Experimental Studies On RGD Peptide Arresting Fibronectin-mediated Drug Resistance In Chemotherapy For Bladder Cancer

Bladder transitional cell carcinoma (BTCC) is the most common cancer of genitourinary tract in China, and does serious harm 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 bladder cancer and the second part is to elucidate the effect of RGD peptide with mitomycin (MMC) on human bladder cancer xenotransplanted model of nude mice. Our objective is to find the experimental evidence on clinical value of RGD peptide as FN-antagonist for patients with BTCC.Part OneExperimental study on RGD peptide arresting fibronectin-mediated drug resistance in chemotherapy for bladder cancer: an in vitro investigationObjectiveTo explore the role of Arg-Gly-Asp ( RGD ) peptide in fibronectin (FN)-mediated drug resistance of chemotherapy for bladder cancer.Materials and MethodsT24 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 only (BO), FN only (FO), RGD only (RO) and FN with RGD (RF) group. Then the plates were incubated for 24h. After exposure to mitomycin C at different concentrations for 2h> 12ru 24h, the bladder cancer cells were detected by MTT cytotoxicity assays at different times. Apoptosis rates of tumor cells were tested by ANNEXIN V apoptotic analysis and fluorescence microscopy.ResultsAfter exposed to mitomycin C at 2h, 12h, 24h respectively, the survival rate of tumor cells in BO group was 33.4%, 27.0%,24.9%,, FO group 56.1%, 46.9%, 47.4%, RO group 28.5%, 23.9%, 22.2%,and RF group 33.6%, 26.6%, 25.5%, respectively. There was a significant different between FO group and RF group (P<0.05). Apoptotic rate of tumor cells in FO group was 7.26%, 9.34%, respectively at 2h, 12h after exposure to mitomycin C, while those of cells in BO group was 11.72%, 15.74%, respectively;in RO group was 15.85%, 18.96%, respectively;and in RF group was 11.68%, 15.40% respectively. Apoptotic rates showed a significant difference between FO and RF group fluorescence microscopy.ConclusionFN-coated T24 bladder cancer cells are less sensitive to mitomycin C and their survival rates increase compared with those of normal T24 bladder cancer cells. RGD peptide may play the role of arresting FN-mediated drug resistance by preventing the combination of FN and its receptor-integrin.Part twoInhibitory effect of RGD peptide and mitomycin on human bladder cancer T24 xenotransplanted model of nude miceObjectiveTo study the effect of FN antagonist-RGD peptide and MMC on primary tumor growth of human bladder cancer in vivo.Materials and MethodsXenotransplanted model of a human bladder cancer cell T24 in the dorsal skin of nude mice was established. Four weeks later, 32 mice were randomly divided into 4 groups: control group(n=8): 0.9%NS injected hypodermically;RGD group(n=8): RGD was injected hypodermically at dose of 6ug/g every third day;MMC group(n=8): MMC was injected hypodermically at dose of 2ug/g every third day;RGD+MMC group (n=8): RGD was injected hypodermically at dose of 6ug/g every third day combined with MMC (2ug/g).Five weeks after treatment, tumor weight, size and tumor growth inhibition rate were evaluated respectively.ResultsCompared with the untreated controls, growth of the tumor implanted subcutaneously was significantly reduced in size in the mice treated with MMC and RGD+MMC, with an inhibition rate of67.4% and 77.6%, respectively (P<0.05). Tumor size in MMC group and MMC+RGD group was (520.6±121.9)mm3 and (351.4±86.5) mm3 respectively, significantly smaller than that in control group:(1613.9±460.2)mm3,(P<0.05).Tumor weight in MMC group and MMC+RGD group was (534.7±135.6)mg and (366.5±93.4)mg respectively.significantly lighter than that in control group:(1639.5±479.2)mg,(P<0.05).There are no statictically significant difference between control group and RGD group in tumor weight and size (P>0.05, P>0.05 respectively).ConclusionRGD, as one kind of FN-antagonist, can induce apoptosis in bladder cancer and has strong inhibitory effect on tumor growth of human bladder cancer implanted in nude mice combined with MMC. The treatment of RGD combined with MMC supplies with a potentially clinical prospect for the treatment of BTCC.