RESUMO
BACKGROUND: Thyroid cancer is the most common endocrine malignancy. Current therapies are successful, however some patients progress to therapeutically refractive disease. The immunotherapeutic potential of the CXCL8-chemokine/CXCR2-chemokine-receptor system is currently being explored in numerous human cancers. This study aimed to evaluate if the targeting of CXCR2 by its selective antagonist, AZD5069, could modulate CXCL8-mediated pro-tumorigenic effects in thyroid-cancer (TC) cells in vitro. METHODS: Normal human primary thyroid cells (NHT) and TC cell lines TPC-1 (RET/PTC), BCPAP, 8505C and 8305C (BRAFV600e) were treated with AZD5069 (100 pM-10 µM) over a time-course. Viability and proliferation were assessed by WST-1 and crystal violet assays. CXCL8 and CXCR2 mRNA were evaluated by RT-PCR. CXCL8-protein concentrations were measured in cell culture supernatants by ELISA. CXCR2 on cell surface was evaluated by flow-cytometry. Cell-migration was assessed by trans-well-migration chamber-system. RESULTS: AZD5069 exerted negligible effects on cell proliferation or viability. AZD5069 significantly reduced CXCR2, (but not CXCL8) mRNAs in all cell types. CXCR2 was reduced on the membrane of some TC cell lines. A significant reduction of the CXCL8 secretion was found in TPC-1 cells (basal-secretion) and NHT (TNFα-induced secretion). AZD5069 significantly reduced basal and CXCL8-induced migration in NHT and different TC cells. CONCLUSIONS: Our findings confirm the involvement of the CXCL8/CXCR2-axis in promoting pro-tumorigenic effects in TC cells, further demonstrating its immunotherapeutic significance in human cancer.
RESUMO
Diabetic retinopathy (DR), one of the most common complications of diabetes mellitus, is characterized by degeneration of retinal neurons and neoangiogenesis. Until today, the pharmacological approaches for DR are limited and focused on counteracting the end-stage of this neurodegenerative disease, therefore efforts should be carried out to discover novel pharmacological targets useful to prevent DR development. Hyperglycemia is a major risk factor for endothelial dysfunction and vascular complication, which subsequently may trigger neurodegeneration. We previously demonstrated that, in the rat retina, hyperglycemia activates a new molecular cascade implicating, up-stream, protein kinase C ßII (PKC ßII), which in turn leads to a higher expression of vascular endothelial growth factor (VEGF), via the mRNA-binding Hu-antigen R (HuR) protein. VEGF is a pivotal mediator of neovascularization and a well-known vasopermeability factor. Blocking the increase of VEGF via modulation of this cascade can thus represent a new pharmacological option to prevent DR progression. To this aim, proper in vitro models are crucial for drug discovery, as they allow to better identify promising effective molecules. Considering that endothelial cells are key elements in DR and that hyperglycemia triggers the PKCßII/HuR/VEGF pathway, we set up two distinct in vitro models applying two different stimuli. Namely, human umbilical vein endothelial cells were exposed to phorbol 12-myristate 13-acetate, which mimics diacylglycerol whose synthesis is triggered by diabetic hyperglycemia, while human retinal endothelial cells were treated with high glucose for different times. After selecting the optimal experimental conditions able to determine an increased VEGF production, in search of molecules useful to prevent DR development, we investigated the capability of troxerutin, an antioxidant flavonoid, to counteract not only the rise of VEGF but also the activation of the PKCßII/HuR cascade in both in vitro models. The results show the capability of troxerutin to hinder the hyperglycemia-induced increase in VEGF in both models through PKCßII/HuR pathway modulation. Further, these data confirm the key engagement of this cascade as an early event triggered by hyperglycemia to promote VEGF expression. Finally, the present findings also suggest the potential use of troxerutin as a preventive treatment during the early phases of DR.
RESUMO
The last decade has come across an increasing demand for theranostic biocompatible nanodevices possessing the double ability of diagnosis and therapy. In this work, we report the design, synthesis and step-by-step characterization of rationally coated gold nanostars (GNSs) for the SERS imaging and photothermal therapy of HeLa cancer cells. The nanodevices were realized by synthesizing GNSs with a seed growth approach, coating them with a controlled mixture of thiols composed of a Raman reporter and a polyethylene glycol with a terminal amino group, and then reacting these amino groups with folic acid (FA), in order to impart selectivity towards cancer cells which overexpress folate receptors on their membranes. After a complete characterization, we demonstrate that these FA-functionalized GNSs (FA-GNSs) are able to bind selectively to the membranes of HeLa cells, acting as SERS tags and allowing SERS imaging. Moreover, we demonstrate that once bound to HeLa cell membranes, FA-GNSs exhibit photothermal effect which can be exploited to kill the same cells in vitro using laser irradiation in the NIR at a very low and safe irradiance. We thus demonstrate that the FA-GNSs designed following the described approach are an efficient prototype of theranostic nanodevices.
RESUMO
Surface modification of noble metal nanoparticles with mixed molecular monolayers is one of the most powerful tools in nanotechnology, and is used to impart and tune new complex surface properties. In imaging techniques based on surface enhanced Raman spectroscopy (SERS), precise and controllable surface modifications are needed to carefully design reproducible, robust and adjustable SERS nanoprobes. We report here the attainment of SERS labels based on gold nanostars (GNSs) coated with a mixed monolayer composed of a poly ethylene glycol (PEG) thiol (neutral or negatively charged) that ensure stability in biological environments, and of a signalling unit 7-Mercapto-4-methylcoumarin as a Raman reporter molecule. The composition of the coating mixture is precisely controlled using an original method, allowing the modulation of the SERS intensity and ensuring overall nanoprobe stability. The further addition of a positively charged layer of poly (allylamine hydrocloride) on the surface of negatively charged SERS labels does not change the SERS response, but it promotes the penetration of GNSs in SH-SY5Y neuroblastoma cells. As an example of an application of such an approach, we demonstrate here the internalization of these new labels by means of visualization of cell morphology obtained with SERS mapping.
Assuntos
Ouro/química , Nanopartículas Metálicas , Nanotecnologia , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortisol, have been obtained; 6D6 is used in an assay kit for cortisol. The antibodies also recognize other, structurally related steroids present in the sample assayed. To improve the specificity of the assay, we aimed to minimize the recognition of non-cortisol steroids by the two anti-cortisol mAbs. Our strategy consisted in constructing an efficient expression vector in E. coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space. We demonstrated that temperature and inducer concentration of the bacterial culture influenced dramatically the yield of active scFv. From the nucleotide sequence we constructed a three-dimensional model of the two variable fragments in order to understand why related steroids are, or are not recognized by the antibody. For both antibodies, we have identified chemical groups which are probably involved in the binding of the steroid haptens and the antibodies. The hydrophobic pocket formed by the antibody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking. The serine at position 35 of the heavy chain is buried in the back of the pocket and can form a hydrogen bond with the 20-keto group of the cortisol. The stacking interactions and the hydrogen bond orient the steroid in the pocket. This reactivity of the binding site is sustained by the analysis of the cross-reactions of related steroids with the mAbs.