Arg-gly-asp Polypeptide Mediated The Truncated Tissue Factor Therapy For Colorectal Carcinoma

Objective The tumor vasculature is increasingly recognized as a target for cancer therapy. In recent years,a fusion protein consisting of the extracellular domain of tissue factor (truncated tissue factor,tTF) was fused to the antibody(Ab) selectively binding to tumor vasculature. Antibody-truncated tissue factor(Ab-tTF) fusion protein specifically induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in some types of solid tumors. However, there were some disadvantages in the above approach. A (RGD)3-tTF fusion protein with peptides arginine-glycine-aspartic acid (Arg-Gly-Asp,abbr. RGD) repeat sequence as the carrier of tTF was expressed to explore its therapy in the colorectal carcinoma bearing mice.Materials and Methods (1)The (RGD)3-tTF fusion gene consisting of the tTF which was fused to three series-wound peptides RGD was expressed in Escherichia coli BL21 (DE3). The fusion protein was purified through Nickel affinity chromatography column. (2)The coagulation activity of the (RGD)3-tTF fusion protein was detected by clotting assay in vitro. (3)Mice colorectal cancer cells of the line CT26 were inoculated subcutaneously into mice to establish model of colorectal cancer .Four mice were randomly divided into two groups to be injected with the (RGD)3-tTF or tTF fusion protein labeled with Rhodamine B Isothiocyanate(RBITC) at a single dose of 50μg respectively. The location of the (RGD)3-tTF fusion protein in the colorectal carcinoma bearing mice tissue was analyzed by the optical in vivo imaging one hour after the injection and confocal microscopy twenty-four hours after the injection. (4)Fifteen mice beared colorectal carcinoma were randomly divided into three groups for injection with the (RGD)3-tTF , tTF fusion protein or phosphate buffered saline(PBS) at a single dose of 50μg respectively.The tumor size was measured to calculate the tumor volume everyday.Five days after the injection, the mice were killed for their tumor tissues, hearts, livers, spleens, lung,kidneys and brains to undergo microscope to observe valid thrombogenesis and tumor necrosis.Results With increasing concentration of the (RGD)3-tTF fusion protein, the clotting time was shorten correspondingly. Under the conditions of Ca2+, the clotting time was (8.6±0.2 )min when the concentration was 6μmol / L(compared with the group of 0μmol / L concentration,P< 0.05). The coagulation activity of (RGD)3-tTF and tTF fusion protein was alike(P > 0.05).The optical in vivo imaging and confocal microscopy analyses showed that RBITC fluorescence labeling (RGD)3-tTF fusion protein was assembling in the tumor vasculature of the colorectal carcinoma bearing mice. On the first,third,fifth day after injection,the tumor volume of (RGD)3-tTF fusion protein ground was(120.8±4.8)mm3,(93.8±3.4)mm3,(132.2±7.7)mm3 respectively,which was significantly less than that of the tTF and PBS grounds (both P< 0.05),however,there was no significant difference in the tumor volume between the latter two grounds (P>0.05).Conclusions The (RGD)3-tTF fusion protein which retained tissue factor thrombogenic had the capability of targeting to tumor vasculature and inducing thrombogenesis to suppress the tumor growth in the colorectal carcinoma mice model,which suggests that it is expected to become the treatment of colorectal cancer in a new way.