Screening And Identification Of The Differential Proteins And Genes Between Platinum-resistant And Platinum-sensitive Cells Of Human Ovarian Epithelial Cancer

ovarian epithelial cancer is a malignant carcinoma which has remained the highest mortality in all gynecologic tumor.And the combination chemotherapy based on platinum compounds is the most important and common therapeutic regimen in clinic.Despite a good response to the initial chemotherapy,unfortunately,ovarian epithelial cancer is not eradicated by chemotherapy due to the emergence of multidrug resistance during therapy,and hence 5-year survival rate of women afflicted with this disease is just 30-50%.To better understand the mechanisms of multidrug resistance in ovarian cancer,we established the carboplatin-resistant cell line(SKOV3/CB)derived from the carboplatin-sensitive human ovarian serous adenocarcinoma cell lines(SKOV3)by challenging SKOV3 with a large dose of carboplatin (50ug/ml).And the differential molecules between the SKOV3 and SKOV3/CB were screened and identified both at protein level and gene level.Part one:detection and Validation of some biological features for SKOV3/CBObjective:Detect and validate some correlated biological features to determine whether the SKOV3/CB is suitable for the next protein and gene experiments.Methods:The resistant index(RI)to carboplatin and cross-resistance to other anticancer drugs(CTX,5-FU,ADM,VP-16,taxol and CDDP)were evaluated by MTT assay;The cell growth curve was painted by cell counting assay and the doubling time was accounted;The cycle distribution and cell apoptosis rate were measured by flow cytometry(FCM).Results:The carboplatin-resistant cell line SKOV3/CB indicated drug-resistance to carboplatin- and the RI was 3.09.SKOV3/CB showed an different degrees of cross-resistance to CTX,VP-16,taxol and CDDP,but still sensitive to 5-FU and ADM.The growing rate of SKOV3/CB was obviously slower than that of the parental cells.The apoptosis rate and S phase cell distribution of the resistant cells was significantly higher than that of the parental cells. Conclusion:carboplatin-resistant ovarian cancer cells line,SKOV3/CB,shows atypical multidrug-resistant cell characters and the model of drug-resistance is steady enough to performed the next protein and gene experiments.Part two:Screening and identification of the differential expression proteins between SKOV3/CB and SKOV3Objective:To perform comparative proteomic analysis of human ovarian epithelial cancer cell lines for detecting platinum-resistance associated proteins.Methods:The total proteins of sensitive(SKOV3)and resistant(SKOV3/CB)human ovarian epithelial cancer cell lines were isolated by protein fractionation-2 dimensional liquid phase chromatography(ProtemeLab^TMPF-2D);then the differentially expressed proteins were screened by Proteovue and Deltavue analysis software and identified using electrospray ionization-tandem mass spectrometry(ESI-MS/MS)and database searching.Results:A total of 54 differential proteins were isolated and screened,in which 34 proteins were up-regulated in SKOV3/CB and 20 proteins were up-regulated in SKOV3.And 15 differential proteins up-regulated in SKOV3/CB were identified and well characterized by bioinformation,including human putative magnesium transporter protein(HPT),Leucine zipper-like protein,Superoxide dismutase(SOD-1),Thioredoxin(Trx),Immunoglobulin heavy chain variable region,SMYD2,cyclin-dependent kinase 6 inhibitor,Myosin regulatory light polypeptide 9(MYL9),pituitary adenylate cyclase-activating polypeptide(PACAP),stanniocalcin homolog,human regulator of G-protein signalling(RGS),T cell receptor beta chain,TADA1 protein and AX887247 NID.These proteins are associated with ionic transport,signal transduction,oxidoreduction,proliferation,differentiation,methylation,cycle and apoptosis.Conclusion:ProtemeLab^TMPF-2D coupled with ESI-MS/MS is a effective proteomic approach for screening and identifying the differential proteins.The identified proteins may be involved in modulating response to platinum in the combined pathway and have potential as markers of treatment response or new treatment targets. Part three:Screening and identification of the differential expression genes between SKOV3/CB and SKOV3Objective:To study the gene differential expression of platinum-resistance cell line of ovarian epithelial cancer compared with the parent cell line.Methods:Gene expression profiles were screened and analyzed by using whole genome oligonucleotide microarrays for carboplatin-sensitive ovarian cancer cell line(SKOV3)and its carboplatin-resistant derivative(SKOV3/CB),and the results were validated by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Results:A total of 3506 differential genes were screened out,of which up-and down-regulated genes were 1712 and 1794 respectively in SKOV3/CB.And 163 genes and 70 genes were up-regulated more than 10 times in SKOV3/CB and SKOV3 separately,which were related to many kinds of gene.And the expression level of 6 genes,ANXA6,UGDH,ZNF198,ERCC5,TWIST2 and BIRC3,selected from the more than 10 times in SKOV3/CB be verified by RT-PCR,in two cell line were conformed to the microarrays outcome.Conclusion:The data provided from the oligonucleotide microarrays is believable and the microarray is a powerful and high-throughput tool to analyze gene differential expression of cancer.Moreover,our findings demonstrate that there is an intricate molecular network that controls the sensitivity of ovarian cancer cell to the chemotherapeutic agents and many different kinds of genes are involved in the mechanism of mutidrug resistance.