RESUMO
In this study, we have utilized a novel strategy based upon the use of dimethyl sulfoxide (DMSO) and gas chromatography-mass spectrometry (GC-MS) for the detection and identification of spin-trapped free radicals. Hydroxymethyl (.CH2OH) radicals, generated by Fenton-type chemistry, have been trapped by N-tert-butyl-α-phenylnitrone (PBN) or one of its derivatives in the presence of DMSO to form a 1,3-diadduct [PBN-(CH2OH)(CH3)], which may be detected directly in the reaction mixture following chloroform extraction or in the reaction vial headspace by sampling with SPME. Separation and identification have been carried out by capillary gas chromatography coupled to electron-ionization mass spectrometry (EI-MS). The results demonstrate that using DMSO aids GC-MS analysis of spin-trapped free radicals via the formation of radical-methyl di-adducts that are sufficiently volatile to be sampled both in the headspace or by an extracting solvent without the need for a derivatization step using silylating agents.
Assuntos
Dimetil Sulfóxido , Microextração em Fase Sólida , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , SolventesRESUMO
The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines. Here we sought to identify the galectin-3-binding molecule(s) on the endothelial cell surface responsible for the galectin-3-mediated cytokine secretion. Using two different galectin-3 affinity purification processes, we extracted four cell membrane glycoproteins, CD146/melanoma cell adhesion molecule (MCAM)/MUC18, CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1), CD144/VE-cadherin, and CD106/Endoglin, from vascular endothelial cells. CD146 was the major galectin-3-binding ligand and strongly co-localized with galectin-3 on endothelial cell surfaces treated with exogenous galectin-3. Moreover, galectin-3 bound to N-linked glycans on CD146 and induced CD146 dimerization and subsequent activation of AKT signaling. siRNA-mediated suppression of CD146 expression completely abolished the galectin-3-induced secretion of IL-6 and G-CSF cytokines from the endothelial cells. Thus, CD146/MCAM is the functional galectin-3-binding ligand on endothelial cell surfaces responsible for galectin-3-induced secretion of metastasis-promoting cytokines. We conclude that CD146/MCAM interactions with circulating galectin-3 may have an important influence on cancer progression and metastasis.