Objective: To explore the therapy effects of (arginine-glycine-aspartic, RGD)(3)-truncated tissue factor (tTF) fusion protein on colorectal carcinoma in mice.
Methods: The (RGD)(3)-tTF fusion gene, constructed with tTF and three series-wound peptides RGD, was expressed in Escherichia coli BL21 (DE(3)). The fusion protein was purified through Nickel affinity chromatography column. The coagulation activity of the (RGD)(3)-tTF fusion protein was detected by clotting assay in vitro. Mice colorectal cancer cells line CT26 were inoculated subcutaneously into mice to establish colorectal cancer model. 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 50 microg respectively. The location of the (RGD)(3)-tTF fusion protein in the colorectal carcinoma bearing mice tissue was analyzed by using in vivo optical imaging one hour after the injection and confocal microscopy twenty-four hours after the injection. Fifteen mice bearing 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 50 microg respectively. The tumor size was measured daily to calculate the tumor volume. Five days after the injection, the mice were killed to harvest tumor tissues, hearts, livers, spleens, lung, kidneys and brains to observe valid thrombogenesis and tumor necrosis.
Results: With the concentration of the (RGD)(3)-tTF fusion protein increased, the clotting time was shorten correspondingly under the conditions of Ca(2+), and the clotting time was (8.6 +/- 0.2) min when the concentration was 6 micromol/L, and it was >30 min in the group of 0 micromol/L (P < 0.05). The coagulation activity of (RGD)(3)-tTF and tTF fusion protein was alike (F = 0.09, P > 0.05). The in vivo optical imaging and confocal microscopy analyses showed that RBITC fluorescence labeling (RGD)(3)-tTF fusion protein was assembled in the tumor vasculature. On the first, third, fifth day after injection, the tumor volume of (RGD)(3)-tTF fusion protein group was (120.8 +/- 4.8) mm(3), (93.8 +/- 3.4) mm(3), (132.2 +/- 7.7) mm(3) respectively, which was significantly smaller than that of the tTF group [(181.4 +/- 13.8) mm(3), (333.0 +/- 32.0) mm(3), (514.0 +/- 11.5) mm(3)] and PBS group [(182.6 +/- 11.5) mm(3), (332.8 +/- 21.0) mm(3), (524.2 +/- 16.7) mm(3)] (both P < 0.05). However, there was no significant difference in the tumor volume between the latter two groups (P > 0.05).
Conclusion: The (RGD)(3)-tTF fusion protein is capable of targeting to tumor vasculature and inducing thrombogenesis for suppressing the tumor growth in the colorectal carcinoma mice model, and it's expected to be a new therapy for colorectal cancer.