Cisplatin is an effective antitumor agent for the treatment of several solid tumors including human ovarian carcinoma. However, the development of cisplatin resistance represents a serious clinical problem; therefore a great deal of investigation has concerned the mechanisms by which tumor cells become resistant to this chemotherapeutic agent. 2008 and C13 cells are human ovarian carcinoma cells sensitive and resistant to cisplatin, respectively. The resistant subline, isolated after prolonged exposure to low drug doses, is approximately 20-fold more resistant to cisplatin. In the present study we investigate the protein profiles of both cell lines, searching for differentially expressed proteins as possible molecular bio-markers of resistance to the drug. To this aim we use two proteomic approaches: two-dimensional liquid chromatography (2D LC) and two-dimensional electrophoresis. 2D LC is performed on PF-2D platform (Beckman Coulter®) where protein separation takes place on a chromatofocusing column, followed by a reverse phase C18 column. Absorbance signal, monitoring the second chromatographic step is analyzed to generate the differential protein pattern by a specific software (Delta Vue™), Selected chromatographic peaks (containing proteins over or under expressed in cisplatin resistant cells with respect to cisplatin sensitive ones) are further analyzed for protein identification by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). About eighty chromatographic peaks generated from 2D LC show a differential expression consisting in at least 100% variation between the two cell line and 85% of peaks were successfully identified by MALDI-MS analysis. 2D EF was applied to compare the results obtained with the former technology. Among differentially expressed proteins we identified: Glyceraldeide-3-phosphate dehydrogenase, phosphoglycerate kinase1, enolase1, HSP27, and ATP-syntase witch are under-expressed in C13, cisplatin resistant cells. Complete differential expression profile could be useful to identify down-/up-regulated pathways in cisplatin resistant cells and, hopefully, some of related proteins could be further investigated as candidates to predict/monitor response to cysplatin treatment in serum and tumor tissue.

Identification of proteins associated with cisplatin resistence in ovarian cancer by two proteomic approaches

ROVERI, ANTONELLA;ZACCARIN, MATTIA;RIGOBELLO, MARIA PIA;BINDOLI, ALBERTO;MARZANO, CRISTINA;GANDIN, VALENTINA;URSINI, FULVIO;
2007

Abstract

Cisplatin is an effective antitumor agent for the treatment of several solid tumors including human ovarian carcinoma. However, the development of cisplatin resistance represents a serious clinical problem; therefore a great deal of investigation has concerned the mechanisms by which tumor cells become resistant to this chemotherapeutic agent. 2008 and C13 cells are human ovarian carcinoma cells sensitive and resistant to cisplatin, respectively. The resistant subline, isolated after prolonged exposure to low drug doses, is approximately 20-fold more resistant to cisplatin. In the present study we investigate the protein profiles of both cell lines, searching for differentially expressed proteins as possible molecular bio-markers of resistance to the drug. To this aim we use two proteomic approaches: two-dimensional liquid chromatography (2D LC) and two-dimensional electrophoresis. 2D LC is performed on PF-2D platform (Beckman Coulter®) where protein separation takes place on a chromatofocusing column, followed by a reverse phase C18 column. Absorbance signal, monitoring the second chromatographic step is analyzed to generate the differential protein pattern by a specific software (Delta Vue™), Selected chromatographic peaks (containing proteins over or under expressed in cisplatin resistant cells with respect to cisplatin sensitive ones) are further analyzed for protein identification by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). About eighty chromatographic peaks generated from 2D LC show a differential expression consisting in at least 100% variation between the two cell line and 85% of peaks were successfully identified by MALDI-MS analysis. 2D EF was applied to compare the results obtained with the former technology. Among differentially expressed proteins we identified: Glyceraldeide-3-phosphate dehydrogenase, phosphoglycerate kinase1, enolase1, HSP27, and ATP-syntase witch are under-expressed in C13, cisplatin resistant cells. Complete differential expression profile could be useful to identify down-/up-regulated pathways in cisplatin resistant cells and, hopefully, some of related proteins could be further investigated as candidates to predict/monitor response to cysplatin treatment in serum and tumor tissue.
2007
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2464293
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