Study On The Anti-Angiogenic Activity Of Tumstatin Related Peptide T3 Mediated By Short Peptide That Home Specially To Osteosarcoma Vascular

BackgroundOsteosarcoma is a malignant bone tumor originated from mesenchymal tissue, mainly in the children and adolescents. At present, High dose chemotherapy is the main treatment for osteosarcoma, which is effective but nonselective. There is also of serious toxic effect for the normal tissue by chemotherapy. Therefore, it is an urgent problem to enhance the effect of targeting of the chemotherapy for osteosarcoma. Tumor angiogenesis is fundamental for the development of osteosarcoma, because osteosarcoma needs the new vascular system to provide adequate nutrition for its diffusion and metastasis. The researchers have been paying more and more attention to some specific molecules of the tumor endothelium, which are expected to play an considerable role in the new drug targets.In order to get the above-mentioned specific binding ligands, an effective screening tool is needed. Phage display technology is one of the most effective one. Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s. It describes a selection technique in which a peptide or protein turns into fusion protein with a coat protein of a bacteriophage. The fused protein will be displayed on the surface of the bacteriophage, while the DNA encoding it resides within the virion. One of the most significant feature of phage-display technology is that it links phenotype with genotype successfully. According to this, the expression of a particular phenotype on phage is directly associated with the genetic information contained within the phage particle. The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome.In 2009, our team member had inoculated murine osteosarcoma cells UMR-106 in nude mice, we had established the osteosacoma-bearing animal models successfully. we have screened the endothelial cell of tumor vasculture of nude mouse for four rounds by in vivo phage display technique without knowing the target. According to the result of experiment in vivo and immunohistochemical staining, we could get the conclusion that the TKPDKGY phage can effectively target to the endothelial cell of tumor vessel of nude mouse. The TKPDKGY peptide will be the targeting peptide of vascular targeting therapy, which has laid a good foundation for the following study. Another member of our team manufacture the TKPDKGY-FITC peptide and againly demonstrate the targeting of the TKPDKGY to the vasculture of the osteosarcoma in vivo and in vitro, which had laid a good foundation for the following study.Tumstatin is the latest protein of macromolecular derived from the NCI domain of type IVcollagen & 3 chain of endothelial cells. Tumstatin binds to aVb3 integrin and inhibits cap-dependent protein synthesis in the proliferating endothelial cells. In 2001,Maeshima had demonstrate that peptide T3 and T7 derived from the tumstatin could inhibited proliferation and induced apoptosis specifically in endothelial cells.The other experiments also had firmed the general inhibition of peptide T3 and T7 to neoplasms and endothelial cell. Howevers,Tumstatin is the protein of gaint molecule and the autoantigens of hemorrhage syndrome of lung and kidney,it may lesion the normal vascular system of organism.For the acquirement of micromolecule and hyperactivity protein to inhibited the proliferation of endothelial cell of cancer,we synthesis recombinant tumstatin related T3 peptide mediated by short peptide that home specially to osteosarcoma mentioned above.In this experiment, we observe the inhibitory effect of tumstatin related peptide T3 mediated by short peptide that home specially to osteosarcoma vascular and suppose that this recombinant tumstatin related T3 peptide mediated by TKPDKGY peptide could specifically concentrate to the vasculture of osteosarcoma and inhibit the proliferation of osteosarcoma effectively.ObjectiveTo observe the inhibitory effect of tumstatin related peptide T3 mediated by short peptide that home specially to osteosarcoma vascular and to demonstrate that this recombinant tumstatin related T3 peptide mediated by TKPDKGY peptide could specifically concentrate to the vessel of osteosarcoma and inhibit the proliferation of osteosarcoma effectively.Method1.Synthesis of the Tumstatin T3 peptide and recombinant targeting-T3 peptide and the preparation of nude mice model bearing osteosarcoma.On the advanced microwaves polypeptide synthesizer, Tumstatin T3 peptide and recombinant targeting-T3 peptide were synthesis according to the sequence of Amino Acid. After that,the polypeptide was dissected and purified by the method of high performance liquid phase.2. Indication of inhibition of recombinant targeting-T3 peptide in vitro.To observe indication of inhibition of the recombinant targeting-T3 peptide and T3 peptidethe by the methods of MTS and Scarification assay. 3 Indication of inhibition of recombinant targeting-T3 peptide in vivo.After the preparation of nude mice model bearing osteosarcoma,the nude mice was treated in four group by T3 peptide, recombinant targeting-T3 peptide,CTX and PBS in equal-volume. The indication of inhibition on osteosarcoma was measured by the index of volume, quality, expression of VEGF and the percentage of metastasis of lung.Result1. Synthesis of the Tumstatin T3 peptide and recombinant targeting-T3 peptide and the preparation of nude mice model bearing osteosarcoma.After the synthesis and depuration of the Tumstatin T3 peptide and recombinant targeting-T3 peptide,it was checked out by the System Gold HPLC. The results mentioned that the depuration of the Tumstatin T3 peptide and recombinant targeting-T3 peptide is 95.11% and95.19% respectively.The osteosarcoma cell was inoculated in the subcutaneous of the nude mice to preparation of nude mice model bearing osteosarcoma. After two weeks of vaccination, we measured the volume of neoplasms by caliper rule. The nude mices whose volume is from the 80mm3 to 120mm3 could be the qualified mice for this assay.2. Indication of inhibition of recombinant targeting-T3 peptide in vitro.Inhibtion assay of cell increment (MTS):The endothelial cell of the human umbilical veins could be suppressed respectively by the Tumstatin T3 peptide and the recombinant targeting-T3 peptide. The inhibition ability of these peptides was-homogeneous and dependent form of does respectively.Inhibtion assay of cell migration (assay of Scarification):The migration of endothelial cell of the human umbilical veins could be suppressed apparently and homogeneously by the Tumstatin T3 peptide and the recombinant targeting-T3 peptide.3. Indication of inhibition of recombinant targeting-T3 peptide in vivo.Increment of volume of osteosarcoma:After the treatment of two weeks, the volume of neoplasms of nude mice which was treated by CTX decreased apparently. the volume of neoplasms of the other three group still increased, howevers, the neoplasms volume of group of the Tumstatin T3 peptide group and recombinant targeting-T3 peptide group increased more slowly than the group of PBS. In the same does of treatment, the effectiveness of treatment of recombinant targeting-T3 peptide is better than the Tumstatin T3 peptide.Inhibition ratio of the recombinant targeting-T3 peptide:After the treatment of two weeks, the nude mice was killed and the neoplasms was dissected absolutely. Compared to the PBS group, Inhibition ratio of every group was apparently different statistically. The inhibition ratio in the osteosarcoma of the Tumstatin T3 peptide is 28.6%, which is more worse than the inhibition ratio of the recombinant targeting-T3 peptide.Empression of VEGF of osteosarcom tissues:The empression of VEGF of osteosarcoma tissues of treatment group was lower than the PBS group. In the same does of treatment, the empression of VEGF of the recombinant targeting-T3 peptide group was lower than the Tumstatin T3 peptide statistically.Rate of metastasis in the lung:Rate of metastasis in the lung of the recombinant targeting-T3 peptide group is 33%, which is lower than the group of CTX and the Tumstatin T3 peptide. The rate of metastasis in the lung of PBS group is 100%。ConclusionAfter the acquirement and qualification of the TKPDKGY peptide which own the specifically binding activity to vascular endothelium in osteosarcoma.,we systhesis the targeting-T3 peptide which is the combinant of the TKPDKGY peptide and Tumstatin T3 peptide. We had research the The inhibition ability of this targeting-T3 peptide in vitro and vivo. Because of the vehicle of the TKPDKGY peptide, the targeting-T3 peptide could concentrate in the vascular endothelium of osteosarcoma and have more inhibition ability in the osteosarcoma than the Tumstatin T3 peptide. Taken together, we draw the following conclusions:1. We had successfully established the tumor-bearing animal models of osteosarcoma;2. The specifically binding activity of the targeting-T3 peptide to vascular endothelium of osteosarcoma was identified,we had demonstrated that endothelium cell of the osteosarcoma is the target point of the recombinant targeting-T3 peptide;3. Because of the vehicle of the TKPDKGY peptide, the Tumstatin T3 peptide could apparently inhibit the enhancement and metastasis of osteosarcoma;4. Compared to the Tumstatin T3 peptide, the recombinant targeting-T3 peptide could scarcely damage the normal vascular system and decrease the toxic and side-effect.