Screening And Identification Of The Differential Proteins And Genes Between Taxol Resistance And Taxol 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 Taxol 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 taxol-resistant cell line(SKOV3/TS)derived from the taxol-sensitive human ovarian serous adenocarcinoma cell lines(SKOV3)by challenging SKOV3 with a large dose of taxol(0.5ug/ml).And the differential molecules between the SKOV3 and SKOV3/TS were screened and identified both at protein level and gene level.Part one:detecting and validating associated biological features of platinum- resistance cell line of ovarian epithelial cancerObjective:Detect and validate some associated biological features to of platinumresistance cell line of ovarian epithelial cancer(SKOV3/TS)Methods:The resistant index(RI)to taxol and cross-resistance to other anticancer drugs(gemcitabine,oxaliplatin, etoposide,ifosfamide,mitoxantrone)were evaluated by MTT assay;The cell growth curve was painted and the doubling time was accounted,by 21-day of cell counting assay Results: The taxol-resistant cell line SKOV3/TS indicated drug-resistance to taxol and the RI was 2.5.SKOV3/TS showed different degrees of cross-resistance to gemcitabine,VP-16,oxaliplatin and ifosfamide.The growing rate of SKOV3/TS was obviously slower than that of the parental cells.Conclusion:taxol-resistant ovarian cancer cells line,SKOV3/TS,shows a typical 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 proteins expression by SKOV3/TS and SKOV3Objective:To compare the proteins expression by SKOV3/TS and SKOV3,screen and identify the associated proteins.Methods:The total proteins of sensitive(SKOV3)and resistant(SKOV3/TS)human ovarian epithelial cancer cell lines were isolated by protein fractionation-2 dimensional liquid phase chromatography(ProtemeLabTMPF-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 12 differential proteins were isolated and screened,in which 12 proteins were up-regulated in SKOV3/TS and no proteins were up-regulated in SKOV3.And 11 differential proteins up-regulated in SKOV3/TS were identified,there are Immunologulin kappa light chain variable region(Fragment),protein DKFZp686F0970 (Fragment),MAPK13 protein variant,AJ972290,RNF4 protein,Cancer/testis antigen CT45-5,AX888106 NID,Homo sapiens(clone E04)gene from CpG-enriched DNA,partial cds,Pyrin-domain containing protein,complement C4A - human,CXorf2 protein。.These proteins relate with 15 genes such as oncology genes,DNA synthesis,repairing,cell signals and transmission of protein,protein synthesis translation category,as well as the cytoskeleton and exercise,ion channels and transport proteins,metabolites.Conclusion: ProtemeLabTMPF-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 Taxol in the combined pathway and have potential as markers of treatment response or new treatment targets.Part three:Screening and identification of the differential genes expression by SKOV3/TS and SKOV3Objective:To probe the differential gene expression by Taxol-resistant and not Taxol-resistant cell lines of ovarian epithelial cancer.Methods:Screening and identification the differential gene expression by SKOV3/TS and the parental cells using the whole genome oligonucleotide microarrays,and the results were validated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).Results:A total of 5098 differential genes were screened out,of which up- regulated genes were2482(425 genes up- regulated over 10 times),and down-regulated genes 2616(409 genes down- regulated over 10 times)in SKOV3/TS comparing with SKOV3,SKOV3/TS than SKOV3 up more than 10 times the three different genes and down 10 times the three different genes.The 6 genes mentioned-above expressed by resistant cells were higher than sensitive cells using RT-PCR,which indicate the result of gene chip is reliable.Conclusion: Gene chip is a powerful and high-throughput tool to analyze gene differential expression of carcinoma.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.