Laboratory of Gene Transfer

Brasília, Brazil

Laboratory of Gene Transfer

Brasília, Brazil

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Bajetto A.,University of Genoa | Porcile C.,University of Molise | Pattarozzi A.,University of Genoa | Scotti L.,University of Chieti Pescara | And 4 more authors.
Journal of Biological Regulators and Homeostatic Agents | Year: 2013

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs. Copyright © by BIOLIFE, s.a.s.


Wurth R.,University of Genoa | Pattarozzi A.,University of Genoa | Gatti M.,University of Genoa | Bajetto A.,University of Genoa | And 12 more authors.
Cell Cycle | Year: 2013

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. © 2013 Landes Bioscience.


PubMed | IRCCS AOU San Martino IST, Laboratory of Gene Transfer, University of Genoa and University of Milan
Type: | Journal: Molecular neurobiology | Year: 2016

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.


Murad A.M.,Laboratory of Gene Transfer | Souza G.H.M.F.,Research Applications Corporation | Garcia J.S.,Federal University of Alfenas | Rech E.L.,Laboratory of Gene Transfer
Journal of Separation Science | Year: 2011

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS E analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50μg can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Rech E.L.,Laboratory of Gene Transfer
Seed Science Research | Year: 2012

There is a worldwide consensus that the production of food, biomolecules and bioenergy should be sustainably intensified within the next decades. To achieve these goals will depend on the intensification of current practices in agricultural systems fused with a wider understanding and acceptance of genomics, metabolomics, synthetic biology and metabolic engineering. However, genetically modified-derived crops, including commodities and non-commodities, may contribute to establishing a new parameter in the agricultural, pharmaceutical and industrial sectors. Seeds will also play an important role in allowing large-scale production of innovative molecules prospected from biodiversity. © 2012 Cambridge University Press.


PubMed | Laboratory of Gene Transfer
Type: Journal Article | Journal: British journal of pharmacology | Year: 2012

The incidence of human malignant pleural mesothelioma (hMPM) is still increasing worldwide. hMPM prognosis is poor even if the median survival time has been slightly improved after the introduction of the up-to-date chemotherapy. Nevertheless, large phase II/III trials support the combination of platinum derivatives and pemetrexed or raltitrexed, as preferred first-line schedule. Better understanding of the molecular machinery of hMPM will lead to the design and synthesis of novel compounds targeted against pathways identified as crucial for hMPM cell proliferation and spreading. Among them, several receptors tyrosine kinase show altered activity in subsets of hMPM. This observation suggests that these kinases might represent novel therapeutic targets in this chemotherapy-resistant disease. Over these foundations, several promising studies are ongoing at preclinical level and novel molecules are currently under evaluation as well. Yet, established tumour cell lines, used for decades to investigate the efficacy of anticancer agents, although still the main source of drug efficacy studies, after long-term cultures tend to biologically diverge from the original tumour, limiting the predictive potential of in vivo efficacy. Cancer stem cells (CSCs), a subpopulation of malignant cells capable of self-renewal and multilineage differentiation, are believed to play an essential role in cancer initiation, growth, metastasization and relapse, being responsible of chemo- and radiotherapy refractoriness. According to the current carcinogenesis theory, CSCs represent the tumour-initiating cell (TIC) fraction, the only clonogenic subpopulation able to originate a tumour mass. Consequently, the recently described isolation of TICs from hMPM, the proposed main pharmacological target for novel antitumoural drugs, may contribute to better dissect the biology and multidrug resistance pathways controlling hMPM growth.


PubMed | Laboratory of Gene Transfer
Type: Evaluation Studies | Journal: Journal of separation science | Year: 2011

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS(E) analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50 g can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26 ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein.

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