The Effect Of Lewis Y Antigen On Regulating The Gene Expression Of Partial Drug Resistance Associated Protein

ObjectiveOvarian carcinoma is the leading cause of mortality among gynecological cancers in the world.The high mortality rate is associated with lack of early diagnosis and development of drug resistance.Most patients with primary epithelial ovarian cancer after initial surgery will relapse between one to two years,and primary or acquired drug resistance is a major cause of treatment failure.Understanding the mechanisms of drug resistance may improve therapeutic strategy and the survival of ovarian cancer patients.Multiple mechanisms have been proposed for the drug resistance,and assosiations between the alterations of glycans and glycosylation in cells and chemoresistance were reported recently.The Lewis y antigen is a blood group-related difucosylated oligosaccharide on the cellular membrane.Transfection of humanα1,2-fucosyltransferases(α1,2-FT) gene into human ovarian carcinoma-devrived RMG-I cells resulted in the increase of Lewis y antigen on cell surface,and involvement ofα1,2-FT and surface-expressed Lewis y in the endothelial cancer cells during angiogenesis,invasion and adhesion had been reported as well as drug resistance.While the viability of the transfections in the medium containing the anticancer drug 5-fluorouracil(5-FU) was signignificantly elevated compared with that of RMG-I cells,we also had observed the acquisition of resistance to carboplatin and docetaxel in the RMG-I-H cells.Thus,these findings strongly indicated the possible involvement of Lewis y antigen of cancer cells in the anticancer drug-resistance.To further characterize the molecular alteration in RMG-I-H able to survive in the presence of anticancer drugs,in this study,we will examine the expression of partial drug resistance associated protein:multidrug resistance 1(MDR-1), multidrug resistance-associated protein-1(MRP-1),multidrug resistance-associated protein-2(MRP-2),protein kinase C-α(PKC-α),topoismeraseⅠ(topoⅠ) between RMG-I-H and RMG-I. Materials and Methods1.Ovarian cancer cell linesRMG-I was clear cell carcinoma of the ovary cell lines.Cell lines RMG-I-H were obtained from RMG-I which was transfected withα1,2-fucosyltransferase gene.2.RT-PCR was used to determine the gene expression of partial drug resistance associated proteinTotal RNAs were extracted from exponentially growing monolayers of RMG-I and RMG-I-H.4ug total RNA of RMG-I and RMG-I-H were reverse transcribed to obtain cDNA,respectively,at 42℃for 1h,70℃for 15 min.The resultant cDNA was amplified together with Taq polymerase using specific sets of primers at 94℃for 3 min,and then with an optimal number of cycles at 94℃for 1 min,optimal annealing temperature for 1 min,and 72℃for 1 minute,followed by incubation at 72℃for 7 min.The PCR products were electrophoresed on a 3%agarose gel.β-actin expression was used as a control for the amount of RNA.The relative intensities of the bands were determined,and the ratios toβ-actin were calculated.Each experiment was performed in triplicate.3.SP immuocytochemical method was used to detect the expression of P-gp in RMG-I and RMG-I-HExponentially growing monolayers of RMG-I and RMG-I-H were harvested and seeded in 35 mm dishes with coverslips.After 2 days' incubation with DMEM containing 10%FBS,the cells were washed with PBS twice and then fixed in 4% paraformaldehyde.P-gp was detected using SP immuocytochemical method according to the kit direction.Assessment of the results:yellow brown particles in the cytoplasm and cytomembrane represented positive reaction.Each experiment was performed in triplicate.With Meta Morph microscopic image analysis system for quantitative analysis of morphology,we detect integral optical density(IOD) and record average data.4.Make transplanted tumor model in nude miceExponentially growing monolayers of RMG-I and RMG-I-H were digested,then counted,and the cell density was adjusted(1×10~6/mL).Ten 4-6-week-old healthy female mice were randomized into two groups,and 0.3 ml suspension of either RMG-I or RMG-I-H cells(1×10~6/L) was injected subcutaneously on their back.Nude mice were fostered under the condition of SPF,after five weeks all the mice were sacrificed and samples were harvested.Tumor tissue blocks were created for histological examinations.The subcutaneously transplanted tumor in nude mice was confirmed poorly differentiated clear-cell carcinoma throughout pathological examination.5.SP immunohistochemistry method was used to detect the expression of P-gp in nude mice tissueParaffin-embedded tissues were cut up into Section which are 4-mm-thick,then were converntional dewaxed,hydrated,and experiencing antigentic revival.P-gp was detected using SP immuocytochemical method according to the kit direction. Assessment of the results:yellow brown particles in the cytoplasm represented positive reaction.With Meta Morph microscopic image analysis system for quantitative analysis of morphology,we detect integral optical density(IOD) and record average data.Each experiment was performed in triplicate.6.Antibody incubation with cellsExponentially growing monolayers RMG-I-H were plated at 2×10~5 cells per dish in seven 35 mm dishies.After 36 hours of incubation,the medium containing 10%FBS was changed with DMEM containing 2%FBS.Total RNA was extracted from RMG-I-H in one dish,and another six dishies were randomly divided into two groups: the control group which were cultured in DMEM medium only(Control group);and the group which were cultured in DMEM medium containing 10ug/ml anti-Lewis y monoclonal antibody(Lewis y mAb treated group).The time of mAb addition was set up as zero.For time-course experiments,MDR-1,MRP-1,MRP-2,PKC-α, topoⅠmRNA was measured at 6h,12h and 24h in each group.7.Statistical analysisThe data shown were mean values of at least three different experiments and expressed as mean±SD.Student's t test and ANOVA(analysis of variance) and q test was used for comparison.P＜0.05 is considered statistically significant.All data were analyzed with SPSS11.5 for Windows. Results1.The gene expression of MDR-1,MRP-1,MRP-2,PKC-α,topoⅠin RMG-I and RMG-I-HThe relative intensity of mRNA expression of PKC-α,TopoⅠ,MRP-1 and MRP-2 in RMG-I-H were 0.46±0.02,0.82±0.08,0.66±0.07 and 0.44±0.08,respectively,which were higher than 0.27±0.05,0.52±0.04,0.34±0.12 and 0.23±0.05 in RMG-I(P＜0.05); However,the gene expression of MDR-1 was 0.26±0.05,which was lower than that (0.45±0.08) in RMG-I-H(P＜0.05).2.P-gp expression in RMG-I and RMG-I-HP-gp positive granules in RMG-I-H were brown or yellow brown,with integral optical density 29.75±2.01,while P-gp positive granules in RMG-I were pale brown granules with integral optical density:17.05±0.56.Staining intensity in RMG-I-H was signifancently higher than that in RMG-I(P＜0.05).3.P-gp expression in in nude mice tissueIn the tissue of the subcutaneously transplanted tumor of RMG-I-H,P-gp positive granules were brown or yellow,with integral optical density:49.58±6.93,while P-gp positive granules in the non-tranfection group were pale yellow with integral optical density:20.09±4.09.Staining intensity in transfection group was signifancently higher than that in non-tranfection group(P＜0.05).4.Effect of anti-Lewis y monoclonal antibody on the gene expression of MDR-1,MRP-1,MRP-2,PKC-α,topoⅠExpression of MDR-1,MRP-1,MRP-2,PKC-α,TopoⅠmRNA were decreased by the time in RMG-I-H treated with anti-Lewis y monoclonal antibody(P＜0.05),while expression of that in the control group were not found to be statistically different for all times(P＞0.05).Additionally,the relative intensity of mRNA expression of MDR-1, MRP-1,MRP-2,PKC-α,TopoⅠwere 0.36±0.05,0.18±0.02,0.15±0.04,0.40±0.09,0.42±0.03,respectively,which were lower than 0.70±0.07,0.80±0.04,0.51±0.08,0.74±0.13,0.77±0.13 in the group treated with anti-Lewis y monoclonal antibody in the control group at 6h(P＜0.05),and the inhibition ratios were 48.55%,77.50%,70.18%, 45.86%and 46.13%,respectively. ConclusionThere is a close relationship between the increased Lewis y antigen and the regulation on the gene expression of partial drug resistance associated proteins in human ovarian cancer cells.