The Effect Of Lewis(y) Antigenic Content On Drug Resistance To Carboplatin In Ovarian Cancer Line RMG-I

ObjectiveAmong female genital organ tumors, ovarian cancer had the highest mortality rate. The drug resistance of the cancer cells was one of most significant factors. Lewis(y) antigen was one kind of significant carbohydrate antigens on cancer cell surface, more over, Lewis(y)-related tumor-associated antigens abundantly expressed (200,000 molecules/cell) on the majority of human carcinomas. Some researches had found that the cells of the epithelial ovarian cancers expressed antigenic structures, such as Lewis(y) antigen, that often were not shared by normal tissues, as well as, the antigenic content of Lewis(y) was dependent on the malignancy degree of the cancers, which means there were more Lewis(y) antigens if the cancer cells had higher malignancy. Further more, some study also had demonstrated no significant deviation in the expression of Lewis(y) antigen between primary and metastatic lesions. We had found the same consequence in the study which we had finished. Then, tumor marker CA-125 mainly contains type 2 blood group antigenic structure and Lewis(y) was one of them. For the traditional therapeutics such as surgery operation and chems could not increase manifestly the long-term survival of the ovarian cancer patients. Now, looking for antigens as the new biochemistry targets to study the more effective methods to therapy ovarian cancer had become a hot direction, and Lewis(y) antigen was one of them.On the contrary, whether there was some contribution of the Lewis(y) antigen on the malignancy degree and drug resistance of ovarian cancers, we had found very limited correlated literatures. As we had found, Masao et al had transfected α1,2-fucosyltransferase gene into serous epithelial ovarian cancer RMG-I. Then, they found the fucosyltransferase activity increases 20-30 times in the new cells RMG-I-H, and Lewis(y) antigen also increased about 10 times on cell surface. At the same time, the cancer cell adhesion attenuated, but adhesion with epithelial cells increased and the drug resistance to 5-fluorouracil (5-FU) also increased. However, fewer people used this drug as the routine method in ovarian chemo therapy. So, in this study, we decided to choose carboplatin to study if the drug resistance of epithelial ovarian cancer to carboplatin would also increase.Materials and Methods1. Ovarian cancer cell lines.RMG-I, RMG-I-C and RMG-I-H were provided by Professor LIN Bei (Department of Gynecology and Obstetrics, The Second Affiliated Hospital of China Medical University, Shenyang China). RMG-I was one kind of epithelial ovarian cancer cell lines. Cell lines RMG-I-C and RMG-I-H were obtained from RMG-I which were transfected with keno-plasmid andα1,2-fucosyltransferase gene, respectively.2. The method of methyl thiazolyl tetrazolium(MTT) was used to determine the fractional inhibition ratio(IR).Exponentially growing monolayers of RMG-I, RMG-I-C and RMG-I-H were incubated in 10% FCS DMEM, harvested and seeded in 96 orifices for 24h, culture fluid changed. Add carboplatin, 6 holes, negative and blank control holes for every concentration, incubate for 72h, add 20μl MTT(5mg/ml), pour supematant after 4h, add 150μl DMSO, shock for 10min and measure OD with 490nm wave. Repeat 3 times. Calculate IR. According to the above, we chose 30μg/ml, 60μg/ml carboplatin and got IR at 24h, 48h, 72h, 96h and 120h.3. flow cytometry(FCM) was use to measured the apoptotic ratios.Exponentially growing monolayers of RMG-I, RMG-I-C and RMG-I-H were incubated in 10% FCS DMEM till covering 30% undersurface. Add carboplatin to 30μg/ml, 60μg/ml and negative control group did not include carboplatin. Incubate for 72h, operate according to the caption of Annexin-V-FICT/PI kit and detect. 10,000 cells were analyzed per sample, calculate the percentage of apoptotic cells.4. The apoptosis conditions were observed by the method of fluorescent staining.Exponentially growing monolayers of RMG-I, RMG-I-C and RMG-I-H were incubated in 10% FCS DMEM, harvested and seeded in 6 orifices till covering 30% undersurface. Add carboplatin to 30μg/ml, 60μg/ml and negative control group did not include carboplatin. Incubated for 72h, pour the supematant, wash with 4℃PBS for 2 times. Away from light, add Buffer300μl, add Annexin-V-FICT 5μl, stete for 15min, add PI 10μl and observe with inv-fluorescence microscope.Results1. The consequences of MTTThe three cell lines had different IR. We could find that the IC₅₀ of RMG-I-H was higher than RMG-I and RMG-I-C (P<0.001). However, no statistics deviation was found between the IC₅₀ of the next two cell lines (P>0.05). We chose 30μg/ml, 60μg/ml carboplatin concentration to detect the IR on different time, the IR of RMG-I-H was lower than the one of RMG-I and RMG-I-C (P<0.001). However, no statistics deviation was found between the IR of the next two cell lines (P>0.05).We could say that the drug resistance deviation among these three cell lines was only dependent on the types of cell lines concerning the IC50 concentration.2. The rate of cell apoptosisWe chose 30μg/ml, 60μg/ml carboplatin concentration to detect the rate of cell apoptosis with flow cytometry. The ratio of RMG-I-H was lower than the one of RMG-I and RMG-I-C (P<0.05). However, no statistics deviation was found between the next two cell lines (P>0.05).3. The change of cellular morphologyThe normal cell growed unilayly as oval-shap or polygon, unstained. The apoptotic cells had the following changes: cell substance concentrating, bulk shrinking, surface presenting green fluorescent light, cell nucleus presenting red fluorescent light, cystoskeleton disassembling, and we could observe cell chip and apoptotic bodies in some pictures. The apoptotic extent of RMG-I and RMG-I-C was more serious than the one of RMG-I-H at the same carboplatin concentration.ConclusionThe drug resistance to carboplatin increased after transfected withα1,2-fucosyltransferase gene. Maybe the content increase of Lewis(y) antigen was the main reason.