Cancer Platinum Drug Resistance-associated Protein For The Ovarian Comparative Proteomics Analysis Of Resistance-associated Protein Annexin A3, Function

Background:Ovarian cancer is the leading cause of gynecological cancer mortality.Despite first-line platinum-based chemotherapy is effective in reducing tumor burden following an optimal cytoreductive surgery,the 5-year survival rate for stageⅢandⅣdisease is about 15%～20%.One of the major reasons causing the low 5-year survival rate is due to the development of drug resistance in cancer cells.Early detection of drug resistance of tumor followed by novel therapeutic strategies may further improve the treatment outcome.Although a number of genes have been associated with platinum resistance in ovarian cancer,no single gene is capable to be used as the biomarker for determining the development of drug resistance.In view of the existence of posttranslational processing and modification and the inconsistency between the expression of gene and protein,it is very necessary to perform high-throughput studies at protein level for which proteomic techniques rightly answers.Proteomics have become a rapidly developing subject since Wilkins and Williams who came from Australia proposed the conception.At present,most studies have focused on early detection and early diagnosis of ovarian cancer.However,so far, seldom proteomic study was processed in drug resistance and further screening resistant biomarkers of ovarian cancer.In our study,platinum-resistance associated proteins were searched for the first time using proteomics-based approaches in multiple resistant cell lines.To screen differentially expressed proteins,two human ovarian cancer cell lines and their respective cisplatin-resistant and carboplatin-resistant clones were analyzed by 2-DE and MALDI-TOF-MS.Five proteins were found to express differentially in at least three resistant lines that may be involved in the mechanism of platinum resistance for ovarian cancer.The roles of the candidate protein in platinum resistance were further explored.Methods:1.The cisplatin-resistant sublines were induced from epithelial ovarian cancer cell line SKOV3 respectively by pulse and intermittent incremental exposures to cisplatin during 16 months.The biological characteristics of all the cell lines were determined using light microscopy and cell counting.Drug sensitivity was monitored by MTT assay.The cell cycle was detected by flow cytometry.We obtained other three platinum resistant sublines:A2780/CDDP,A2780/CBP and SKOV3/CBP.The mRNA expression of MDR1,MRP1,LRP and GST-πwas determined by RT-PCR.The stability of drug resistance of all resistant cell lines was examined at monthly intervals by MTT assay.2.The total proteins of all sensitive and resistant human ovarian cancer cell lines were separated by 2-DE.The differentially expressed proteins were analyzed using image analysis software,stained with Coomassie Brilliant Blue,then identified using MALDI-TOF-MS and database searching.The mRNA and protein levels of the differentially expressed protein which was significant in more than three resistant cell lines were validated by quantitative PCR and Western blot,respectively.3.The plasmid containing sense and antisense cDNA of the target protein were constructed and stable transfection of the sense and antisense plasmids was carried out. The effects on growth and proliferation and drug sensitivity of the transfected cells were detected.4.The relationship between the target protein and platinum resistance was further validated by tumorigenicity assay in nude mice.5.Tumor tissues from 21 sensitive and 21 resistant ovarian cancer patients were evaluated for the expression of the interesting protein by immunohistochemistry.The Mann-Whitney U test was used to determine differences.Disease-free survival and 95%confidence intervals were calculated and evaluated by the Kaplan-Meier method; Disease-free survival between high expression and low expression group of the target protein compared using the log-rank test.6.Drug resistance to Taxol and epirubicin of the transfected cells was examined by MTT assay.Localization of the target protein was detected by immunofluorence staning.Intracellular uptake and efflux of cisplatin and Pt-DNA binding were tested by Agilent 7500c inductively coupled plasma Mass Spectrometry(ICP-MS) apparatus. Western blot were performed to detect p53 level and the extracellular target protein.Results:1.The resistance indexes of SKOV3/CDDP-P and SKOV3/CDDP-80 were 4.1±2.0 and 11.5±3.2,respectively.Both the two resistant cell lines had some changes in biological characteristics in which SKOV3/CDDP-80 showed more distinct changes. The expression of the well-known drug resistance-associated genes had significant differences between SKOV3/CDDP-P and SKOV3/CDDP-80.The resistance indexes of SKOV3/CBP,A2780/CDDP and A2780/CBP were 3.0±0.2,3.5±0.8,3.5±0.7, respectively.The resistant phenotype of all resistant cells was very stable because the values of IC50 and RI had no significant change in 4 months' drug-free medium.2.Sixty-two protein spots in all samples were found to be significantly different in spot intensity by statistical analysis,fifty-seven of which were successfully identified by MALDI-TOF-MS.Five proteins were found to be significant coexisting in four cell lines.Annexin A3 and destrin were up-regulated and NADP-dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was no change. However,cofilin 1 represented a different trend.In the two resistant sublines of SKOV3 cofilin 1 had an up-regulation,but it had an up-regulation in the cell lines induced from SKOV3.The expression of Annexin A3 up-regulated 3-20-fold and the results of RT-PCR and Western blot showed completely consistency with 2-DE in the expression of Annexin A3.3.We defined the final candidate proteins through two standards.One was that the alteration of protein expression was identical in all the four resistant sublines and the other was that the alteration was the most significant.Therefore,we selected annexin A3 as the target protein in the next studies.4.Transfection of Annexin A3-expressing plasmid into drug sensitive cells showed 2-4-fold increase in resistance of transfected cells to cisplatin and carboplatin and inhibition of growth and proliferation.Conversely,transfection of reverse Annexin A3 plasmid into drug resistant cells showed about 2-fold decrease in resistance to these drugs and increased cell growth.All the transfected cells had no significant changes in morphology.5.When cisplatin treatment was administrated in tumorigenicity assay,Annexin A3 transfected cells tumors showed an increased resistance to CDDP.The tumor growth inhibition of SKOV3 tumor was(76.9±22.5)%.It was significantly higher than that of Annexin A3-overexpressed tumor,which was only(28.5±13.7)%(P =0.002).6.Immunohistochemical staining of the platinum-sensitive and platinum-resistant group showed the expression of Annexin A3 increased significantly in the resistant tissues compared with the sensitive group(P=0.035).The mean DFS for the high expression of Annexin A3 was only 11.3 months(95%CI = 4.0 to 18.0 months).Mean DFS for the low expression group of Annexin A3 was 16.0 months (95%CI = 5.9 to 26.1 months).In the low expression group,DFS was significantly longer than for the high expression group patients(P=0.012).7.When testing the drug sensitivity of these transfected cells to Taxol and epirubicin,the sensitivity to Taxol and epirubicin had no significant alteration between target gene-transfected cells and control cells.The intracellular concentration of cisplatin and Pt-DNA binding in Annexin A3-overexpressing cells was low and that in Annexin A3-depleted cells was high.The efflux of cisplatin in the Annexin A3-overexpressing cells increased significantly compared with that in the control cells. Annexin A3 was distributed both in the cytoplasm and in the nucleus,and could be secreted to the outside of the cells.Along with the enhancement of intracellular Annexin A3,the secreted Annexin A3 increased.When the cells were treated with cisplatin,the expression of Annexin A3 had no change in cellular lysates,whereas extracellular Annexin A3 increased compared with the untreated cells.Conclusion:1.The results suggest that there are many differences between the two resistant cell lines which involved in multi-gene changes.The intermittent administration has greater trend to develop resistance.Due to the similar pattern with the clinical administration,the resistant sublines selected by pulse method may serve as appropriate models for the study of mechanisms of drug resistance in ovarian cancer.2.The differentially expressed proteins are involved in energy metabolism, redox state regulation,molecular chaperone,nucleotide metabolism,cytoskeleton regulation,signal transduction,transcription regulation,suggesting that the mechanism of platinum resistance may be associated with multiple signal transduction pathways.The results that five proteins coexist in more than three resistant cell lines show destrin,IDHc,GSTOI-1,cofilin 1 and especially Annexin A3,may keep close connection with platinum resistance.3.We selected annexin A3 as the target protein in the next studies through two standards.One was that the alteration of protein expression was identical in all the four resistant sublines and the other was that the alteration was the most significant.4.The increased or decreased drug resistance in transfected cells indicates there is a direct correlation between Annexin A3 levels and platinum resistance in cell lines in vitro.5.The increased or decreased drug resistance in transfected cells indicates there is a direct correlation between Annexin A3 levels and platinum resistance in cell lines in vivo.6.Immunohistochemical staining of the clinical samples shows a direct correlation between Annexin A3 levels and platinum resistance in clinical response and an inverse correlation between Annexin A3 expression levels and DFS.Annexin A3 was demonstrated to be a platinum resistance-associated protein.It may be a hopeful predictor for clinical platinum resistance and prognosis and a candidate for overcoming platinum resistance.7.Annexin A3 may be a specific protein associated with platinum resistance. Annexin A3 may play a role in decreasing the intracellular platinum concentration accompanied with Annexin A3 externalization,and finally lead to specific platinum resistance.