RESUMO
Tumour pathogenesis in multiple myeloma (MM) correlates with a high vascular index. Therefore, targeting angiogenesis is an important therapeutic tool to reduce MM progression. This study aimed to investigate the role of invariant natural killer T (iNKT) cells in angiogenesis and the mechanisms behind the stimulation by α-Galactosylceramide (α-GalCer). We have previously found that α-GalCer could increase the survival of 5T33MM mice and here we demonstrate that α-GalCer reduces the microvessel density. We performed both in vivo and in vitro angiogenic assays to confirm this observation. We found that conditioned medium of α-GalCer stimulated iNKT cells reduced neovascularization in the chick chorioallantoic membrane and in matrigel plug assays. Moreover, we observed a reduction in proliferation, migration and network formation and an induction of apoptosis upon exposure of murine endothelial cell lines to this conditioned medium. We furthermore observed that the JAK-STAT signaling pathway was highly activated in endothelial cells in response to stimulated iNKT cells, indicating the possible role of IFN-γ in the anti-angiogenic process. In conclusion, these results highlight the possibility of recruiting iNKT cells to target MM and angiogenesis. This gives a rationale for combining immunotherapy with conventional anti-tumour treatments in view of increasing their therapeutic potential.
Assuntos
Galactosilceramidas/farmacologia , Janus Quinases/imunologia , Mieloma Múltiplo/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Fatores de Transcrição STAT/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Imunoterapia/métodos , Interferon gama/imunologia , Camundongos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Células T Matadoras Naturais/patologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Transdução de Sinais/imunologiaRESUMO
Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.
Assuntos
Arseniato Redutases/metabolismo , Arsênio/toxicidade , Corynebacterium glutamicum/efeitos dos fármacos , Corynebacterium glutamicum/enzimologia , Estresse Fisiológico , Sequência de Aminoácidos , Arseniato Redutases/genética , Arsênio/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Cinética , Redes e Vias Metabólicas/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.