The Study Of Reversal Of Multidrug Resistance In Human Bladder Cancer By Glycosylation Of Ceramide

Bladder cancer is most common malignant tumor of urinary system tumors in our country, and the relapse rate after operation is high. It is acutely harm to the health. The multidrug resistance of the tumor cells against anticancer drugs is accepted as the reason of the treatment failure and tumor recurrence. Recently the researches about this item are more but the research about the mechanism of the ceramide glycosylation is few.Cer is a lipid molecular which is generated by the hydrolysis of sphingomyelin on the cell membrane. As the second messenger in the process of cell apoptosis, it is proved that cer can mediate the apoptosis of many tumor cells including the bladder cancer cell by the stimulating factors such as radioactive ray, heat shock, chemotherapeutics. First Obeid induced the apoptosis of human leukemic cell in vitro by exogenous cer. After this there were the experiments to prove that cer can enhance mitochondrion apoptosis factor releasease to activate the pathway of caspases to induce the apoptosis of human bladder cancer cells. Interesting, some scholars found that in the research of MDR cells cer couldn't make the cell apotosis, but make the cell proliferation quickly. In 1996, Lavie et al. found that the content of GluCer increased obviously in the KB-V-1 cell line of drug fast vincristine, but there was no this matter in KB-3-lcell line of sensitive to the drug. The study determined that under the catalysis of GCS, saccharide group on the UDP-glucose combined cer withβ-glucosidic bond to form GluCer, but the function of GluCer in vivo was opposite with Cer to promote the cell proliferation. Further research showed that it is a universal phenomenon that cer had glycosylation to turn to GluCer in MDR cells. The related research of the American Cabot laboratory determined that the activity of GCS in MDR cells increased, which turned Cer to GluCer and decreased the therapeutic efficacy of anticancer drugs. If the process of the glycosylation of Cer of MDR cells was inhibited, the MDR of tumor cells could be deteriorated. As yet, the research about this field is not clear in urinary system.ObjectiveWe used the transgenic technology and SiRNA to import the GCS gene into bladder cancer cells and examined the expression of GluCer and the corresponding index after chemotherapeutics, and analysed the correlation between them by the statistics. Thereby, it suggested the relationship between the glycosylation of Cer and the MDR of bladder cancer, which provide the new idea to therapy the MDR of bladder cancer.Method1. MaterialsHuman bladder cancer cell line T-24 was purchased from the cell bank of China Shanhai Cell Institute;rabbit anti-human GCS polyclone antibody was purchased from Imgenex company; HRP-tagged second antibody,PBS buffer, DAB, hematoxylin, dimethyl benzene, confining liquid, acridine orange were purchased from Roche company; Genefinder nucleic acid dye and EXTAQ enzyme were purchased from Takara company; Thermo Scropt RT-PCR Syst kit was purchased from Invitrogen company; Caspase-3 shade selection kit was purchased from Bio Vision company; QIA quick gel recovery kit was purchased from QIAGEN company; plasmid extraction kit, DNA fast linkage kit, TRIzol kit, MTT kit, Lipofectamine 2000 transfection kit and pcDNA3.1(+)were purchased from Invitrogen company.Major instrument:freezing microtome,-80℃profound hypothermia refrigerator, Tissue homogenator, desk centrifuge, high speed deepfreeze centrifuge, multifunction electrophoretic apparatus, image analysator, ultraviolet spectrophotometer, GeneAmp PCR System 9700 amplificator were purchased from PE company.2. Experimental methods:(1).The expression of the membrane and cytoplasm the tissue of bladder cancer and adjacent normal tissue.Surgical specimens were obtained from 50 patients (39 men and 11 women; age range 33 to 75 years) with newly diagnosed primary SBC treated with transurethral resection or segmental cystectomy and 20 patients with resection of prostatic hypertrophy. All patients provided informed consent for use of their bladder specimens. Among them recurrence occurred on 22 patients(follow-up survey for two years), and according to this, the samples of bladder cancer were divided into two groups: recurrence group and non recurrence group. Immunohistochemistry and Western blot were used to examine the expression of GCS in bladder cancer and normal tissue.(2).Cell cultureThe cell was maintained in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37℃in a humidified,5% CO2 incubator. The cell was passaged every two or three days. The cell on logarithmic growth phase was used to make the study.(3).Construct pcDNA3.1-His-asGCS expression vector.(4).Gene tansfectionT-24 cell was cultured and transfected pcDNA3.1-His-asGCS expression vector. The primers were designed according to human GCS gene sequence on Genebank and amplified it by PCR, and reconstructed it into between EcoR I and Xho I of pcDNA3.1(+)/GCS. They were transfected into bladder cancer cell line by Lipofectamin2000.(5).The screening and identification of pcDNA3.1(+)/GCS cell line After screening, the cell line with stable high expression was built. The expression was obtained. A. The fluorescence inverted microscope was used to observe the stable expression of green fluorescence protein; B. The cells were collected for Western blot, RT-PCR to examine the GCS expression; C. The change of protein level of Bcl-2 before or after transfection and related index.(6). After the cell lines of every group were treated with adriamycin, the change of cell survival rate (MTT assay), Bcl-2 protein MDR1 level (Western blot) were examined and analyzed the difference between them.(7). After RNAi was transfected into the drug-resistant cell line with GCS high expression, the related apoptosis index was examined and compared the difference between before tansfection and after transfection. (8). Statistic analysis All the quantitative data were showed asχ±SD and the multi factor variance analysis was used. All the data were analyzed by SPSS11.5 and when the value of p was less than 0.05, it was statistical significance. The enumeration data were analyzed by Fisher definite probability methods or Pearson X2 test.Result1. Immunohistochemistry SP assay and Western blot were used to analysize the difference of the GCS expression of bladder cancer (recurrence group and non-recurrence group) and normal bladder tissue. Every group has the expression, but there is obviously different in every group. The GCS expression of bladder cancer is obviously higher than that of normal tissue, and the GCS expression of recurrence group is higher than that of non-recurrence group.2. The figures showed the result of the culture of bladder cancer.3. The plasmid of pcDNA3.1-His-asGCS is constructed as the sense expression vestor of GCS gene.4. The expression of green fluorescence protein proved that the vector was successfully transfected into cell line after the eukaryotic expression vector was transfected by liposome.5. The obvious difference of transcription level and expression level before or after the GCS transfection was examined by RT-PCR and Western blot. There were the transcription and translation in the cells before the GCS transfection, but it was obviously lower than those after the GCS transfection.6. The cell survival rate, apoptosis index, apoptosis ratio and the change of Bcl-2 and MDR1 protein content by MTT assay, Western blot, and the difference in every group was compared. The protein content of Bcl-2 after the transfection was obviously higher than that before the transfection. Every apoptosis index was significantly lower.7. After the multidrug resistance bladder cancer cell line with GCS high expression was transfected by RNAi, the related apoptosis index was examined and every apoptosis index was significantly increased compared with that before the interference. 1. The expression of GCS was associated with the ontogenesis, development and prognosis.2. GCS gene could be successfully transfected into human bladder cancer cells and stably expressed.3. There was obviously different of the transcription level and protein expression level of GCS in bladder cancer cell line before or after the transfection. There were the transcription and translation of GCS in bladder cancer cell line before the transfection, but it was obviously lower than that after transfection.4. The high expression of GCS could obviously increase the glycosylation level, thus improve the drug-resistance of cancer cells.5. After the cells were treated with the pro-apoptotic drug, the multidrug resistance of bladder cancer cells that were transfected with GCS gene was obviously increased.6. The mechanism of glycosylation of ceramide was the important factor that lead to the multidrug resistance against anticancer drugs of bladder cancer cells.7. RNA interference could reverse the drug resistance against ADM of bladder cancer.