| Cisplatin is known to target DNA in which it forms both interstrand and intrastrand cross-links. As such, cisplatin is a cytotoxic agent which causes cell death via genotoxic damage and interference with DNA functions. Cisplatin is successfully used for the treatment of ovarian and testicular carcinomas as well as for squamous cell carcinomas. Despite its effectiveness, a major limitation of its clinical use is the development of resistance. Resistance to Platinum compounds, one of the most effective drugs in the fight against ovarian cancer, is the major barrier to the successful long-term treatment of this disease. Understanding the mechanisms involved is a first step towards rational strategies to overcome platinum resistance and is an area of intense research effort.Although a number of mechanisms for ovarian cancer cisplatin resistance have been reported, such as reduced drug accumulation, increased detoxification through cellular thiols and reduced DNA platination, no study on the overall protein expression has been reported so far in ovarian cancer resistant cells. In the present study, we have applied highly sensitive two-dimensional differential gel electrophoresis(2-D DIGE) coupled with Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/pro) for the identification of protein differentially expressed in cisplatin sensitive and cisplatin resistance ovarian cancer cell lines. Potential markers of such a chemoresistance were identified based on the outlined protein patterns.The C0C1 and C0C1/DDP cell line were purchased from China Center for Type Culture Collection. C0C1 cell lines were grown and maintained in IMDM medium, supplemented with 10% fetal calf serum. The drug-resistant variants were grown by adding 0.5 ug/ml cisplatin to the medium. The drug resistance characters of C0C1/DDP clones were 6.5-fold. The C0C1/DDP clone (resistant to cisplatin) was obtained after a six months'selection during which the cells were treated with increasing concentrations of cisplatin reaching 0.5ug/ml. The cells were solubilized and separated by DIGE systerm, then the certain proteins were identified by MALDI-TOF MS and protein database searching.DIGE enable the incorporation of the same internal standard on every 2D gel. No other systerms use an internal standard, which is a pool of all the samples within the experiment, and therefore contains every protein from every sample. The internal standard is used to match the protein patterns across gels thereby eliminating the problem of iter-gel variation, a common problem with standard 2D assays. This allows accurate quantitation of differences between samples, with an associated statistical significance. For the first time, we use DIGE systerm and MALDI-TOF MS to research the proteomic changes in platinum chemo-resistance cells and platinum sensitive cells of ovarian cancer, and achieved reliable and precise outcomes. 29 proteins showed significant increase or decrease in cisplatin resistant ovarian carcinoma cell line, and 13 proteins and their isoforms were identified by MALDI-TOF MS. Among them several functional groups of proteins were affected, including regulators of transcription, signal transduction proteins, modulation of chaperones, and those involved in apoptosis. Some of them such as 14-3-3 protein theta,14-3-3 protein zeta and NASP have not been previously reported to be associated to platinum resistance in ovarian cancer. The sorting of differentially expressed proteins into several functional groups will permit the development of hypothesis concerning their potential involvement in drug resistance.14-3-3 protein theta and 14-3-3 protein zeta were among the up-regulated proteins found in C0C1/DDP cell clones. Recent studies have revealed 14-3-3 theta and 14-3-3 zeta belong to the 14-3-3 protein family, which is a class of highly conserved proteins involved in regulating signal transduction pathways, apoptosis, adhesion, cellular proliferation, differentiation and survival. 14-3-3 proteins play an active role in delaying and suppressing apoptosis, and in allowing time for DNA repair after cellular damage. 14-3-3 regulates apoptosis through its interactions with the two pro-apoptotic proteins Bax and BAD.NASP was among the down-regulated proteins found in drug resistant cell lines. The study has demonstrated that the histone-binding protein NASP is present in dividing somatic cells and coupled to the cell cycle.In the second of this experiment, the 14-3-3 protein zeta protein was investigated by Western blot analysis, by utilizing specific polyclonal antibodies. We found after SDS-PASE followed by Western blot and specific immuno-detection, 14-3-3 protein zeta appeared to be up-regulated in C0C1/DDP cells.The findings have implications in the explanation of platinum resistance in ovarian cancer and some novel markers have been selected. Future investigation will be necessary to test the function of the identified proteins.
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