RESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Metástase Neoplásica/prevenção & controle , Neovascularização Patológica/prevenção & controle , Talidomida/análogos & derivados , Junções Aderentes/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Lenalidomida , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Camundongos , Microcirculação/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Talidomida/farmacologia , Artérias Umbilicais/efeitos dos fármacos , Artérias Umbilicais/crescimento & desenvolvimento , beta Catenina/metabolismoRESUMO
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.
Assuntos
Nitrilas/farmacologia , Tubulina (Proteína)/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Neoplasias/patologia , Transplante Heterólogo , Moduladores de Tubulina/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Fatores Imunológicos/farmacologia , Interleucina-2/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Talidomida/análogos & derivados , Fatores de Transcrição/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ionomicina/farmacologia , Ionóforos/farmacologia , Células K562 , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Proteína Quinase C-theta , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Talidomida/farmacologiaRESUMO
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.
Assuntos
Fatores Imunológicos/farmacologia , Interleucina-10/imunologia , Isoenzimas/antagonistas & inibidores , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Talidomida/análogos & derivados , Northern Blotting , Western Blotting , Ciclo-Oxigenase 2 , Dinoprostona/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-10/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/imunologia , Lenalidomida , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfotoxina-alfa/imunologia , Linfotoxina-alfa/farmacologia , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/imunologia , RNA/química , RNA/genética , Talidomida/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/sangue , Adjuvantes Imunológicos/química , Talidomida/sangue , Talidomida/química , Adjuvantes Imunológicos/farmacocinética , Antineoplásicos/sangue , Antineoplásicos/química , Antineoplásicos/farmacocinética , Humanos , Técnicas In Vitro , Neoplasias/tratamento farmacológico , Cloreto de Sódio , Estereoisomerismo , Talidomida/análogos & derivados , Talidomida/farmacocinéticaRESUMO
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.