The Study On The Effects And Mechanisms Of RGD Peptide Arresting Fibronectin-mediated Drug Resistance In Chemotherapy For Human Bladder Carcinoma Cells

Bladder transitional cell carcinoma (BTCC) is the most common cancer of genitourinary tract in China, and it's a threat to people's health. Despite of the improvement of operational skills and intravesical chemotherapeutic agents, the recurrent rate of bladder cancer still keeps highly at 50～70% after initial treatment. To increase the sensitivity of chemotherapeutic agents is the key to elongate the survival time free of tumor and improve the life quality of patients suffered from bladder cancer. Our prelimiary in vitro investigation had verified that fibronectin-mediated drug resistance is present in BTCC cell line and this phenomenon attributes to the delayed effect of chemotherapy in bladder cancer.The present studies include two parts: the first one is to explore the role of Arg-Gly-Asp (RGD) peptide in fibronectin (FN)-mediated drug resistance of chemotherapy for human BTCC 5637 cell; the second part is to elucidate the expression of the cell apoptosis related protein in drug resistance of chemotherapy for human bladder carcinoma cells which was induced by the fibronectin and Arg-Gly-Asp(RGD) peptide.Our objective is to find the experimental evidence on clinical value of RGD peptide as FN-antagonist for patients with BTCC.Part IExperimental Study on RGD Peptide ArrestingFibronectin-mediated Drug Resistance in Chemotherapy forHuman Bladder Carcinoma 5637 CellsObjective: The present study was undertaken to examine the effects of the FN and RGD peptide in drug resistance of chemotherapy for human BTCC 5637 cells.Methods: 5637 bladder cancer cells were attached to FN-coated and bovine serum albumin (BSA)- coated plates, respectively. According to the administration of RGD peptide, the groups was subdivided into BSA group, FN group, RGD group and FN+RGD group. Then the plates incubated for 24h. After exposure to mitomycin C at different concentrations for 2h, the bladder cancer cells were detected by MTT cytotoxicity assays at different times. The induction of apoptosis was measured by acridine orange/ethidium bromide assays. The extent of apoptosis was quantified by flow-cytometric analysis of cells labeled with PI and annexin V.Results:1. The survival rates of tumor cells , FN group were 56.1%, 46.9%, 47.4% at 2h, 12h, 24h after exposed to mitomycin C, while those of tumor cells in BSA group were 33.4%, 27.0%, 24.9%, in RGD group were 28.5%, 23.9%, 22.2% and in FN+RGD group were 33.6%, 26.6%, 25.5% at the same time. The survival rates of tumor cells in FN group is much more than that of other group (P<0.05). And the rate of RGD group is much less than that of other group (P<0.05).2. After exposured to MMC (concentration=50mg/L) for 2h, the apoptosis cell ratio of RGD group is much more than that ofFN+RGD group and BSA group. And the apoptosis cell ratio of FN group is the least one.3. As shown by PI staining and the annexin V method, after exposured to MMC (concentration=50mg/L) for 2h, the number of early apoptotic cells (LR) in FN group is 0.7%, and the number of late apoptotic cells (UR) is 1.14%. The numbers are 2.29% and 2.49% in RGD group, 1.47% and 2.04% in BSA group, 1.66% and 1.87% in FN+RGD group, respectively. There was a significant different between FN group and other groups (P<0.05). And RGD group has higher UR and LR ratio than that of other group (P<0.05). The total percent of apoptotic cells (UR + LR) is 4.767% in RGD group, 3.533% in FN+RGD group, 3.513% in BSA group, and 1.847% in FN group.4. As shown by PI staining and the annexin V method, 12h after exposured to MMC (concentration=50mg/L), the number of early apoptotic cells (LR) in FN group is 10.30%, and the number of late apoptotic cells (UR) is 0.63%. The numbers are 26.33% and 1.32% in RGD group, 15.47% and 0.83% in BSA group, 14.20% and 1.10% in FN+RGD group, respectively. There was a significant different between FN group and other groups (P<0.05). And RGD group has higher UR and LR ratio than that of other group (P<0.05).The total percent of apoptotic cells (UR + LR) is 27.65% in RGD group, 15.30% in FN+RGD group, 16.29% in BSA group, and 10.94% in FN group.Conclusions: Our data suggested that the human bladder carcinoma 5637 cells attached to FN-coated plates had less sensitivity to mitomycin C and the survival rates increased, and RGD peptide could arrest FN-mediated adhesion drug resistance.Part IIThe Experimental Study of the Mechanism ofRGD Peptide Arresting Fibronectin-mediated Drug Resistance inChemotherapy for Human Bladder Carcinoma CellsObjective: The present study was undertaken to examine the expression of the cell apoptosis related protein in drug resistance of chemotherapy for human bladder carcinoma cells which was induced by the fibronectin and Arg-Gly-Asp(RGD) peptide. Methods: The bladder cancer cells were attached to FN-coated and BSA-coated plates, respectively. According to the administration of RGD peptide, the groups was subdivided into BSA group, FN group, RGD group and FN+RGD group. Then the plates incubated for 24h. After exposure to mitomycin C (concentration=50mg/L) for 2h, the bladder cancer cells were incubated for 24h. Then the poly(ADP-ribose)polymerase (PARP), p53, bcl-2 and bax protein of human bladder carcinoma T-24 cells and 5637 cells were determined in Western blot analysis. Results:1. After exposure to MMC, the expression of the cleaved PARP (89kb) was down-regulated in FN group, and RGD peptide can up-regulate the expression of the cleaved PARP.2. The expression of P53 protein was down-regulated by FN and RGD peptide has a conversion effects to P53.3. After exposure to MMC, the increase of Bcl-2 protein and the reduce of Bax protein was induced by FN, and the administration of RGD peptide can up-regulate the expression of Bax protein and down-regulate the expression of Bcl-2 protein.4. The ratio of Bax/Bcl-2 was down-regulated by FN and RGDpeptide has a conversion effects to the ratio.Conclusions: Our results sugguested that fibronectin can up-regulate the expression of Bcl-2 protein and down-regulate the expression of the cleaved PARP, Bax and P53 protein to mediated the drug resistance of BTCC. RGD peptide could has a conversion effects to that of induced by fibronectin.