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Please use this identifier to cite or link to this item: http://hdl.handle.net/10805/2603

Title: GROWTH AND APOPTOSIS PATHWAYS IN HUMAN CUTANEOUS MELANOMA: IN VITRO AND IN VIVO STUDIES BY USING BIOLOGICAL AND PROTEOMICS APPROACHES
Authors: ROSSI, STEFANIA
Tutor: FACCHIANO, FRANCESCO
Keywords: MELANOMA
AGGRESSIVENESS
PROTEOMICS
CYTOKINES
Issue Date: 16-Jan-2013
Abstract: Purpose. Melanoma is the most aggressive cutaneous cancer without effective treatment. Diagnosis is achieved very often too late and prognosis is poor. Aim of this work is to identify proteomic pathways potentially involved in melanoma aggressiveness. Materials and Methods. We analyzed 5 human metastatic melanoma cell lines and human keratinocyte and melanocyte cell lines as a control. Their proliferation and apoptotic behaviors under serum stimulation and starvation were analyzed in order to identify the most aggressive one. Melanoma cell proteome from the most aggressive cell line (A375), compared to the less aggressive one (SK mel 28), was analyzed by means of two complementary approaches: 1) multiplexed assay to measure the levels of 27 cytokines both in cell extracts and in conditioned media; 2) proteomic study through LC-MS/MS analysis of cell extracts. Data obtained were analyzed using bioinformatic analysis. Results. A375 cells were found to possess the highest growth rate both under serum stimulation and under serum starvation, while SK mel 28 cells under similar conditions were significantly less aggressive, confirmed also by invasion assays. The effect was markedly cell-density dependent, suggesting that cell-cell interaction and/or secretory signals dependent phenomena are important. Proteome analyses indicated that several proteins are differentially expressed and possibly related the aggressiveness. Some of these proteins have been identified as transport and proteasome components. The Bio-Plex analysis of the melanoma cell lines under study indicated that melanoma cells contain significantly (p<0.001) different levels of some inflammatory cytokines and angiogenic growth factors: the most significantly modified factors were IL-6, Il-7, RANTES and VEGF, suggesting a novel interesting viewpoint to explain melanoma cell aggressiveness. Conclusions. The reported results show that transport, proteasome components and an altered cytokine balance may be responsible for human melanoma aggressiveness.
URI: http://hdl.handle.net/10805/2603
Research interests: Study of human melanoma cells growth, invasion and metastatic dissemination; study of cytokines/chemokine profile and immunity response in biological samples from cancer models; cancer stem cells as a model to develop new therapeutic approaches against cancer
Skills short description: - Cell culture, optical and fluorescence microscopy, FACS analysis - In vivo and In vitro metastatic assays (aggregation, wound healing migration, adhesion, Boyden chamber invasion and migration, 3D invasion and circular invasion) - Biochemistry (i.e. enzymatic activity assays, melanogenesis induction etc.): HPLC. Gel electrophoresis (SDS-PAGE), Western Blotting. Dot Blotting. - DNA/RNA extraction and purification techniques, PCR and RT-PCR, agarose gel electrophoresis; - Multiplex Analysis for cytokine/chemokine profiling (Bio-Plex platform: Certified Experience (*) set-up and analysis of biological samples (human and murine) like cell/tissue lysates and conditioned media, sera, vitreous, saliva, CSF and tears obtained from patients; - Use of Animal Models (e.g B16-F10/C57BL6, and others). Mouse healthcare management. - Computer skills: Windows platform and Office package (Word, Excel, Access, PowerPoint, FrontPage and Outlook). In silico: bioinformatic analysis of datasets derived from mass spectrometry, protein and gene arrays (KEGG, GEO, DAVID, PANTHER, ProTeinOn).
Personal skills keywords: Melanoma
Cytokines
Proteomics
Bio-Plex
Bioinformatic
Appears in PhD:SCIENZE E TECNOLOGIE CELLULARI

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