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Porto, Portugal

Goncalves R.,NEWTherapies Group | Martins M.C.L.,NEWTherapies Group | Oliveira M.J.,NEWTherapies Group | Almeida-Porada G.,University of Nevada, Reno | And 2 more authors.
Journal of Biomedical Materials Research - Part A

Last trends in Biomaterials focus the mimic of cellular environments capable to control cellular responses. Epidermal growth factor (EGF) is a pleiotropic cytokine known to regulate cell proliferation, differentiation, and death. This study aims to optimize the immobilization of EGF on 11-mercapto-1-undecyl-tetra(ethylene)glycol (EG4) - self-assembled monolayers (SAMs) and to establish a new model surface to study EGF-mediated signaling. Gold substrates were modified with a monolayer of EG4 and N,N′- carbonyldiimidazole (CDI) was used to activate hydroxyl terminated groups of EG4-SAMs. EGF was then immobilized on activated EG4-SAMs at pH 7.4, 4°C, and 100 rpm. Different immobilization reaction times were tested as well as different CDI concentrations to optimize the reaction conditions and obtain a range of immobilized EGF concentrations on the surfaces. Surface characterization of EGF-SAMs was performed using radiolabeling, water contact angle measurements, X-ray photoelectron spectroscopy, and ELISA. Phosphorylation of EGFR on BT-20 breast cancer cell line by EGF-SAMs was tested by immunostaining. EGF was successfully immobilized on EG4-SAMs, at 4°C and pH 7.4 in a range of concentrations from 3.6 ± 0.8 to 17.6 ± 1.5 ng/cm2. The concentration of EGF increases with immobilization time and with the CDI concentration reaching the maximum for surfaces activated with 30 mg/mL of CDI after 48 h. The bioactivity of EGF-SAMs was confirmed by immunostaining of phospho-EGFR of BT-20 cells. This study described EGF immobilization on EG4-SAMs at different concentrations, which could be important surface models to study specific protein interactions at the molecular level evolving EGF-family of proteins. © 2010 Wiley Periodicals, Inc. J. Source

Ribeiro R.,Portuguese Institute of Oncology | Ribeiro R.,Abel Salazar Biomedical Sciences Institute | Monteiro C.,Portuguese Institute of Oncology | Cunha V.,Portuguese Institute of Oncology | And 16 more authors.
Journal of Experimental and Clinical Cancer Research

Background. Obesity is associated with prostate cancer aggressiveness and mortality. The contribution of periprostatic adipose tissue, which is often infiltrated by malignant cells, to cancer progression is largely unknown. Thus, this study aimed to determine if periprostatic adipose tissue is linked with aggressive tumor biology in prostate cancer. Methods. Supernatants of whole adipose tissue (explants) or stromal vascular fraction (SVF) from paired fat samples of periprostatic (PP) and pre-peritoneal visceral (VIS) anatomic origin from different donors were prepared and analyzed for matrix metalloproteinases (MMPs) 2 and 9 activity. The effects of those conditioned media (CM) on growth and migration of hormone-refractory (PC-3) and hormone-sensitive (LNCaP) prostate cancer cells were measured. Results. We show here that PP adipose tissue of overweight men has higher MMP9 activity in comparison with normal subjects. The observed increased activities of both MMP2 and MMP9 in PP whole adipose tissue explants, likely reveal the contribution of adipocytes plus stromal-vascular fraction (SVF) as opposed to SVF alone. MMP2 activity was higher for PP when compared to VIS adipose tissue. When PC-3 cells were stimulated with CM from PP adipose tissue explants, increased proliferative and migratory capacities were observed, but not in the presence of SVF. Conversely, when LNCaP cells were stimulated with PP explants CM, we found enhanced motility despite the inhibition of proliferation, whereas CM derived from SVF increased both cell proliferation and motility. Explants culture and using adipose tissue of PP origin are most effective in promoting proliferation and migration of PC-3 cells, as respectively compared with SVF culture and using adipose tissue of VIS origin. In LNCaP cells, while explants CM cause increased migration compared to SVF, the use of PP adipose tissue to generate CM result in the increase of both cellular proliferation and migration. Conclusions. Our findings suggest that the PP depot has the potential to modulate extra-prostatic tumor cells' microenvironment through increased MMPs activity and to promote prostate cancer cell survival and migration. Adipocyte-derived factors likely have a relevant proliferative and motile role. © 2012 Ribeiro et al; licensee BioMed Central Ltd. Source

Ribeiro R.J.T.,Portuguese Institute of Oncology | Ribeiro R.J.T.,Abel Salazar Biomedical Sciences Institute | Ribeiro R.J.T.,NEWTherapies Group | Monteiro C.P.D.,Portuguese Institute of Oncology | And 24 more authors.
Cellular Physiology and Biochemistry

Background/Aims: The microenvironment produces important factors that are crucial to prostate cancer (PCa) progression. However, the extent to which the cancer cells stimulate periprostatic adipose tissue (PPAT) to produce these proteins is largely unknown. Our purpose was to determine whether PCa cell-derived factors influence PPAT metabolic activity. Methods: Primary cultures of human PPAT samples from PCa patients (adipose tissue organotypic explants and primary stromal vascular fraction, SVF) were stimulated with conditioned medium (CM) collected from prostate carcinoma (PC3) cells. Cultures without CM were used as control. We used multiplex analysis and ELISA for protein quantification, qPCR to determine mitochondrial DNA (mtDNA) copy number and zymography for matrix metalloproteinase activity, in order to evaluate the response of adipose tissue explants and SVFs to PC3 CM. Results: Stimulation of PPAT explants with PCa PC3 CM induced adipokines associated with cancer progression (osteopontin, tumoral necrosis factor alpha and interleukin-6) and reduced the expression of the protective adipokine adiponectin. Notably, osteopontin protein expression was 13-fold upregulated. Matrix metalloproteinase 9 activity and mitochondrial DNA copy number were higher after stimulation with cancer CM. Stromovascular cells from PPAT in culture were not influenced by tumor-derived factors. Conclusion: The modulation of adipokine expression by tumor CM indicates the pervasive extent to which tumor cells command PPAT to produce factors favorable to their aggressiveness. © 2012 S. Karger AG, Basel. Source

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