RESUMEN
A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.
Asunto(s)
Talidomida/análogos & derivados , Administración Oral , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Semivida , Humanos , Interleucina-2/metabolismo , Lenalidomida , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratas , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Talidomida/química , Talidomida/farmacocinética , Talidomida/toxicidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A series of 1,1-diarylalkene derivatives were prepared to optimize the properties of CC-5079 (1), a dual inhibitor of tubulin polymerization and phosphodiesterase 4 (PDE4). By using the 3-ethoxy-4-methoxyphenyl PDE4 pharmacophore as one of the aromatic rings, a significant improvement in PDE4 inhibition was achieved. Compound 28 was identified as a dual inhibitor with potent PDE4 (IC(50)=54 nM) and antitubulin activity (HCT-116 IC(50)=34 nM and tubulin polymerization IC(50) â¼1 µM). While the nitrile group at the alkene terminus was generally required for potent antiproliferative activity, its replacement was tolerated if there was a hydroxyl or amino group on one of the aryl rings. Conveniently, this group could also serve as a handle for amino acid derivatization to improve the compounds' solubility. The glycinamide analog 45 showed significant efficacy in the HCT-116 xenograft model, with 64% inhibition of tumor growth upon dosing at 20 mg/kg qd.
Asunto(s)
Alquenos/química , Alquenos/farmacología , Antineoplásicos/química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/química , Moduladores de Tubulina/química , Alquenos/síntesis química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Derivados del Benceno/síntesis química , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células HCT116 , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacología , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/farmacologíaRESUMEN
INTRODUCTION: CC-5079, a small molecule inhibitor of tubulin polymerization and phosphodiesterase-4 activity, was evaluated for antiangiogenic and antitumor activities. MATERIALS AND METHODS: First, CC-5079 in vitro activity on human umbilical vein endothelial cells (HUVECs), fibroblasts, and MC38 were evaluated by proliferation, migration, and invasion assays. Second, CC-5079 effect on microvessel formation was evaluated ex vivo by chick chorioallantoic membrane (CAM), rat aortic rings assays, and with directed in vivo angiogenesis assay (DIVAA). Third, CC-5079 antitumor effect was determined in treatment of C57BL/6 mice with MC38 tumors. Finally, CC-5079 modulation of MKP1 in HUVECs, human fibroblast, and MC38 were determined by RNA isolation for qRT-PCR. RESULTS: At the 0.1 µM concentration CC-5079 significantly inhibited HUVEC, fibroblast, and MC38 proliferation and migration (all P < 0.001). At the 0.1 µM concentration, CC-5079 also inhibited HUVEC invasion (P < 0.05) but not fibroblast. In the CAM and rat aortic ring assays, CC-5079 at 0.1 µM inhibited microvessel formation (P < 0.05). By DIVAA, CC-5079 at 1 mg/kg/d continuous delivered inhibited microvessel formation (P < 0.05). Intraperitoneal CC-5079 was well tolerated and inhibited the growth of subcutaneous MC38 at 100 mg/kg/d (P < 0.01). By qRT-PCR, CC-5079 stimulated MKP1 expression in HUVEC and fibroblast. CONCLUSION: CC-5079 demonstrated stimulation of MKP1, antiangiogenic, and antitumor properties.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Fosfatasa 1 de Especificidad Dual/metabolismo , Nitrilos/farmacología , Inhibidores de la Angiogénesis/química , Animales , Antineoplásicos/química , Aorta/citología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Pollos , Membrana Corioalantoides/citología , Neoplasias del Colon/metabolismo , Fosfatasa 1 de Especificidad Dual/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Ratones , Nitrilos/química , Ratas , Venas Umbilicales/citologíaRESUMEN
Lenalidomide and pomalidomide have both been evaluated clinically for their properties as anticancer agents, with lenalidomide being available commercially. We previously reported that both compounds cause cell cycle arrest in Burkitt's lymphoma and multiple myeloma cell lines by increasing the level of p21(WAF-1) expression. In the present study, we unravel the molecular mechanism responsible for p21(WAF-1) up-regulation using Namalwa cells as a human lymphoma model. We show that the increase of p21(WAF-1) expression is regulated at the transcriptional level through a mechanism independent of p53. Using a combination of approaches, we show that several GC-rich binding transcription factors are involved in pomalidomide-mediated up-regulation of p21(WAF-1). Furthermore, we report that p21(WAF-1) up-regulation is associated with a switch from methylated to acetylated histone H3 on p21(WAF-1) promoter. Interestingly, lysine-specific demethylase-1 (LSD1) silencing reduced both pomalidomide and lenalidomide up-regulation of p21(WAF-1), suggesting that this histone demethylase is involved in the priming of the p21(WAF-1) promoter. Based on our findings, we propose a model in which pomalidomide and lenalidomide modify the chromatin structure of the p21(WAF-1) promoter through demethylation and acetylation of H3K9. This effect, mediated via LSD1, provides GC-rich binding transcription factors better access to DNA, followed by recruitment of RNA polymerase II and transcription activation. Taken together, our results provide new insights on the mechanism of action of pomalidomide and lenalidomide in the regulation of gene transcription, imply possible efficacy in p53 mutated and deleted cancer, and suggest new potential clinical uses as an epigenetic therapy.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Linfoma/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Oxidorreductasas N-Desmetilantes/metabolismo , Talidomida/análogos & derivados , Antineoplásicos/farmacología , Línea Celular Tumoral , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Demetilasas , Histonas/genética , Histonas/metabolismo , Humanos , Lenalidomida , Linfoma/genética , Linfoma/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Talidomida/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-alpha inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Talidomida/análogos & derivados , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Humanos , Inhibidores de Fosfodiesterasa/administración & dosificación , Inhibidores de Fosfodiesterasa/química , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Talidomida/administración & dosificación , Talidomida/química , Talidomida/farmacologíaRESUMEN
OBJECTIVES: Pancreatic cancer is one of the leading causes of cancer related deaths in the western world. It is also resistant to most chemotherapeutic modalities. Phosphodiesterase-4 inhibitors (PDE4is) have found applications in the treatment of respiratory diseases. The aim of this study is to investigate the cytotoxic effect of 2 novel PDE4is, the CC-8075 and CC-8062 compounds in pancreatic cancer cells. METHODS: Cell proliferation was measured using the sulforhodamine B protein dye. Induction of apoptosis was detected using enzyme-linked immunosorbent assay. Regulation of proteins and posttranslational modifications were determined using immunoblotting. RESULTS: Treatment of pancreatic cancer cells with CC-8075 and CC-8062 reduces their proliferation and increases apoptosis that is caspase dependent in T3M4 cells. Furthermore, PDE4is increase phosphorylation of p38MAPK, mitogen-activated protein kinase (MAPK) kinase 3/6,MAPKYactivated protein kinase 2, Atf2, and Hsp27. The use of thep38MAPK-specific inhibitors SB202190 and SB203580 results in a modest reduction in PDE4i-induced apoptosis in T3M4 cells. Also, retinoids enhance apoptosis induced by CC-8075 and CC-8062 in GER cells. CONCLUSIONS: These results highlight the antiproliferative effects of the phosphodiesterase inhibitors CC-8075 and CC-8062 in pancreatic cancer cells and suggest that activation of p38MAPK signaling pathway may be associated with this process.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/farmacología , Neoplasias Pancreáticas/patología , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Alitretinoína , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/farmacología , Neoplasias Pancreáticas/enzimología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Metástasis de la Neoplasia/prevención & control , Neovascularización Patológica/prevención & control , Talidomida/análogos & derivados , Uniones Adherentes/efectos de los fármacos , Animales , Antígenos CD/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula , Línea Celular Tumoral , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Técnicas In Vitro , Lenalidomida , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Melanoma Experimental/tratamiento farmacológico , Ratones , Microcirculación/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Talidomida/farmacología , Arterias Umbilicales/efectos de los fármacos , Arterias Umbilicales/crecimiento & desarrollo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Ocular melanoma is the leading intraocular malignancy. There is no effective treatment for metastatic ocular melanoma. We sought a treatment targeting the tumor microenvironment as well as the tumor cells. METHODS: Migration of HUVEC cells, the ability of HUVEC cells to form tubes, and proliferative capacity of a human ocular melanoma cell line were tested in the presence of lenalidomide and sorafenib alone and in combination. The compounds were also tested in a rat aortic ring assay and were tested in a highly aggressive human ocular melanoma xenograft model. RESULTS: Lenalidomide and Sorafenib inhibit HUVEC ability to migrate and form tubes and when used in combination the inhibition is increased. The agents alone and in combination inhibit outgrowth in the rat aortic ring model. The combination of the agents improved the inhibition over either single agent. In a xenograft model, combination therapy inhibited tumor growth over inhibition by single agent alone in a significant fashion (p < 0.004: lenalidomide and p < 0.0035: sorafenib). Furthermore, spontaneous lung metastasis development was completely inhibited in the combination treated animals. Sixty percent of vehicle treated animals developed lung metastases compared to 50% of lenalidomide treated animals, and 33% of sorafenib treated animals. CONCLUSION: Lenalidomide and sorafenib are effective at targeting endothelial cells, inhibiting growth of ocular melanoma cells and can inhibit growth of tumors in a xenograft model as well as inhibit development of metastases. Combining these agents works in an additive to synergistic way to inhibit the growth of tumors and development of metastases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Ojo/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencenosulfonatos/uso terapéutico , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Neoplasias del Ojo/irrigación sanguínea , Neoplasias del Ojo/patología , Humanos , Técnicas In Vitro , Lenalidomida , Melanoma/irrigación sanguínea , Melanoma/patología , Neovascularización Patológica/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Sorafenib , Talidomida/análogos & derivados , Talidomida/farmacología , Talidomida/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clinical studies involving patients with myelodysplastic syndromes or multiple myeloma have shown the efficacy of lenalidomide by reducing and often eliminating malignant cells while restoring the bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide and a structurally related analogue, CC-4047, on the proliferation of two different human hematopoietic cell models: the Namalwa cancer cell line and normal CD34+ progenitor cells. Both compounds had antiproliferative effects on Namalwa cells and pro-proliferative effects on CD34+ cells, whereas p21WAF-1 expression was up-regulated in both cell types. In Namalwa cells, the up-regulation of p21WAF-1 correlated well with the inhibition of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest, whereas in CD34+ progenitor cells the increase of p21WAF-1 did not inhibit proliferation. Similarly, antiproliferation results were observed in two B lymphoma cell lines (LP-1 and U266) but interestingly not in normal B cells where a protection of apoptosis was found. Finally, CC-4047 and lenalidomide had synergistic effects with valproic acid [a histone deacetylase (HDAC) inhibitor] by increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus normal cells and could explain, at least in part, the reduction of malignant cells and the restoration of bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, proposes new potential clinical uses, such as bone marrow regeneration, and suggests that the combination of lenalidomide or CC-4047 with certain HDAC inhibitors may elevate the therapeutic index in the treatment of hematologic malignancies.
Asunto(s)
Antígenos CD34/biosíntesis , Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Linfoma de Células B/tratamiento farmacológico , Talidomida/análogos & derivados , Linfocitos B/patología , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Lenalidomida , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Fosforilación/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Talidomida/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Accumulating evidence suggests that inflammation plays a major role in the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (ALS). Important mediators of inflammation such as the cytokine tumor necrosis factor-alpha (TNF-alpha) and its superfamily member fibroblast-associated cell-surface ligand (FasL) have been implicated in apoptosis. We found increased TNF-alpha and FasL immunoreactivity in lumbar spinal cord sections of ALS patients and G93A transgenic mice. Both increased TNF-alpha and FasL immunostaining in the lumbar spinal cord of the G93A SOD1 transgenic mice occurred at 40-60 d, well before the onset of symptoms and loss of motor neurons. We tested the neuroprotective effect of thalidomide and its analog lenalidomide, pharmacological agents that inhibit the expression of TNF-alpha and other cytokines by destabilizing their mRNA. Treatment with either thalidomide or lenalidomide attenuated weight loss, enhanced motor performance, decreased motor neuron cell death, and significantly increased the life span in G93A transgenic mice. Treated G93A mice showed a reduction in TNF-alpha and FasL immunoreactivity as well as their mRNA in the lumbar spinal cord. Both compounds also reduced interleukin (IL)-12p40, IL-1alpha, and IL-1beta and increased IL-RA and TGF-beta1 mRNA. Therefore, both thalidomide and lenalidomide bear promise as therapeutic interventions for the treatment of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Modelos Animales de Enfermedad , Inmunosupresores/uso terapéutico , Sobrevida , Talidomida/análogos & derivados , Talidomida/uso terapéutico , Adulto , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/mortalidad , Análisis de Varianza , Animales , Recuento de Células , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Proteína Ligando Fas , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Lenalidomida , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Neuronas/patología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Prueba de Desempeño de Rotación con Aceleración Constante/métodos , Superóxido Dismutasa/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC(50) value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G(2)-M phase, increased phosphorylation of G(2)-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [(3)H]colchicine for binding to tubulin; however, it does not compete with [(3)H]paclitaxel (Taxol) or [(3)H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC(50), 270 nmol/L). This inhibitory effect on TNF-alpha production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy.
Asunto(s)
Nitrilos/farmacología , Tubulina (Proteína)/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Neoplasias/patología , Trasplante Heterólogo , Moduladores de Tubulina/farmacología , Células Tumorales CultivadasRESUMEN
Immunomodulatory drugs (IMiDs) are orally available small molecules that potently inhibit tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (HuPBMCs) but enhance secretion of such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by stimulated T cells. The mechanism of cytokine regulation by IMiDs has not yet been determined. In the present study, we investigated the effects of one of the IMiDs, CC-4047 (Actimid, Celgene, Warren, NJ), on synthesis of IL-2 protein and mRNA and on the activity and expression of transcription factors. Treatment with CC-4047 enhances the secretion of IL-2 protein and the expression of IL-2 mRNA in a dose-dependent and time-dependent manner. In T cells stimulated with phorbol myristate acetate (PMA)/ionomycin, CC-4047 enhanced the DNA-binding activity of activated protein-1 (AP-1) but not NF-kappaB, Octomer-1 (OCT-1), or NFAT by 2-fold and 4-fold after an incubation time of 1 and 3 h, respectively. Luciferase reporter assays in Jurkat cells showed similar effects on transcription factor activity. Using in vitro kinase activity assays, we also showed that CC-4047 enhances the activity of protein kinase C-theta (PKC-theta) in stimulated T cells. The secreted IL-2 from HuPBMCs was shown to activate natural killer (NK) cells to lyse their target cell line K562. Taken together, our results demonstrate that the IMiDs exert their effects at least in part by activating PKC-theta and acting on AP-1 DNA-binding activity in T cells, resulting in augmented IL-2 synthesis and activation of IL- 2-dependent downstream effectors, such as NK cells.
Asunto(s)
Factores Inmunológicos/farmacología , Interleucina-2/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Linfocitos T/enzimología , Talidomida/análogos & derivados , Factores de Transcripción/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Células K562 , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Proteína Quinasa C-theta , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Talidomida/farmacologíaRESUMEN
Immunomodulatory drugs (IMiDs) are potent inhibitors of TNF-alpha and IL-1beta and elevators of IL-10 production in LPS-stimulated human PBMC. They are currently in clinical trials for various diseases, including multiple myeloma, myelodysplastic syndrome, and melanoma. In the present study, we have investigated the effects of thalidomide, CC-5013 and CC-4047 on the expression of COX-2 by stimulated PBMC. Our results show that thalidomide and IMiDs inhibited the expression of COX-2 but not the COX-1 protein in LPS-TNF-alpha and IL-1beta stimulated PBMC and shortened the half-life of COX-2 mRNA in a dose-dependent manner. They also inhibited the synthesis of prostaglandin E2 from LPS-stimulated PBMC. While anti-TNF-alpha or IL-1beta neutralizing antibodies had no effect on COX-2 expression, anti-IL-10 neutralizing antibody elevated the expression of COX-2 mRNA, and protein from treated PBMC. These data suggest that the anti-inflammatory and anti-tumor effects of IMiDs may be due in part to elevation of IL-10 production and its subsequent inhibition of COX-2 expression.
Asunto(s)
Factores Inmunológicos/farmacología , Interleucina-10/inmunología , Isoenzimas/antagonistas & inhibidores , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Talidomida/análogos & derivados , Northern Blotting , Western Blotting , Ciclooxigenasa 2 , Dinoprostona/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-10/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/inmunología , Lenalidomida , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfotoxina-alfa/inmunología , Linfotoxina-alfa/farmacología , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/inmunología , ARN/química , ARN/genética , Talidomida/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Reliable quantitative evaluation of molecular pathways is critical for both drug discovery and treatment monitoring. We have modified the CAM assay to quantitatively measure vascular density, endothelial proliferation, and changes in protein expression in response to anti-angiogenic and pro-angiogenic agents. This improved CAM assay can correlate changes in vascular density with changes seen on a molecular level. We expect that these described modifications will result in a single in vivo assay system, which will improve the ability to investigate molecular mechanisms underlying the angiogenic response.
RESUMEN
Selective cytokine inhibitory drugs (SelCIDs) are a novel class of phosphodiesterase 4 inhibitors discovered during a thalidomide analog discovery program. These analogs were evaluated for their ability to inhibit tumor angiogenesis, vascularity, and growth. Two analogs (CC-7034 and CC-9088) were identified that had enhanced antiangiogenic activity in Matrigel assays compared with parental thalidomide. These analogs also inhibited the growth of established K1735 and RENCA murine tumors. Tumors whose growth was suppressed by SelCID treatment exhibited decreased vessel density together with increased tumor cell hypoxia and death. The decrease in vascularity produced by SelCID treatment is attributed to a selective loss of vessels devoid of pericyte coverage, suggesting that these agents target immature tumor vessels. That tumor cell death was localized to relatively avascular or hypoxic areas, coupled with the fact that none of the analogs was cytotoxic in vitro against the tumor cells, demonstrates that these analogs are novel antivascular agents with potent antitumor activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Citocinas/antagonistas & inhibidores , Endotelio Vascular/efectos de los fármacos , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Talidomida/análogos & derivados , Animales , División Celular/efectos de los fármacos , Hipoxia de la Célula/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Talidomida/farmacologíaRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is an antivascular drug that induces tumor necrosis factor (TNF) in mice. Thalidomide inhibits TNF induction by DMXAA and also potentiates its antitumor activity. We investigated whether these effects were enantiomer specific, using the R- or S-enantiomers of two nonracemizable thalidomide analogues. Racemic 3-fluorothalidomide (3FThal) and racemic 3-methylthalidomide (3MeThal) were separated into enantiomers of greater than 98% optical purity using preparative chiral column chromatography. C57Bl/6 mice implanted with subcutaneous Colon 38 tumors were treated with DMXAA (25 mg/kg) alone or together with the pure R- or S-enantiomers by a single i.p. injection. TNF levels in the serum or tumor tissues 3 h after treatment were measured using ELISAs and tumor growth was also measured. 3FThal and 3MeThal, at their respective single maximum tolerated doses (MTD) of 15 and 50 mg/kg, were more toxic in mice than thalidomide (100 mg/kg). The R- and S-enantiomers of either 3FThal or 3MeThal, at their respective MTD, inhibited DMXAA-induced TNF activity in serum and tumor tissue, but no significant differences were observed between the enantiomers. Coadministration of racemic or enantiomers of 3FThal or 3MeThal at their respective MTD did not potentiate the antitumor responses above that obtained with DMXAA alone, and no enantioselectivity was apparent. We conclude that there is no advantage in using the nonracemizable thalidomide analogues to improve the antitumor activity of DMXAA.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Talidomida/uso terapéutico , Factor de Necrosis Tumoral alfa/biosíntesis , Xantonas/uso terapéutico , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Neoplasias del Colon/metabolismo , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Inyecciones Intraperitoneales , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Trasplante de Neoplasias , Estereoisomerismo , Relación Estructura-Actividad , Talidomida/administración & dosificación , Talidomida/análogos & derivados , Talidomida/química , Resultado del Tratamiento , Xantonas/administración & dosificaciónRESUMEN
Thalidomide, (1), has made a remarkable comeback from its days of a sedative with teratogenic properties due to its ability to selectively inhibit TNF-alpha, a key pro-inflammatory cytokine and its clinical benefit in the treatment of cancer. Thalidomide contains one chiral center and is known to be chirally unstable under in vitro and in vivo conditions. It has been hypothesized that different biological properties are associated with each isomer. Thus, chirally stable analogues of thalidomide, alpha-fluorothalidomide, (3) and alpha-fluoro-4-aminothalidomide (4) were prepared by electrophilic fluorination. Analogue 3 was found to be cytotoxic and did not inhibit TNF-alpha production in LPS stimulated hPBMC below toxic concentrations. On the other hand, 4 was non-cytotoxic at the tested concentrations and was found to be 830-fold more potent than thalidomide as TNF-alpha inhibitor.
Asunto(s)
Flúor/química , Talidomida/química , Células Cultivadas , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Talidomida/farmacología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
CC-4047 is a racemic second-generation immunomodulatory drug currently in clinical development for various oncologic indications. It has potent effects on key cytokines including tumor necrosis factor-alpha, interleukin (IL-10), and interferon (IFN-gamma). The S-isomer of CC-4047 has been reported to be the more potent enantiomer of the racemate. In this article we report on the rapid racemization of the S-isomer of CC-4047 in human plasma and phosphate-buffered saline. These results support the further development of the racemate instead of the S-isomer.
Asunto(s)
Adyuvantes Inmunológicos/sangre , Adyuvantes Inmunológicos/química , Talidomida/sangre , Talidomida/química , Adyuvantes Inmunológicos/farmacocinética , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Humanos , Técnicas In Vitro , Neoplasias/tratamiento farmacológico , Cloruro de Sodio , Estereoisomerismo , Talidomida/análogos & derivados , Talidomida/farmacocinéticaRESUMEN
CC-4047 (Actimid) and CC-5013 (Revimid) belong to a class of thalidomide analogs collectively known as the immunomodulatory drugs (IMiDs), which are currently being assessed in the treatment of patients with multiple myeloma and other cancers. IMiDs potently enhance T cell and natural killer cell responses and inhibit tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-12 production from LPS-stimulated peripheral blood mononuclear cells. However, the molecular mechanism of action for these compounds is unknown. Herein, we report on the ability of the IMiDs to up-regulate production of IL-2 from activated human CD4+ and CD8+ peripheral blood T cells, production of IL-2 and IFN-gamma from T helper (Th)1-type cells, and production of IL-5 and IL-10 from Th2-type cells. Elevation of IL-2 production from Jurkat T cells was observed as early as 6 h poststimulation and correlated with an increase in IL-2 promoter activity that was dependent upon the proximal but not the distal AP-1 binding site. The IMiDs enhanced AP-1-driven transcriptional activity 2- to 4-fold after 6 h of T cell stimulation, and their relative potencies for AP-1 activation correlated with their potencies for increased IL-2 production in Jurkat T cells and in CD4+ or CD8+ human peripheral blood T cells. The most potent of these IMiDs, CC-4047, had no effect on nuclear factor of activated T cells transcriptional activity, calcium signaling, or phosphorylation of extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase 1/2, p38 mitogen-activated protein kinase, or c-Jun/Jun D in Jurkat T cells. These data suggest that IMiDs increase T cell cytokine production by potentiating AP-1 transcriptional activity.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citocinas/biosíntesis , Proteínas Proto-Oncogénicas , Linfocitos T/efectos de los fármacos , Talidomida/farmacología , Factor de Transcripción AP-1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Calcineurina/metabolismo , Señalización del Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Interleucina-2/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Janus Quinasa 2 , Células Jurkat , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/metabolismo , Células TH1/metabolismo , Células Th2/metabolismo , Talidomida/análogos & derivados , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Thalidomide is clinically useful in a number of cancers. Antitumor activity may be related to a number of known properties, including anti-tumor necrosis factor (TNF)-alpha and T-cell costimulatory and antiangiogenic activities. However, it may also involve direct antitumor effects. A series of second generation thalidomide analogues have been separated into two distinct groups of compounds, each with enhanced therapeutic potential, i.e., SelCIDs, which are phosphodiesterase (PDE) type IV inhibitors, and IMiDs, which have unknown mechanism(s) of action. We report here our efforts to determine direct antitumor effects of thalidomide and compounds from both groups. We found that one of the SelCID analogues (SelCID-3) was consistently effective at reducing tumor cell viability in a variety of solid tumor lines but had no effect on non-neoplastic cells. The antitumor activity was independent of known PDE4 inhibitory activity and did not involve cAMP elevation. Growth arrest was preceded by the early induction of G(2)-M cell cycle arrest, which led to caspase 3 mediated apoptosis. This was associated with increased expression of pro-apoptotic proteins and decreased expression of antiapoptotic bcl-2. Furthermore, extensive apoptosis in vivo was detected during SelCID-3-mediated inhibition of tumor growth in a murine xenotransplantation cancer model. Our results suggest that SelCID-3 represents a novel antitumor agent distinct from thalidomide and from previously characterized analogues with therapeutic potential against a range of solid tumors. This effect appears to be mediated via alterations in the expression of bcl-2 family proteins.