RESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.
Asunto(s)
Atrofia Muscular Espinal/terapia , Oligonucleótidos/genética , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Exones/genética , Fibroblastos , Dosificación de Gen , Terapia Genética/métodos , Humanos , Ratones , Terapia Molecular Dirigida/métodos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Mutación Puntual , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Activación Transcripcional/genética , Regulación hacia ArribaRESUMEN
PURPOSE: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. EXPERIMENTAL DESIGN: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. RESULTS: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. CONCLUSIONS: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Triazoles/farmacología , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones SCID , Prostaglandina-E Sintasas , Unión Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Triazoles/uso terapéutico , Triazoles/toxicidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vascular disrupting agents (VDAs) are an emerging class of therapeutics targeting the existing vascular network of solid tumors. However, their clinical progression has been hampered because of limited single-agent efficacy, primarily caused by the persistence of surviving cells at the well perfused "viable rim" of tumors, which allows rapid tumor regrowth to occur. In addition, off-target adverse events, including cardiovascular toxicities, underscore a need for compounds with improved safety profiles. Here, we characterize the mechanism of action, antitumor efficacy, and cardiovascular safety profile of (S)-2-amino-N-(2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl)-3-phenylpropanamide hydrochloride (STA-9584), a novel tubulin-binding VDA. In vitro, 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)aniline (STA-9122) (active metabolite of STA-9584) displayed increased potency relative to other tubulin-binding agents and was highly cytotoxic to tumor cells. STA-9584 induced significant tumor regressions in prostate and breast xenograft models in vivo and, in an aggressive syngeneic model, demonstrated superior tumor growth inhibition and a positive therapeutic index relative to combretastatin A-4 phosphate (CA4P). It is noteworthy that histological analysis revealed that STA-9584 disrupted microvasculature at both the center and periphery of tumors. Compared with CA4P, STA-9584 induced a 73% increase in central necrotic area, 77% decrease in microvasculature, and 7-fold increase in tumor cell apoptosis in the remaining viable rim 24 h post-treatment. Ultrasound imaging confirmed that STA-9584 rapidly and efficiently blocked blood flow in highly perfused tumor regions. Moreover, cardiovascular effects were evaluated in the Langendorff assay and telemetered dogs, and cardiovascular toxicity was not predicted to be dose-limiting. This bioactivity profile distinguishes STA-9584 from the combretastatin class and identifies the compound as a promising new therapeutic VDA candidate.
Asunto(s)
Inhibidores de la Angiogénesis , Antineoplásicos , Isoxazoles/farmacología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Fenilalanina/análogos & derivados , Animales , Antineoplásicos Fitogénicos/farmacología , Bibencilos/farmacología , Supervivencia Celular/efectos de los fármacos , Perros , Femenino , Corazón/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microcirculación/efectos de los fármacos , Neovascularización Patológica/patología , Fenilalanina/farmacología , Conejos , Flujo Sanguíneo Regional/efectos de los fármacos , Moduladores de Tubulina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Cobre/metabolismo , Hidrazinas/farmacología , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Cobre/química , Humanos , Mitocondrias/efectos de los fármacos , Neoplasias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Interleucina-12/antagonistas & inhibidores , Interleucina-23/antagonistas & inhibidores , Morfolinas/uso terapéutico , Psoriasis/tratamiento farmacológico , Piel/efectos de los fármacos , Triazinas/uso terapéutico , Administración Oral , Adolescente , Adulto , Anciano , Antígenos CD/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Femenino , Humanos , Hidrazonas , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Interleucina-23/biosíntesis , Interleucina-23/inmunología , Masculino , Persona de Mediana Edad , Morfolinas/administración & dosificación , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Psoriasis/inmunología , Psoriasis/patología , Pirimidinas , Piel/inmunología , Piel/patología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Triazinas/administración & dosificaciónRESUMEN
Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 µm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Triazoles/farmacología , Animales , Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Benzoquinonas/efectos adversos , Benzoquinonas/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Cristalografía por Rayos X , Femenino , Células HL-60 , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Células K562 , Lactamas Macrocíclicas/efectos adversos , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Conejos , Ratas , Ratas Sprague-Dawley , Triazoles/efectos adversos , Triazoles/química , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors. METHODS AND FINDINGS: This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25). CONCLUSIONS: This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias/veterinaria , Profármacos/uso terapéutico , Triazoles/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Cromatografía Líquida de Alta Presión , Perros , Femenino , Indoles , Masculino , Neoplasias/tratamiento farmacológico , Espectrometría de Masas en Tándem , Triazoles/química , Triazoles/farmacocinética , Triazoles/toxicidadRESUMEN
There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2(V617F) mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Neoplasias Experimentales/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Triazoles/farmacología , Animales , División Celular/efectos de los fármacos , Genes cdc , Ratones , Neoplasias Experimentales/patología , Células Tumorales CultivadasRESUMEN
The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Leucemia Mieloide/genética , Proteínas WT1/genética , Animales , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Benzoquinonas/uso terapéutico , Línea Celular Tumoral , Etopósido/farmacología , Femenino , Silenciador del Gen , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Humanos , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Ratones , Ratones SCID , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Triazoles/uso terapéutico , Proteínas WT1/química , Proteínas WT1/metabolismoRESUMEN
Elesclomol is a small-molecule investigational agent that selectively induces apoptosis in cancer cells by increasing oxidative stress. Elesclomol plus paclitaxel was shown to prolong progression-free survival compared with paclitaxel alone in a phase II clinical trial in patients with metastatic melanoma. However, the therapeutic potential of elesclomol in human breast cancer is unknown, and the signaling mechanism underlying the elesclomol effect is unclear. Here, we show that elesclomol alone modestly inhibited the growth of human breast cancer cells but not normal breast epithelial cells. Elesclomol potentiated doxorubicin- or paclitaxel-induced apoptosis and suppression of breast cancer cell growth. While both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were activated by elesclomol, elesclomol-induced apoptosis was only in part mediated by JNK1. The additive effect of elesclomol on chemotherapy drug-induced apoptosis was associated with increases in cleaved caspase-3, p21(Cip1), and p27(Kip1) and decreases in the Inhibitor of Apoptosis Protein levels and NF-kappaB activity. We also found that Akt/Hsp70 survival signaling was induced by elesclomol, which may reflect a cellular feedback mechanism. Blockade of Akt activation using a small-molecule inhibitor enhanced elesclomol-elicited apoptosis, while expression of a hyperactive Akt abolished the elesclomol effect. These data suggest that elesclomol's interaction with conventional chemotherapeutic and Akt-targeting agents may be exploited to induce apoptosis in breast cancer cells, and clinical trials of combined treatment of elesclomol and chemotherapy drugs or Akt-targeting agents in breast cancer patients, especially the estrogen receptor negative subgroup, may be warranted.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Hidrazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Separación Celular , Doxorrubicina/farmacología , Sinergismo Farmacológico , Femenino , Citometría de Flujo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Paclitaxel/farmacologíaRESUMEN
Osteosarcoma (OSA), the most common malignant bone tumor in dogs and children, exhibits a similar clinical presentation and molecular biology in both species. Unfortunately, 30-40% of children and 90% of dogs still die of disease despite aggressive therapy. The purpose of this study was to test the biologic activity of a novel heat shock protein 90 (HSP90) inhibitor, STA-1474, against OSA. Canine and human OSA cell lines and normal canine osteoblasts were treated with STA-1474 and evaluated for effects on proliferation (CyQuant), apoptosis (Annexin V, PARP cleavage, caspase 3/7 activation) and known HSP90 client proteins. HSP90 was immunoprecipitated from normal and malignant osteoblasts and Western blotting for co-chaperones was performed. Mice bearing canine OSA xenografts were treated with STA-1474, and tumors samples were evaluated for caspase-3 activation and loss of p-Akt/Akt. Treatment with STA-1474 promoted loss of cell viability, inhibition of cell proliferation and induction of apoptosis in OSA cell lines. STA-1474 and its active metabolite STA-9090 also demonstrated increased potency compared to 17-AAG. STA-1474 exhibited selectivity for OSA cells versus normal canine osteoblasts, and HSP90 co-precipitated with co-chaperones p23 and Hop in canine OSA cells but not in normal canine osteoblasts. Furthermore, STA-1474 downregulated the expression of p-Met/Met, p-Akt/Akt and p-STAT3. Finally, STA-1474 induced tumor regression, caspase-3 activation and downregulation of p-Met/Met and p-Akt/Akt in OSA xenografts. Together, these data suggest that HSP90 represents a relevant target for therapeutic intervention in OSA.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Óseas/patología , Proliferación Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Osteosarcoma/patología , Triazoles/farmacología , Animales , Western Blotting , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Perros , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Inmunoprecipitación , Indoles , Ratones , Ratones SCID , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Hidrazinas/farmacología , Estrés Oxidativo , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Perfilación de la Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
OBJECTIVE: Mutations of the receptor tyrosine kinase Kit occur in several human and canine cancers. While Kit inhibitors have activity in the clinical setting, they possess variable efficacy against particular forms of mutant Kit and drug resistance often develops over time. Inhibitors of heat shock protein 90 (HSP90), a chaperone for which Kit is a client protein, have demonstrated activity against human cancers and evidence suggests they downregulate several mutated and imatinib-resistant forms of Kit. The purpose of this study was to evaluate a novel HSP90 inhibitor, STA-9090, against wild-type (WT) and mutant Kit in canine bone marrow-derived cultured mast cells (BMCMCs), malignant mast cell lines, and fresh malignant mast cells. MATERIALS AND METHODS: BMCMCs, cell lines, and fresh malignant mast cells were treated with STA-9090, 17-AAG, and SU11654 and evaluated for loss in cell viability, cell death, alterations in HSP90 and Kit expression/signaling, and Kit mutation. STA-9090 activity was tested in a canine mastocytoma xenograft model. RESULTS: Treatment of BMCMCs, cell lines, and fresh malignant cells with STA-9090 induced growth inhibition, apoptosis that was caspase-3/7-dependent, and downregulation of phospho/total Kit and Akt, but not extracellular signal-regulated kinase (ERK) or phosphoinositide-3 kinase (PI-3K). Loss of Kit cell-surface expression was also observed. Furthermore, STA-9090 exhibited superior activity to 17-AAG and SU11654, and was effective against malignant mast cells expressing either WT or mutant Kit. Lastly, STA-9090 inhibited tumor growth in a canine mastocytoma mouse xenograft model. CONCLUSIONS: STA-9090 exhibits broad activity against mast cells expressing WT or mutant Kit, suggesting it may be an effective agent in the clinical setting against mast cell malignancies.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Leucemia de Mastocitos/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-kit/genética , Triazoles/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cartilla de ADN , Enfermedades de los Perros/patología , Perros , Mastocitoma/patología , Mastocitoma/veterinaria , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member, IL-23, shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17-producing T cells. We introduce a novel compound, STA-5326, that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level, and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-gamma (IFN-gamma) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326, indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis.
Asunto(s)
Interleucina-12/antagonistas & inhibidores , Interleucina-23/antagonistas & inhibidores , Morfolinas/farmacología , Células TH1/efectos de los fármacos , Triazinas/farmacología , Animales , Artritis Reumatoide/tratamiento farmacológico , Colon/patología , Enfermedad de Crohn/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Haplorrinos , Humanos , Hidrazonas , Inflamación/tratamiento farmacológico , Interleucina-12/genética , Interleucina-23/genética , Ratones , Ratones SCID , Morfolinas/administración & dosificación , Subunidades de Proteína , Pirimidinas , Células TH1/inmunología , Transcripción Genética/efectos de los fármacos , Triazinas/administración & dosificaciónRESUMEN
Preclinical in vitro and in vivo studies have demonstrated synergistic interactions between 5-fluorouracil (5-FU) and type I and II IFNs against human colorectal cancer cells. Despite these activities, randomized human trials have failed to identify a clinical benefit for this combination treatment. These limited clinical results may be secondary to the short half-life of recombinant IFN protein and the increased systemic toxicities of 5-FU/IFN combinations. We have previously reported an adenoviral-mediated IFN-beta gene therapy strategy, which may circumvent the pitfalls of recombinant IFN therapy. However, a dose-dependent toxicity and acute inflammatory response to systemically administered adenovirus vectors may limit the clinical application of this therapy. The combination of adenoviral-mediated IFN-beta gene therapy and 5-FU resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model. These therapeutic effects were observed at a significantly lower vector dose than we had previously reported and with limited toxicity. This approach may allow for an effective clinical application of this therapy and warrants additional investigation.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Fluorouracilo/farmacología , Terapia Genética , Interferón beta/genética , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Adenoviridae/genética , Animales , Apoptosis , Línea Celular Tumoral , Neoplasias Colorrectales/mortalidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Inflamación , Interferón beta/sangre , Interferón beta/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas/mortalidad , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Factores de Tiempo , Resultado del TratamientoRESUMEN
Type I IFNs are known to inhibit tumor cell growth and stimulate the immune system. However, little is known of the mechanism of type I IFN-induced apoptosis in human cancer cells. In this study, we have IFN-beta treatment of a human colorectal cell line (KM12L4) and a resistant clone of this cell line, L4RIFN. We demonstrate the induction of apoptosis in the parent cell line. This process was associated with the induction of the Jak-Stat signaling pathway, induction of the proapoptotic mediator tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activation of procaspase-3, -8, -9, and -10. Additionally, we evaluated the role of Stat1 in mediating IFN-beta induction of these proapoptotic signals in a fibrosarcoma cell line (2ftgh) and a Stat1-deficient clone (U3A). Our results demonstrate that IFN-beta induction of apoptosis and the induction of proapoptotic mediator TRAIL is Stat1 dependent. Evaluation of a stable transfectant of the KM12L4 cell line expressing c-FLIP supports the role of TRAIL and the cell-surface death signaling pathways in IFN-beta induction of apoptosis. Studies evaluating the TRAIL promoter indicate induction of TRAIL promoter activity by IFN-beta. These results may represent a novel pathway by which IFN-beta may induce therapeutic effects.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas de Unión al ADN/fisiología , Interferón Tipo I/farmacología , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/biosíntesis , Transactivadores/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/biosíntesis , Inhibidores de Caspasas , Caspasas/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Recombinantes , Factor de Transcripción STAT1 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Interferon-beta (IFN-beta) induces various antiproliferative activities. In solid tumor cells, IFN-beta inhibits cell cycle progression, which mainly occurs as S phase accumulation. The IFN-beta-induced cell cycle effect has been implicated in the antitumor effect of combinations of IFN-beta and chemotherapeutic drugs. In this report, we characterized the viability of various human tumor cells in vitro after combination treatment with IFN-beta protein and the chemotherapeutic drugs, cis-platinum (II) diamine dichloride (cisplatin), 5-fluorouracil (5-FU), paclitaxel (Taxol) and gemcitabine. IFN-beta could significantly potentiate the cytotoxicity of these chemotherapeutic drugs. The potentiating effect was observed after pretreatment of tumor cells with IFN-beta but did not require the constant presence of IFN-beta. The potentiating effect correlated with the sensitivity of the tumor cells to the IFN-beta-induced cytotoxicity. Furthermore, chemotherapeutic drugs also potentiated the cytotoxicity of IFN-beta. We conclude that the cell cycle effect per se did not determine the ability of IFN-beta to potentiate the cytotoxicity of chemotherapeutic drugs. We suggest that the combination of local IFN-beta gene therapy with chemotherapy could be an effective cancer treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Desoxicitidina/análogos & derivados , Interferón beta/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/patología , Carcinoma/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/farmacología , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Sinergismo Farmacológico , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacología , Humanos , Interferón beta/administración & dosificación , Neoplasias Pulmonares/patología , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Neoplasias del Cuello Uterino/patología , GemcitabinaRESUMEN
Previously, we provided evidence that adenovirus-mediated interferon-beta (IFN-beta) gene therapy inhibits tumor formation and causes dramatic regression of established tumors in immunodeficient mice. We suggested that local IFN-beta gene therapy with adenoviral vectors could be an effective treatment for cancer. In this report, the actions of murine IFN-beta (MuIFN-beta) gene delivery on both subcutaneous and metastatic tumors were evaluated in syngeneic immunocompetent mice. We found that the antitumor response mediated by MuIFN-beta gene delivery relied on CD8(+) T cells but was completely independent of CD4(+) T cells. In fact, depletion of CD4(+) T cells appeared to enhance the effect on tumor inhibition and animal survival induced by adenovirus-MuIFN-beta gene delivery. Therefore, adenovirus-MuIFN-beta gene therapy can bypass CD4(+) T helper (Th) cells and activate an effective CD8(+) T cell-dependent antitumor immunity in immunocompetent mice. Furthermore, we found that depletion of macrophages but not natural killer (NK) cells suppressed the antitumor response induced by MuIFN-beta gene therapy. These data, together with our previous results, suggest that in the clinical setting, local adenovirus-mediated IFN-beta gene therapy may lead to an efficient and long-lasting eradication of tumors by a direct antitumor effect and via activation of the innate and the adoptive immune systems.
Asunto(s)
Linfocitos T CD4-Positivos , Terapia Genética , Interferón beta/genética , Interferón beta/uso terapéutico , Neoplasias Experimentales/terapia , Adenoviridae/genética , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular , Femenino , Vectores Genéticos , Interferón beta/administración & dosificación , Depleción Linfocítica , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Neoplasias Experimentales/genética , Análisis de SupervivenciaRESUMEN
Interferon-beta (IFN-beta) induces aberrant cell cycle progression as well as cytotoxicity and apoptosis. However, the relationship between the cell cycle alteration and the induction of cytotoxicity/apoptosis is unknown. Here, we report the first demonstration that the IFN-beta-induced direct cytotoxic/apoptotic effect can be separated functionally from its cell cycle effect. By using lentiviral transduction, we generated human tumor cells that stably expressed IFN-beta and were resistant to its direct cytotoxic/apoptotic effect. Despite this resistance to apoptosis, these cells showed significant S phase accumulation as measured by both FACS analyses and bromodeoxyuridine (BrdU) incorporation. Although the cells proliferated in the presence of high levels of IFN-beta, they had lost their tumorigenicity in mice. A portion of these cells was observed to undergo a tumor cell-specific senescence. Therefore, our study revealed a direct tumor-suppressor function of IFN-beta. This tumor-suppressor function was independent of IFN-beta-induced direct cytotoxic effect. It was also distinct from the IFN-beta-induced immunologic antitumor response, an indirect effect of IFN-beta. We conclude that the antiproliferative effect on human tumor cells is the collective activities of the direct cytotoxic/apoptotic effect, the cell cycle alteration that occurs as predominantly S phase accumulation, and less frequently other cell cycle effects, such as G(1) arrest, and the promotion of tumor cells into a senescent-like state.