic carcinoma cell lines DU145 and LNCaP that are resistant to treatment with TNF family death-inducing ligands, we have shown that different, albeit still unidentified, inhibitory factors are responsible for the resistance to TRAIL-, Fas-, and TNF-a-mediated apoptosis. Our data also suggest that the turnover of apoptosis suppressor factors is much faster in DU145 compared to LNCaP. Lastly, we have found that Bis IX can override the apoptosis-inhibitory effects of Bcl-2 overexpression. In conclusion, Bis IX could be used as a drug to facilitate apoptosis of cancer cells that are resistant to treatment with death-inducing ligands as well as a valuable tool to discern the factors that mediate resistance to different death-inducing ligands.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Reguladoras de Apoptose , Northern Blotting , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Ligantes , Masculino , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Tempo , Transcrição Gênica , Células Tumorais Cultivadas , Receptor fas/metabolismoRESUMO
TPCK is widely used as an inhibitor of chymotrypsin-like proteases but has recently been identified as an inhibitor of the PDK1/Akt pathway. In this study, we show that TPCK inhibits TRAIL-induced caspase activity but potentiates wortmannin-dependent caspase activity in prostatic carcinoma cell lines. The inhibitory activity of TPCK was found to be death ligand-specific since TPCK inhibits TRAIL-mediated caspase activity but does not affect Fas-induced caspase activity. Our data also show that impaired TRAIL-DISC formation in the presence of TPCK is responsible for caspase inhibition. Further, TPCK induces p53 expression and inhibits the PDK1/Akt pathway resulting in BAD dephosphorylation, and the release of cytochrome c and Smac/DIABLO from mitochondria. TPCK also selectively decreases the levels of androgen receptor and caspase-2 whereas it does not change the levels of other proteins (caspases-3, -7, -8, -9; heat shock proteins 27, 70, 90). Finally, TPCK-induced degradation of caspase-2 is protected by Bcl-2 overexpression, apparently by an adapter protein since direct interaction between caspase-2 and Bcl-2 was not detected. Together, these features suggest that TPCK could be used as a therapeutic agent for treatment of those tumor cells that are resistant to ligand-induced treatment because of aberrant signaling pathways downstream of the DISC.
Assuntos
Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tosilfenilalanil Clorometil Cetona/farmacologia , Androstadienos/farmacologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Caspases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Humanos , Masculino , Glicoproteínas de Membrana/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , WortmaninaRESUMO
We and others have previously described that the androgen-responsive human prostatic carcinoma cell line LNCaP is resistant to TRAIL and that TRAIL-mediated apoptosis in LNCaP is PI3K/Akt-dependent. In this study, we found that LNCaP remained resistant to treatment with TRAIL after androgen deprivation even in the presence of the PI3K/Akt pathway inhibitor wortmannin. This resistance was determined by failure to form the TRAIL-DISC and by decreased TRAIL-R1 and TRAIL-R2 levels after androgen deprivation; the capacity of TRAIL to induce DISC formation was completely restored in the presence of DHT. TRAIL and wortmannin together accelerated processing of caspase-8 on the DISC and apparently the release of caspase-8 from the DISC into the cytoplasm. Surprisingly, we found that wortmannin decreased the total amount of TRAIL-R1, but not TRAIL-R2, in the cells as well as the amount of TRAIL-R1 precipitated by TRAIL. Our data suggest that TRAIL-DISC formation and sensitivity to TRAIL treatment are androgen-dependent in LNCaP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Androgênios/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Glicoproteínas de Membrana/biossíntese , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Androstadienos/farmacologia , Proteínas Reguladoras de Apoptose , Western Blotting , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Primers do DNA/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Proteína de Domínio de Morte Associada a Fas , Genes Dominantes , Humanos , Masculino , Inibidores de Fosfoinositídeo-3 Quinase , Reação em Cadeia da Polimerase , Testes de Precipitina , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Ligante Indutor de Apoptose Relacionado a TNF , Transfecção , Células Tumorais Cultivadas , WortmaninaRESUMO
Mutual regulation of expression between p53 and AR has been reported. To further investigate the role of p53 in the regulation of AR expression, an ARE-Luciferase vector was inserted into LNCaP and into LNCaP-sip53 transfectants, and AR activity was quantitatively estimated after treatment with proteasome inhibitors. LNCaP expresses a mutated form of AR. Therefore, to investigate whether p53 can modulate the expression of wild-type (wt) of AR, we transfected PC3-wtAR with a p53 vector together with ARE-Luc and showed that p53 expression decreased DHT-dependent activity of wtAR. Since proteasomes also participate in AR transcriptional activity, we investigated the role of p53 in proteasome-dependent inhibition of AR activity. More than 80% of AR activity was inhibited by 3 µM of lactacystin in LNCaP whereas no inhibition was noted in LN-sip53. We also found that lactacystin decreased AR-DNA binding 3-fold in LNCaP but no binding decrease was observed in LN-sip53. Taken together, our data show that the inhibitory effects of proteasome inhibitors are dependent on p53 status, at least in prostate cancer. Therefore, the role of p53 during treatment with proteasome inhibitors in different tumors should be further investigated.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Androgênicos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Anilidas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Nitrilas/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Inibidores de Proteassoma , Compostos de Tosil/farmacologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.
Assuntos
Cromatina/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Neoplasias da Próstata/genética , Ligação Proteica , Receptores Androgênicos/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Caspase-2 can induce apoptosis in response to extrinsic and intrinsic signals. Unlike other caspases, this protein is not expressed solely in nonnuclear compartments; a subpopulation is constitutively localized in the nucleus. As one of the most evolutionarily conserved caspases, caspase-2 may have roles in multiple cellular processes. However, its contribution to nonapoptotic processes remains a mystery. In this study, we show that caspase-2 activity is important for proliferation by cells of the androgen-dependent prostate cancer cell line LNCaP. LNCaP cells expressing either a dominant-negative (dn) form of caspase or an siRNA against caspase-2 had lower androgen receptor (AR)-dependent proliferative responses than control cells, and application of the siRNA resulted in downregulation of the expression of both AR-dependent prostate-specific antigen (PSA) and AR-dependent reporter luciferase. Also, caspase-2 formed complexes with the cell cycle regulatory proteins cyclin D3, CDK4, and p21/Cip1, and caspase-2 regulated AR transactivation by inhibiting the repressive function of cyclin D3. Taken together, these results reveal, for the first time, that caspase-2 is involved in cell cycle promotion and AR activation. Given that prostate cancer cells depend on AR activity in order to survive, the fact that our data indicate that caspase-2 positively regulates AR activity suggests that caspase-2 has potential as a target in the treatment of prostate cancer.
RESUMO
A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.
Assuntos
Carcinoma/tratamento farmacológico , Ácidos Graxos/biossíntese , Furanos/farmacologia , Neuropilina-1/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Receptores Androgênicos/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/genética , Carcinoma/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Di-Hidrotestosterona/farmacologia , Genes p53 , Humanos , Íntrons , Lipídeos/biossíntese , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neuropilina-1/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
It has been suggested that the downregulation of AR expression should be considered the principal strategy for the treatment of hormone-refractory prostate cancer. We have previously shown that inhibition of AR induced PI3K-independent activation of Akt that was mediated by CaMKII. In this study, we found that the CaMKII inhibitor KN-93 has a broader effect on apoptosis than just inhibition of CaMKII: first, KN-93 inhibits AR activity and induces cell death in PCa cells after androgen deprivation when many other drugs fail to kill prostate cancer cells; second, KN-93 inhibits expression of the anti-apoptotic protein Mcl-1 and induces expression of the pro-apoptotic protein PUMA; third, KN-93-mediated cell death is p53-independent; and fourth, KN-93 induces the generation of ROS. The ROS induction allows KN-93 to circumvent the activation of Akt, which occurs in prostate cancer cells under androgen deprivation, since Akt could not inhibit ROS-mediated apoptosis. KN-93 also synergistically induces cell death in combination with low doses of doxorubicin and converts the phenotype of prostate cancer cells from TRAIL-resistant to -sensitive. These data suggest that KN-93 could be used for novel therapeutic approaches when hormonal therapy has failed.
Assuntos
Antagonistas de Receptores de Andrógenos , Apoptose/efeitos dos fármacos , Benzilaminas/farmacologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
The majority of breast cancer cell lines are resistant to tumor necrosis factor -related apoptosis inducing ligand (TRAIL) induced apoptosis. TRAIL and Fas receptor death-inducing signaling complex (DISCs) formation are similar and involve ligand-dependent recruitment of FADD and caspase-8. We have found that the breast carcinoma cell line T47D is an unusual example of selective sensitivity to anti-Fas mAb treatment but resistant to TRAIL. Therefore, a detailed comparison of these two signaling pathways in one cell line should provide insight into the mechanism of TRAIL resistance. We observed that only anti-Fas mAb induces caspase activation and cell death in T47D. Further, FADD and caspase-8 interact with both TRAIL-R1 and TRAIL-R2, and that the amount of caspase-8 recruited by Fas-, TRAIL-R1 and TRAIL-R2 are the same. cFLIP(S) and cFLIP(R )isoforms block death receptor-induced apoptosis by inhibiting caspase-8 activation at the DISC; the role of cFLIP(L )at the DISC is still controversial. It has been suggested that the presence of the cleaved form of FLIP(L)-p43 at the DISC prevents caspase-8 cleavage. We found that both TRAIL and anti-Fas mAb-induced DISCs contain the cleaved form of p43 cFLIP(L) and its amount at the Fas DISC was higher compared to the TRAIL DISC. We also found that inhibition of cFLIP(L) expression in T47D cells decreased Fas-mediated caspase-8 activation and activation of effector caspases. We propose that in T47D p43 cFLIP(L) in the Fas-DISC may promote caspase-8 activation. The mechanism by which different amounts of p43cFLIP(L) regulates caspase-8 activation remains to be investigated.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptor fas/antagonistas & inibidores , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Caspase 8/fisiologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína de Domínio de Morte Associada a Fas/fisiologia , Feminino , Humanos , Imunoprecipitação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologiaRESUMO
We investigated whether knocking down AR expression effects apoptosis after treatment with different apoptosis-inducing agents. We found that siRNA AR (si-AR) significantly decreased apoptosis induced by topoisomerase inhibitors doxorubicin (DOX) and camptothecin (Campt). It is known that DNA double-strand break inducing agents leads to activation (phosphorylation) of p53 that in turn regulates the expression of a variety of apoptosis-related genes including microRNA(miR)-34a and 34b/c. We found that DOX induced five phosphorylation sites of p53 (Ser15, 20, 37, 46 and 392); all of these sites were inhibited by si-AR. Subsequently we identified three kinases, SPAK, MDC1 and CaMKII that are under AR control and two of them, MDC1 and CaMKII, apparently participate in p53 upstream events that resulted in p53 inhibition. Using qPCR we showed that the level of miR-34a increased by 3-fold after DOX, but no increase was found with si-AR. MiR-34c expression increased 27 fold after DOX and only by 2.7 times with si-AR. It appears that AR-dependent inhibition of p53 resulted in suppression of miR-34a and -34c expression. Importantly, DOX did not induce miR-34 in LNCaP grown in an androgen free medium or in AR-negative prostate cancer cell lines, DU145 and PC3. To directly investigate the role of miR-34 in DOX-mediated apoptosis, we transfected cells with anti-miR-34 oligonucleotides or with miR-34. We found that inhibition of individual miR-34, either 34a or 34c, or forced overexpression of miR-34a or miR-34c did not modulate DOX-mediated apoptosis. Only simultaneous inhibition or forced overexpression of both miR-34 resulted in modulation of DOX-mediated apoptosis. Taken together, our data indicate that cooperation between miR-34a and 34c plays an important role in AR-dependent p53-mediated apoptosis in prostate cancer.