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Fármacos Dermatológicos/uso terapéutico , Dermatomiositis/tratamiento farmacológico , Oligonucleótidos/uso terapéutico , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism.
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Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/fisiología , Quinasa I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Animales , Activación Enzimática , Células HeLa , Humanos , Ratones , Quinasa de Factor Nuclear kappa BRESUMEN
The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.
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Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Biomimética , Dicroismo Circular , Dimerización , Activación Enzimática , Humanos , Lentivirus/genética , Ratones , Mutagénesis , Unión Proteica , Estructura Terciaria de Proteína , Dispersión de Radiación , Transducción de Señal , Ubiquitina/químicaRESUMEN
PURPOSE: Tilsotolimod is an investigational synthetic Toll-like receptor 9 (TLR9) agonist that has demonstrated antitumor activity in preclinical models. The ILLUMINATE-101 phase I study explored the safety, dose, efficacy, and immune effects of intratumoral (it) tilsotolimod monotherapy in multiple solid tumors. PATIENTS AND METHODS: Patients with a diagnosis of refractory cancer not amenable to curative therapies received tilsotolimod in doses escalating from 8 to 32 mg into a single lesion at weeks 1, 2, 3, 5, 8, and 11. Additional patients with advanced malignant melanoma were enrolled into an expansion cohort at the 8 mg dose. Objectives included characterizing the safety, establishing the dose, efficacy, and immunologic assessment. Blood samples and tumor biopsies of injected and noninjected lesions were obtained at baseline and 24 hours after treatment for immune analyses. RESULTS: Thirty-eight and 16 patients were enrolled into the dose escalation and melanoma expansion cohorts, respectively. Deep visceral injections were conducted in 91% of patients. No dose-limiting toxicities (DLT) or grade 4 treatment-related adverse events were observed. Biopsies 24 hours after treatment demonstrated an increased IFN pathway signature and dendritic cell maturation. Immunologic profiling revealed upregulation of IFN-signaling genes and modulation of genes for checkpoint proteins. In the dose escalation cohort, 12 (34%) of 35 evaluable patients achieved a best overall response rate (ORR) of stable disease (SD), whereas 3 (19%) of 16 evaluable patients in the melanoma cohort achieved stable disease. CONCLUSIONS: Overall, tilsotolimod monotherapy was generally well tolerated and induced rapid, robust alterations in the tumor microenvironment. See related commentary by Punekar and Weber, p. 5007.
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Melanoma , Neoplasias , Neoplasias Cutáneas , Humanos , Receptor Toll-Like 9 , Presentación de Antígeno , Neoplasias/patología , Melanoma/tratamiento farmacológico , Melanoma/genética , Estudios de Cohortes , Microambiente TumoralRESUMEN
Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of bladder tumors are non-muscle invasive at diagnosis, but even after local surgical therapy there is a high rate of local tumor recurrence and progression. Current treatments extend time to recurrence but do not significantly alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein TNF-related apoptosis-inducing ligand (TRAIL) with a small molecule inhibitor of apoptosis proteins (IAP) antagonist to interfere with intracellular regulators of apoptosis in human bladder tumor cells. Our results demonstrate that the IAP antagonist Compound A exhibits high binding affinity to the XIAP BIR3 domain. When Compound A was used at nontoxic concentrations in combination with TRAIL, there was a significant increase in the sensitivity of TRAIL-sensitive and TRAIL-resistant bladder tumor lines to TRAIL-mediated apoptosis. In addition, modulation of TRAIL sensitivity in the TRAIL-resistant bladder tumor cell line T24 with Compound A was reciprocated by XIAP small interfering RNA-mediated suppression of XIAP expression, suggesting the importance of XIAP-mediated resistance to TRAIL in these cells. These results suggest the potential of combining Compound A with TRAIL as an alternative therapy for bladder cancer.
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Apoptosis/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Proteínas Mitocondriales/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis , Caspasas/metabolismo , Sinergismo Farmacológico , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14.
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Descubrimiento de Drogas , Inhibidores Enzimáticos , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Indoles , Piranos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Sitio Alostérico , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Piranos/síntesis química , Piranos/química , Piranos/farmacología , Relación Estructura-ActividadRESUMEN
Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase I/II trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti-PD-1- resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase II dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in noninjected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local IFNα gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase III clinical trial with this combination (NCT03445533) is ongoing. SIGNIFICANCE: Despite recent developments in advanced melanoma therapies, most patients do not experience durable responses. Intratumoral tilsotolimod injection elicits a rapid, local type 1 IFN response and, in combination with ipilimumab, activates T cells to promote clinical activity, including in distant lesions and patients not expected to respond to ipilimumab alone.This article is highlighted in the In This Issue feature, p. 1861.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Ipilimumab , Melanoma , Neoplasias Cutáneas , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Protocolos de Quimioterapia Combinada Antineoplásica , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ipilimumab/administración & dosificación , Ipilimumab/uso terapéutico , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Resultado del Tratamiento , Estados UnidosRESUMEN
We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Indoles/química , Piranos/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/farmacología , Piranos/síntesis química , Piranos/farmacología , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.
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Antivirales/farmacología , Benzofuranos/antagonistas & inhibidores , Farmacorresistencia Viral , Inhibidores Enzimáticos/farmacología , Variación Genética , Hepacivirus/efectos de los fármacos , Replicón/efectos de los fármacos , Antivirales/metabolismo , Línea Celular Tumoral , Clonación Molecular , Inhibidores Enzimáticos/metabolismo , Genotipo , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Modelos Moleculares , Mutación , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.
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Apoptosis/efectos de los fármacos , Caspasa 10/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferón gamma/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Caspasa 10/química , Caspasa 10/genética , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular , Citocina TWEAK/farmacología , Sinergismo Farmacológico , Células HT29 , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Interferón gamma/genética , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Ácidos Pentanoicos/farmacología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Dipéptidos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Animales , Neoplasias de la Mama/patología , Caspasa 8/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones Desnudos , Proteínas Mitocondriales/metabolismo , Receptores del Factor de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.
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Antineoplásicos/farmacología , Proteínas Portadoras/química , Dipéptidos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Indoles/farmacología , Proteínas Mitocondriales/química , Imitación Molecular , Neoplasias Experimentales/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis , Dipéptidos/uso terapéutico , Descubrimiento de Drogas , Indoles/uso terapéutico , Ratones , Modelos MolecularesRESUMEN
PURPOSE: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. EXPERIMENTAL DESIGN: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. RESULTS: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. CONCLUSIONS: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.
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Dipéptidos/farmacología , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melanoma/metabolismo , Proteínas Mitocondriales/metabolismo , Imitación Molecular , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dipéptidos/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Indoles/administración & dosificación , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Proteínas Mitocondriales/química , Esferoides Celulares , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BCG, the current gold standard immunotherapy for bladder cancer, exerts its activity via recruitment of neutrophils to the tumor microenvironment. Many patients do not respond to BCG therapy, indicating the need to understand the mechanism of action of BCG-stimulated neutrophils and to identify ways to overcome resistance to BCG therapy. Using isolated human neutrophils stimulated with BCG, we found that TNF-α is the key mediator secreted by BCG-stimulated neutrophils. RT4v6 human bladder cancer cells, which express TNFR1, CD95/Fas, CD95 ligand/FasL, DR4, and DR5, were resistant to BCG-stimulated neutrophil conditioned medium but effectively killed by the combination of conditioned medium and Smac mimetic. rhTNF-α and rhFasL, but not rhTRAIL, in combination with Smac mimetic, generated signature molecular events similar to those produced by BCG-stimulated neutrophils in combination with Smac mimetic. However, experiments using neutralizing antibodies to these death ligands showed that TNF-α secreted from BCG-stimulated neutrophils was the key mediator of anticancer action. These findings explain the mechanism of action of BCG and identified Smac mimetics as potential combination therapeutic agents for bladder cancer.
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Proteína Ligando Fas/metabolismo , Imitación Molecular , Mycobacterium bovis/inmunología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Oligopéptidos/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Neoplasias de la Vejiga Urinaria/prevención & control , Western Blotting , Proliferación Celular , Medios de Cultivo Condicionados/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Neutrófilos/inmunología , Neutrófilos/microbiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
PURPOSE: inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. RESULTS: single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis. EXPERIMENTAL DESIGN: a panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. CONCLUSION: our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.
Asunto(s)
Antineoplásicos/farmacología , Oligopéptidos/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Immunoblotting , Inmunoprecipitación , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/patología , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Lisosomas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Inhibidores de Caspasas , Catepsinas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Lisosomas/efectos de los fármacos , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Receptor de TWEAKRESUMEN
XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.
Asunto(s)
Apoptosis/fisiología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Comunicación Autocrina , Benzoquinonas/metabolismo , Brefeldino A/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas , Línea Celular , Inhibidores Enzimáticos/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lactamas Macrocíclicas/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Imitación Molecular , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Serpinas/metabolismo , Transducción de Señal/fisiología , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Proteínas Virales/metabolismoRESUMEN
A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique beta-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 microM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sustitución de Aminoácidos , Sitios de Unión , Unión Competitiva , Línea Celular Tumoral , Cristalografía por Rayos X , Farmacorresistencia Viral/genética , Genes Virales , Ingeniería Genética , Variación Genética , Guanosina Trifosfato/metabolismo , Hepacivirus/genética , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Estructura Terciaria de Proteína , ARN Viral/genética , Recombinación Genética , Replicón/genética , Selección Genética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.