Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Androgênios , Glucocorticoides , Humanos , MasculinoRESUMO
The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) is frequently dysregulated in prostate adenocarcinoma (PC), via either somatic mutations or mRNA downregulation, suggesting an important tumour suppressor function. To examine its physiologic role in the prostate epithelium in vivo, we generated mice with prostate-specific biallelic ablation of Spop. These mice exhibited increased prostate mass, prostate epithelial cell proliferation, and expression of c-MYC protein compared to littermate controls, and eventually developed prostatic intraepithelial neoplasia (PIN). We found that SPOPWT can physically interact with c-MYC protein and, upon exogenous expression in vitro, can promote c-MYC ubiquitination and degradation. This effect was attenuated in PC cells by introducing PC-associated SPOP mutants or upon knockdown of SPOP via short-hairpin-RNA, suggesting that SPOP inactivation directly increases c-MYC protein levels. Gene Set Enrichment Analysis revealed enrichment of Myc-induced genes in transcriptomic signatures associated with SPOPMT. Likewise, we observed strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent PC patient cohorts. The core SPOPMT;MYCHigh transcriptomic response, defined by the overlap between the SPOPMT and c-MYC transcriptomic programmes, was also associated with inferior clinical outcome in human PCs. Finally, the organoid-forming capacity of Spop-null murine prostate cells was more sensitive to c-MYC inhibition than that of Spop-WT cells, suggesting that c-MYC upregulation functionally contributes to the proliferative phenotype of Spop knock-out prostates. Taken together, our data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, identify c-MYC as a novel bona fide SPOP substrate, and help explain the frequent inactivation of SPOP in human PC. We propose SPOPMT-induced stabilization of c-MYC protein as a novel mechanism that can increase total c-MYC levels in PC cells, in addition to amplification of c-MYC locus.
Assuntos
Adenocarcinoma/patologia , Proteínas Nucleares/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitinação , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Estudos de Coortes , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Próstata/metabolismo , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Inibidores de Proteassoma/farmacologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/genética , Complexos Ubiquitina-Proteína LigaseRESUMO
MicroRNAs are important epigenetic regulators of protein expression by triggering degradation of target mRNAs and/or inhibiting their translation. Dysregulation of microRNA expression has been reported in several cancers, including prostate cancer (PC). We comprehensively characterized the proteomic footprint of a panel of 12 microRNAs that are potently suppressed in metastatic PC (SiM-miRNAs: miR-1, miR-133a, miR-133b, miR-135a, miR-143-3p, miR-145-3p, miR-205, miR-221-3p, miR-221-5p, miR-222-3p, miR-24-1-5p, and miR-31) using reverse-phase proteomic arrays. Re-expression of these SiM-miRNAs in PC cells suppressed cell proliferation and targeted key oncogenic pathways, including cell cycle, apoptosis, Akt/mammalian target of rapamycin signaling, metastasis and the androgen receptor (AR) axis. However, only 12%, at most, of these observed protein expression changes could be explained by predicted direct binding of miRNAs to corresponding mRNAs, suggesting that the majority of these proteomic effects result indirectly. AR and its steroid receptor coactivators (SRCs; SRC-1, -2 and -3) were recurrently affected by these SiM-miRNAs. In agreement, we identified inverse correlations between expression of these SiM-miRNAs and early clinical recurrence, as well as with AR transcriptional activity in human PC tissues. We also identified robust induction of miR-135a by androgen and strong direct binding of AR to the miR-135a locus. As miR-135a potently suppresses AR expression, this results in a negative feedback loop that suppresses AR protein expression in an androgen-dependent manner, while de-repressing AR expression upon androgen deprivation. Our results demonstrate that epigenetic silencing of these SiM-miRNAs can result in increased AR axis activity and cell proliferation, thus contributing to disease progression. We further demonstrate that a negative feedback loop involving miR-135a can restore AR expression under androgen-deprivation conditions, thus contributing to the upregulation of AR protein expression in castration-resistant PC. Finally, our unbiased proteomic profiling demonstrates that the majority of actual protein expression changes induced by SiM-miRNAs cannot be explained based on predicted direct interactions.
Assuntos
MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Proteômica , Receptores Androgênicos/genética , Transdução de Sinais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Humanos , Masculino , Metástase Neoplásica , Prognóstico , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Regulação para CimaRESUMO
Multiple myeloma (MM) remains fatal despite all available therapies. Initial treatment with conventional drugs such as, Dexamethasone (Dex) effectively induces MM cell death; however, prolonged drug exposures results in the development of chemoresistance. Our prior study demonstrated that 2-Methoxyestradiol (2ME2) induces apoptosis in multiple myeloma (MM) cells resistant to Dexamethasone (Dex). Here, we show the mechanism whereby 2ME2 overcomes Dex-resistance. The oligonucleotide array analysis demonstrates that Heat Shock Protein-27 (Hsp27) is upregulated in Dex-resistant, but not in Dex-sensitive MM cells. Proteomics analysis of Hsp27-immunocomplexes revealed the presence of actin in Dex-resistant, but not in Dex-sensitive cells. Biochemical interaction between Hsp27 and actin was examined by co-immunoprecipitation with anti-Hsp27 or actin Abs. Far western blot analysis using GST-Hsp27 fusion protein showed a direct association with actin both in vitro and in vivo. Importantly, 2ME2- or Bortezomib/Proteasome inhibitor (PS-341)-induced apoptosis in Dex-resistant MM cell lines and MM patient cells is associated with disruption of the Hsp27-actin complexes. Finally, blockade of Hsp27 by anti-sense strategy enhanced anti-MM activity of both 2ME2 and PS-341. These findings provide the clinical application of novel therapeutics targeting Hsp27 to improve patient outcome in MM.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Dexametasona , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Estradiol/farmacologia , Proteínas de Choque Térmico/metabolismo , Mieloma Múltiplo/metabolismo , Inibidores de Proteassoma , Pirazinas/farmacologia , 2-Metoxiestradiol , Actinas/metabolismo , Bortezomib , Humanos , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The tumor necrosis factor (TNF) family comprises several ligands, such as the prototype TNF-alpha, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), which trigger apoptosis in susceptible cells by activating respective cell-surface receptors. The study of these cell death pathways has attracted significant attention in several fields, including that of thyroid cancer, because they participate in immune system function, as an arm of cell-mediated cytotoxicity, and because recombinant ligands are available for pharmacological use. TNF-alpha is a pluripotent cytokine that induces both pro-apoptotic and anti-apoptotic effects on thyroid carcinoma cells. FasL triggers apoptosis in other tumor types, but thyroid carcinoma cells are resistant to this effect. On the other hand, TRAIL potently and selectively kills thyroid carcinoma cells. Consequently, TRAIL is the only member of the family with significant anticancer activity and an acceptable toxicity profile to be used as a novel therapy for thyroid cancer. The caspase inhibitor FLIP plays a significant role in negatively regulating receptor-induced apoptosis. Thelper 1-type cytokines, such as interferon-gamma, TNF-alpha and interleukin-1beta increase the sensitivity of both normal and neoplastic thyrocytes to FasL and TRAIL. On the other hand, IGF-I and other growth/survival factors produced in the local tumor microenvironment activate the phosphatidylinositol 3-kinase/Akt kinase pathway and exert an anti-apoptotic effect by upregulating several apoptosis inhibitors, including FLIP. Pharmacological modulation of apoptosis induced by FasL and TRAIL/Apo2L holds promise of therapeutic applications in human malignancies.
Assuntos
Glicoproteínas de Membrana/fisiologia , Neoplasias da Glândula Tireoide/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas de Bactérias/metabolismo , Comunicação Celular , Proteína Ligante Fas , Humanos , Interferon gama/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapêutico , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Th1/imunologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/imunologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/uso terapêutico , Receptor fas/metabolismoRESUMO
FasL and TRAIL/Apo2L participate in cell-mediated cytotoxicity by inducing apoptosis in susceptible cells via respective cell surface receptors. Normal and neoplastic thyroid tissues are resistant to FasL-induced apoptosis but are sensitized by Th-1-type cytokines. In Hashimoto's thyroiditis, both FasL and its receptor, Fas, are strongly upregulated and their interaction leads to the suicidal/fratricidal death of thyrocytes. In Graves' disease, FasL expression in thyroid follicular cells is induced by thionamides and kills infiltrating lymphocytes. In this condition, Th-2-type cytokines upregulate the anti-apoptotic molecules FLIP and Bcl-x(L) and protect thyrocytes from apoptosis. FasL is expressed by neoplastic thyrocytes and induces apoptosis of infiltrating lymphocytes. TRAIL/Apo2L kills thyroid carcinoma cells but spares normal thyrocytes, thus providing a potential therapy for thyroid cancer.
Assuntos
Apoptose/fisiologia , Glicoproteínas de Membrana/fisiologia , Doenças da Glândula Tireoide/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Proteína Ligante Fas , Doença de Graves/metabolismo , Humanos , Ligante Indutor de Apoptose Relacionado a TNF , Neoplasias da Glândula Tireoide/fisiopatologia , Receptor fas/metabolismoRESUMO
Multiple myeloma (MM) remains incurable and novel treatments are urgently needed. Preclinical in vitro and in vivo evaluations were performed to assess the potential therapeutic applications of human recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) in MM. TRAIL/Apo2L potently induced apoptosis of MM cells from patients and the majority of MM cell lines, including cells sensitive or resistant to dexamethasone (Dex), doxorubicin (Dox), melphalan, and mitoxantrone. TRAIL/Apo2L also overcame the survival effect of interleukin 6 on MM cells and did not affect the survival of peripheral blood and bone marrow mononuclear cells and purified B cells from healthy donors. The status of the TRAIL receptors (assessed by immunoblotting and flow cytometry) could not predict TRAIL sensitivity of MM cells. The anti-MM activity of TRAIL/Apo2L was confirmed in nu/xid/bg mice xenografted with human MM cells; TRAIL (500 microg intraperitoneally daily for 14 days) was well tolerated and significantly suppressed the growth of plasmacytomas. Dox up-regulated the expression of the TRAIL receptor death receptor 5 (DR5) and synergistically enhanced the effect of TRAIL not only against MM cells sensitive to, but also against those resistant to, Dex- or Dox-induced apoptosis. Nuclear factor (NF)-kappaB inhibitors, such as SN50 (a cell-permeable inhibitor of the nuclear translocation and transcriptional activity of NF-kappaB) or the proteasome inhibitor PS-341, enhanced the proapoptotic activity of TRAIL/Apo2L against TRAIL-sensitive MM cells, whereas SN50 reversed the TRAIL resistance of ARH-77 and IM-9 MM cells. Importantly, normal B lymphocytes were not sensitized to TRAIL by either Dox, SN50, or PS-341. These preclinical studies suggest that TRAIL/Apo2L can overcome conventional drug resistance and provide the basis for clinical trials of TRAIL-based treatment regimens to improve outcome in patients with MM. (Blood. 2001;98:795-804)
Assuntos
Apoptose/efeitos dos fármacos , Glicoproteínas de Membrana/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteínas Reguladoras de Apoptose , Linfócitos B/efeitos dos fármacos , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Interleucina-6/farmacologia , Glicoproteínas de Membrana/administração & dosagem , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/farmacologia , Transplante de Neoplasias , Plasmocitoma/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Transplante Heterólogo , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Fas-mediated apoptosis proceeds though mitochondria-dependent or -independent pathways and is deficient in drug-resistant cells. Neuroblastoma, a common pediatric malignancy, often develops drug-resistance and has a silenced caspase 8 (FLICE) gene, which has been associated with Fas- and drug-resistance. We report that besides caspase 8, which was absent in approximately one-third of 26 neuroblastoma cases in this study, other proteins such as bcl-2 and FLICE-inhibitory protein (FLIP), are equally important in conferring Fas-resistance to neuroblastoma cells. Both bcl-2 and FLIP were frequently expressed in neuroblastoma tissues. Our in vitro studies showed that FLIP was recruited to the death-inducing signaling complex and interfered with the recruitment of caspase 8 in neuroblastoma cells. bcl-2 inhibited the activation of the mitochondria; but it also lowered the free cytoplasmic levels of caspase 8 by binding and sequestering it, thus acting through a novel antiapoptotic mechanism upstream of the mitochondria. In vitro down-regulation of bcl-2 with antisense oligonucleotides allowed the release of cytochrome c from mitochondria and the activation of caspases 8 and 3 upon Fas activation as well as sensitized neuroblastoma cells to Fas-mediated apoptosis. Down-regulation of FLIP had only a modest apoptotic effect because of the coexistent mitochondrial block. However, combined treatment with bcl-2 and FLIP antisense oligonucleotides had a statistically significant synergistic effect reversing Fas-resistance in neuroblastoma cells in vitro. These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation. Thus, gene-targeting therapies for bcl-2 and FLIP may reverse Fas-resistance and prove useful in the treatment of drug-resistant neuroblastomas.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/fisiologia , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptor fas/fisiologia , Adolescente , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/genética , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/biossíntese , Caspases/metabolismo , Criança , Pré-Escolar , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Lactente , Recém-Nascido , Neuroblastoma/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
We have previously shown that corticotropin-releasing hormone plays an important proinflammatory role in the induction of experimental autoimmune uveoretinitis. In this study, we examined the role of apoptosis in the destruction of the retina during experimental autoimmune uveoretinitis, and the role of corticotropin-releasing hormone as a local regulator of Fas and Fas Ligand expression in this condition. We evaluated apoptosis by the terminal deoxynucleotidyl transferase dUTP nick end labeling method and Fas and Fas Ligand presence by immunohistochemistry. We examined formalin-fixed, paraffin-embedded eye sections from female Lewis rats or B10.A mice immunized with the major pathogenetic epitope (R16 peptide) of the interphotoreceptor retinoid-binding protein. Female B10.A mice similarly immunized were treated with intraperitoneal injections of the rabbit anti-corticotropin-releasing hormone antibody TS-2 or nonimmune rabbit serum. The percentage of retinal cells undergoing apoptosis and the expression of Fas and Fas Ligand were increased in inflamed retinas in immunized Lewis rats and B10.A mice, compared to controls. Retinas from immunized B10.A mice treated with anti-corticotropin-releasing hormone antibody showed significantly lower apoptosis and Fas and Fas Ligand expression than placebo-treated animals. In conclusion, retinal cells in experimental autoimmune uveoretinitis undergo apoptosis associated with concurrent upregulation of Fas and Fas Ligand. The local presence of corticotropin-releasing hormone appears to be of pivotal importance in this process.
Assuntos
Apoptose/fisiologia , Doenças Autoimunes do Sistema Nervoso/patologia , Hormônio Liberador da Corticotropina/fisiologia , Retinite/patologia , Uveíte/patologia , Receptor fas/fisiologia , Animais , Doenças Autoimunes do Sistema Nervoso/metabolismo , DNA Nucleotidiltransferases/fisiologia , Feminino , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Endogâmicos Lew , Retinite/metabolismo , Regulação para Cima , Uveíte/metabolismoRESUMO
The anti-CD20 chimeric monoclonal antibody rituximab (Rituxan) is used to treat patients with various B-cell tumors, including patients with plasma cell dyscrasias who have CD20+ disease. Many patients with CD20+ disease have either primary unresponsive disease or progress after initially responding to rituximab; therefore, understanding how tumor cells are, or become, resistant to rituximab is of clinical relevance. In this report, we determined whether tumor cells express antigens that block complement-mediated lysis or antibody-dependent cell-mediated cytotoxicity (ADCC) and thereby contribute to rituximab resistance. We demonstrate that expression of the complement regulator CD59 is associated with resistance to rituximab-mediated complement lysis of multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) cell lines. Moreover, neutralization of CD59 using a blocking monoclonal antibody reversed resistance to rituximab-mediated complement lysis of CD20++ CD59++ ARH-77 MM cells. In addition, we demonstrate the presence of CD59 and rituximab binding on viable tumor cells from patients with MM and Waldenstrom's macroglobulinemia with progressive disease despite rituximab therapy. Last, we also examined MM and NHL B-cell lines, as well as patient tumor cells, for the expression of other antigens that may have a role in blocking ADCC activity, such as Fas ligand (FasL), MUCI, or TRAIL. FasL, MUC1, and/or TRAIL were coexpressed with complement regulators on many of these cells. These studies therefore show that complement regulators, particularly CD59 and antigens that may block ADCC, are present on various B-cell tumors and associated with rituximab resistance in patients. A prospective, clinical study is assessing the role of these antigens in mediating rituximab resistance.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD59/metabolismo , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Animais , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/metabolismo , Proteínas Reguladoras de Apoptose , Linfócitos B/imunologia , Proteína Ligante Fas , Humanos , Técnicas In Vitro , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Glicoproteínas de Membrana/imunologia , Camundongos , Mucina-1/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Rituximab , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologia , Macroglobulinemia de Waldenstrom/imunologia , Macroglobulinemia de Waldenstrom/terapiaRESUMO
Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.
Assuntos
Ganglioneuroblastoma/patologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/patologia , Animais , Carbacol/farmacologia , Diferenciação Celular , Agonistas Colinérgicos/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Melanoma/patologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Muscarínicos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Gânglio Trigeminal/química , Células Tumorais CultivadasRESUMO
In this study, we investigated the sensitivity of Ewing's sarcoma family tumors (ESFTs) of children and adolescents to the tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL). TRAIL binds to death receptors (DRs) DR4, DR5, DcR1, and DcR2. Either DR4 or DR5 can induce apoptosis, whereas DcR1 and DcR2 are considered inhibitory receptors. Nine of 10 ESFT cell lines, including several that were Fas resistant, underwent apoptosis with TRAIL through activation of caspase-10, capase-8 (FLICE), caspase-3, and caspase-9. In contrast to the Fas signaling pathway, caspase-10, but not caspase-8 or the Fas-associated death domain-containing molecule, was recruited to the TRAIL receptor-associated signaling complex. We found that 9 of 10 ESFT cell lines expressed both DR4 and DR5 by Western blotting, whereas the TRAIL-resistant line expressed only DR4. However, DR4 was absent from the cell surface in the resistant and two additional lines (three of five tested lines), suggesting that it may have been nonfunctional. On the contrary, DR5 was located on the cell surface in all four sensitive lines tested, being absent only from the cell surface of the resistant line that was also DR5-negative by Western blotting. In agreement with these findings, the resistance of the line was overcome by restoration of DR5 levels by transfection. Levels of DcR1 and DcR2 or levels of the FLICE-inhibitory protein (FLIP) did not correlate with TRAIL resistance, and protein synthesis inhibition did not sensitize the TRAIL-resistant line to TRAIL. Because these data suggested that sensitivity of ESFTs to TRAIL was mainly based on the presence of DR4/DR5, we investigated the presence of these receptors in 32 ESFT tissue sections by immunohistochemistry. We found that 23 of 32 tumor tissues (72%) expressed both receptors, 8 of 32 (25%) expressed one receptor only, and 1 was negative for both. Our finding of wide expression of DR4/DR5 in ESFT in vivo, in combination with their high sensitivity to TRAIL in vitro and the reported lack of toxicity of TRAIL in mice and monkeys, suggests that TRAIL may be a novel effective agent in the treatment of ESFTs.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/biossíntese , Sarcoma de Ewing/patologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Caspases/metabolismo , Membrana Celular/metabolismo , Cicloeximida/farmacologia , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacologia , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Transfecção , Células Tumorais CultivadasRESUMO
Recent evidence suggests that one mechanism whereby cytotoxic drugs, such as doxorubicin, kill tumors is the induction or up-regulation of Fas ligand (FasL) expression on the tumor cell surface. The ensuing engagement of Fas by FasL on adjacent cells leads to apoptosis. However, despite cytotoxic drug-induced FasL expression, Fas-sensitive tumors frequently resist chemotherapy, suggesting that they may possess a mechanism that prevents or inactivates Fas-FasL interactions. In the present work, we addressed the involvement of the FasL/Fas signaling pathway in doxorubicin-induced apoptosis and the ability of matrix metalloproteinases (MMPs) to proteolytically cleave FasL in tumor cells. Doxorubicin-induced apoptosis was inhibited by expression of soluble Fas or incubation of the tumor cells with MMP-7 but not with MMP-2 or MMP-9. Resistance to doxorubicin was also induced by expression in the tumor cells of constitutively active MMP-7 but not of a catalytically inactive mutant. Conversely, inhibition of MMP-7 expression in tumor cells by transfection of MMP-7 cDNA in antisense orientation resulted in sensitization to doxorubicin. MMP-7 efficiently cleaved recombinant FasL in vitro and reduced cell surface FasL expression. Our observations provide evidence that one mechanism whereby MMP-7 may promote tumor survival and resistance to doxorubicin is by cleaving FasL and reducing its effectiveness in triggering Fas-mediated apoptosis.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Metaloproteinase 7 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Resistencia a Medicamentos Antineoplásicos , Proteína Ligante Fas , Humanos , Hidrólise , Immunoblotting , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Sarcoma de Ewing/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Receptor fas/metabolismoRESUMO
Matrix metalloproteinases (MMPs) regulate the turnover of extracellular matrix (ECM) components and play an important role in embryo development, morphogenesis and tissue remodelling, as well as in tumour invasion and metastasis. Synthetic MMP inhibitors (MMPIs) were designed to prevent tumour cell-induced changes in ECM and thereby achieve antitumour activity. Several MMPIs have entered clinical trials but the preliminary results did not meet the expectations. Recent evidence suggests that MMPs may have more diverse roles than originally believed, influencing angiogenesis, cytokine secretion, as well as tumour cell growth and survival. In particular, synthetic MMPIs may directly induce apoptosis of cancer cells via their inhibitory effect on the shedding of Fas Ligand (FasL), a transmembrane member of the TNF superfamily that kills susceptible cells through its receptor, Fas. Several types of cancers have been shown to express FasL and to shed it from their surface as a soluble form, which is significantly less potent in promoting apoptosis. MMP-7 was recently reported to catalyse this process. Conversely, inhibition of FasL-shedding by a synthetic MMPI results in apoptosis of Fas-sensitive cancer cells. More importantly, DNA-damaging anticancer agents, such as adriamycin, kill cancer cells, at least in part, by upregulating FasL. By inhibiting the proteolytic cleavage of FasL, MMPIs can potentiate the killing effect of traditional chemotherapeutic drugs. These studies therefore demonstrate a direct link between DNA-damaging chemotherapeutic drugs, the apoptosis-inducing Fas/FasL system and the proteolytic activity of MMPs and have important therapeutic implications. For example, the proteolytic activity of MMP-7, which is broadly expressed in primary and especially metastatic human malignancies, may contribute to tumour resistance to cytotoxic agents; targeting and inactivating MMP-7 may, therefore, enhance the efficacy of conventional cancer chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Metaloproteinases de Matriz , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/fisiologia , Ensaios Clínicos como Assunto/estatística & dados numéricos , Proteína Ligante Fas , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Receptor fas/metabolismoRESUMO
TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) is a member of the TNF superfamily of death ligands that selectively induces apoptosis in tumour cells of diverse origins. In this report, we have reviewed recent studies examining TRAIL/Apo2L-induced apoptosis in multiple myeloma (MM), a B-cell malignancy which, in spite of its initial sensitivity to steroids, cytotoxic and high-dose chemotherapy, remains incurable. Recently, we demonstrated that TRAIL/Apo2L induces apoptosis of steroid- and chemotherapy-sensitive and resistant MM cell lines. Moreover, TRAIL/Apo2L selectively induced apoptosis of patient MM tumour cells while sparing non-malignant bone marrow and peripheral blood mononuclear cells. In addition, TRAIL/Apo2L inhibited the growth of human plasmacytomas xenografted into mice. Importantly, TRAIL/Apo2L-induced apoptosis was unaffected by IL-6, a potent growth and survival factor for MM cells which, as we and others have previously shown, blocks various pro-apoptotic signals including Fas ligand, which like TRAIL/Apo2L is also a member of the TNF family of ligands. In view of the potential clinical application of TRAIL/Apo2L to the treatment of MM, we have attempted to discern intracellular mechanisms of action and resistance for TRAIL/Apo2L in MM, along with strategies to increase sensitivity and overcome resistance of MM cells to TRAIL/Apo2L. These studies demonstrated that doxorubicin, an agent which is commonly used to treat MM patients, upregulated the expression of the DR5 death-signalling TRAIL receptor and synergistically enhanced the pro-apoptotic effect of TRAIL on MM cells. Moreover, NF-kappaB inhibitors such as SN50 (a cell permeable inhibitor of NF-kappaB nuclear translocation) as well as the proteasome inhibitor PS-341, which is currently in Phase II clinical trials, also enhanced the pro-apoptotic activity of TRAIL/Apo2L in MM cells. Lastly, TRAIL/Apo2L-induced apoptosis in MM cells was dependent on caspase-8 activation and inhibited by the caspase regulatory proteins FLIP and cIAP2. These studies provide a framework for the use of TRAIL/Apo2L as a single agent or as part of combination therapy for the treatment of MM.
Assuntos
Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Neoplasias/tratamento farmacológico , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Inibidores de Caspase , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Humanos , Glicoproteínas de Membrana/farmacologia , Mieloma Múltiplo/patologia , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fas Ligand (FasL) is a member of the TNF superfamily that induces apoptosis in susceptible cells upon cross-linking of its own receptor, Fas (Apo-1/CD95). FasL-induced apoptosis contributes to immune homeostasis and cell-mediated cytotoxicity. Several groups have suggested that it also participates in the mechanism of action of DNA-damaging anticancer drugs. However, others have disputed this hypothesis, based largely on the inability of exogenously added anti-Fas/FasL reagents to attenuate drug-induced apoptosis in their studies. In this minireview, we discuss the most recent evidence for and against the involvement of FasL/Fas in the sensitivity and resistance to chemotherapy in a variety of models. In our own model of Ewing's sarcoma (ES), we have extensively investigated the involvement of the FasL/Fas pathway in doxorubicin (Dox)-induced apoptosis. We have generated clones of the Fas-sensitive, Dox-sensitive ES cell line SK-N-MC that were either Fas-resistant or FasL-deficient, and found that they were significantly resistant to Dox. Cleavage of FasL by MMP-7 (matrilysin) protected the parental SK-N-MC cells from Dox, whereas inhibition of MMP-7 activity increased their sensitivity. Transfection of a construct encoding soluble (decoy) Fas protected SK-N-MC cells from Dox. However, incubation with anti-Fas or anti-FasL neutralizing antibodies or exogenous addition of pre-synthesized recombinant soluble Fas decoy protein had no protective effect. This raises the possibility that the proposed Fas/FasL suicidal interaction may take place in an intracellular compartment and thus is not accessible to exogenously added reagents. Therefore, commercially available Fas/FasL neutralizing reagents may not be a reliable indicator of the involvement of the Fas pathway in anticancer-drug-induced apoptosis and experiments using these agents should be carefully re-evaluated. The combined use of MMP inhibitors with conventional, cytotoxic chemotherapy may hold therapeutic benefit.
Assuntos
Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/fisiologia , Sarcoma de Ewing/tratamento farmacológico , Receptor fas/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Ligante Fas , Humanos , Sarcoma de Ewing/patologiaRESUMO
After its endocytosis from the colloid, some thyroglobulin (Tg) is transcytosed intact across thyrocytes, accounting in part for its presence in the circulation. We previously showed that megalin (gp330), an endocytic Tg receptor, mediates apical to basolateral Tg transcytosis. Here we investigated whether a portion of megalin remains combined with Tg after its transcytosis, using studies with cultured thyroid cells and in vivo observations. FRTL-5 cells, a rat thyroid cell line, cultured on filters in dual chambers form tight junctions and exhibit features of polarity, with expression of megalin exclusively on the upper (apical) surface. After the addition of unlabeled Tg to the upper chamber and incubation at 37 C, some Tg was transcytosed intact across FRTL-5 cells into the lower chamber. Two antimegalin ectodomain antibodies precipitated transcytosed Tg in fluids collected from the lower chamber. After the addition of Tg to surface-biotinylated FRTL-5 cells, an anti-Tg antibody and the two antimegalin ectodomain antibodies precipitated high molecular mass biotinylated material in fluids collected from the lower chamber, corresponding to much of the megalin ectodomain, as well as smaller amounts of lower molecular mass material. The results indicate that Tg transcytosed across FRTL-5 cells remains complexed with megalin ectodomain components, which we refer to as megalin secretory components. In aminotriazole-treated rats, which develop increased megalin-mediated Tg transcytosis, antimegalin antibodies precipitated some of the Tg in the serum. Tg was also precipitated by antimegalin antibodies in sera from patients with Graves' disease, in which we found increased megalin expression on the apical surface of thyrocytes. In contrast, in thyroidectomized patients with metastatic papillary thyroid carcinoma, in whom Tg is directly secreted by neoplastic thyroid cells into the circulation rather than transcytosed, serum Tg was not precipitated by antimegalin antibodies. The detection of Tg-megalin complexes may help identify the source of serum Tg in patients with thyroid diseases.
Assuntos
Autoantígenos/metabolismo , Glicoproteínas de Membrana/metabolismo , Tireoglobulina/sangue , Glândula Tireoide/metabolismo , Adulto , Idoso , Amitrol (Herbicida) , Animais , Western Blotting , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Bócio/induzido quimicamente , Bócio/metabolismo , Doença de Graves/metabolismo , Complexo Antigênico da Nefrite de Heymann , Humanos , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , Ratos , Ratos Endogâmicos Lew , Doenças da Glândula Tireoide/metabolismo , Glândula Tireoide/citologiaRESUMO
Thionamides are used in the treatment of Graves' disease (GD) and act mainly by inhibiting the organification of iodide, but also lower the levels of thyroid autoantibodies, sometimes leading to long-term remission. Fas ligand (FasL) induces apoptosis of susceptible cells by cross-linking its own receptor, Fas. While Fas is present in a wide variety of normal tissues, FasL expression is limited mainly to cells of the immune system, where it acts as an effector molecule of cell-mediated cytotoxicity, and to the placenta, brain, eye, and testis where it presumably contributes to their immune-privileged status by eliminating infiltrating lymphocytes. We examined immunohistochemically the presence of FasL in thyroid tissue from 15 glands of thionamide-treated GD patients and in 8 normal thyroid control specimens. We also investigated the presence of FasL in thionamide-treated thyrocytes in vitro and their ability to induce Fas-mediated apoptosis in lymphocytes. We found that FasL expression was very weak to undetectable in normal thyroid tissue and cultured thyrocytes, whereas it was strong in thionamide-treated GD glands and cultured thyrocytes. Methimazole-treated thyrocytes induced FasL-dependent apoptosis in cocultured lymphocytes, whereas methimazole treatment of lymphocytes grown in the absence of thyrocytes had no such effect. We conclude that FasL is highly expressed in follicular cells of thyroid glands obtained from thionamide-treated Graves' patients and may contribute to the immunomodulatory effect of thionamides in this disease.
Assuntos
Antitireóideos/uso terapêutico , Doença de Graves/metabolismo , Glicoproteínas de Membrana/análise , Glândula Tireoide/química , Adulto , Apoptose/efeitos dos fármacos , Carbimazol/uso terapêutico , Células Cultivadas , Técnicas de Cocultura , Proteína Ligante Fas , Feminino , Doença de Graves/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Células Jurkat , Masculino , Glicoproteínas de Membrana/genética , Metimazol/uso terapêutico , Pessoa de Meia-Idade , RNA Mensageiro/análise , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/patologiaRESUMO
Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.
Assuntos
Apoptose/fisiologia , Glicoproteínas de Membrana/fisiologia , Neoplasias da Glândula Tireoide/patologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologia , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Western Blotting , Antígenos CD8/biossíntese , Antígenos CD8/genética , Carcinoma Papilar/imunologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Caspases/biossíntese , Caspases/genética , Caspases/metabolismo , Caspases/farmacologia , Ativação Enzimática , Feminino , Humanos , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/biossínteseRESUMO
Nuclear factor kappa-B (NF-kappaB) is a heterodimeric transcription factor with a pivotal role in orchestrating immune and inflammatory processes. Inflammatory cytokines and prostanoids activate NF-kappaB, which, in turn, stimulates expression of cytokines, proteases, adhesion molecules and other inflammatory mediators. Caffeic acid phenethyl ester (CAPE) is a compound that modulates nuclear binding of the NF-kappaB p65 subunit (RelA). To determine whether CAPE decreases the viability of cells participating in host defense, we first tested its in vitro effect on a glucocorticoid-sensitive and -resistant cell line of lymphoid origin. CAPE induced apoptotic cell death in a dose-dependent fashion and to a similar extent in both cell lines. Furthermore, a low concentration of CAPE decreased the LD(50) of dexamethasone by 3- to 5-fold. Since therapeutic induction of apoptosis of activated inflammatory cells holds the attraction of destroying effector cells safely without secondary tissue damage, we examined the effects of CAPE in a rat model of carrageenin-induced subcutaneous inflammation. Local administration of CAPE resulted in increased leukocyte apoptosis and marked reduction in exudate leukocyte, neutrophil and monocyte concentrations at the inflammatory site. CAPE decreased expression of cytosolic IkappaBalpha and increased nuclear translocation of p65. These findings may suggest that novel anti-inflammatory therapies can be based upon activation of NF-kappaB-mediated transcription of genes curbing the inflammatory response and that CAPE or its analogs hold therapeutic promise.