Antiangiogenic effect of endostatin-derived peptides on a mouse model of colon cancer
Shohreh Pourmomen jorshari
,1,* Seyed mohsen asghari
,2 Farzad khanipour
1. University of Guilan
2. University of Guilan
3. Max Planck Institute of Molecular Physiology
Colon cancer is among the most common malignancies worldwide. angiogenesis is the formation of new microvasculature through budding from the existing vessels. since demonstration of tumor growth dependence on angiogenesis by folkman et al., its inhibition via antiangiogenic agents has become one of the cancer treatment strategies. endostatin is an endogenous collagen xviii-derived 20 kd protein fragment with an n-terminal zn binding site, which has demonstrated to have antiangiogenic and antitumor activities. in a study by chamani et al. it was demonstrated that a 27 amino acid residue peptide (es-zn) derived from n-terminus of endostatin, preserves the full-length protein’s function. in another variant of this peptide engineered by chamani et al. (es-ss) the n-terminal zn binding site was replaced with a disulfide bond and its function was compared with es-zn. the results showed that the antiangiogenic activity was significantly improved in es-ss compared with es-zn (p<0.05). accordingly, the aim of this study was to assess the comparative ability of es-ss and es-zn to inhibit tumor angiogenesis (through analysis of cd31 antigen expression as a determinant for microvasculature density) and growth in a mouse model of colon cancer (ct26 murine colon cancer cell line in balb/c mice). also, the direct effect of es-ss peptide on ct26 cell viability was assessed in vitro.
Ct26 cell line culture
ct26 murine colon carcinoma cell line, obtained from pasteur institute of iran, was cultured as suspension in t25 flasks containing rpmi-1640 medium, fbs 10, streptomycin, and penicillin to grow under conditions including 37oc incubator with 5% co2 and 90% humidity.
creating the mouse model of colon cancer
female 6-8 week old balb/c mice were inoculated subcutaneously on their left flank with 106 ct26 cells.
peptide treatment of the mice
seven days after cell inoculation and tumor formation the mice were divided in three treatment groups (control, es-ss, and es-zn) and were treated through intracranial injection.
microvasculature density (mvd) analysis (cd31 antigen expression)
mice were sacrificed through spinal cord displacement and the tumors were carefully excised under sterile conditions. tumors were sliced and tissue samples were made for immunohistochemistry experiments to analyze and compare mvds. the experiments were performed in cancer research institute of imam khomeini hospital of tehran.
effects of es-ss and es-zn peptides on viability rates of ct26 cells and human umbilical vein endothelial cells (huvec) were assessed through cell culture and mtt assay in vitro.
all the experiments were performed three times and in vitro tests were analyzed using graphs in excel program. tumor volume comparisons were analyzed using two-way anova test. comparisons of tumor weights and tumor microvasculature densities were analyzed through one-way anova-tukey test.
Assessment of antitumor activity in vivo
the capability of es-ss peptide to inhibit growth of ct26 tumors in balb/c mice was assessed and compared with es-zn peptide. at the end of day 20, the average of tumor volume in control group, es-ss group, and es-zn group were 2469.6 mm3, 312.6 mm3, and 818.3 mm3 respectively (p<0.05).
microvasculature density (mvd) analysis
es-ss treatment significantly reduced mvd of the tumors and showed a significantly stronger activity compared with es-zn peptide treatment in this respect. the average mvd values in control group, es-ss group, and es-zn group were 34±11.58, 6.2±2.13, and 15.2±1.4 respectively (p<0.05).
human umbilical vein endothelial cell (huvec) viability inhibition assessment
effects of es-ss and es-zn peptides on huvec cell proliferation were analyzed and compared in a concentration range of 0-600 ng/ml. the results showed 50% inhibition concentrations (ic50) of es-ss and es-zn peptides to be 600±28 ng/ml and 500±22 ng/ml respectively.
ct26 cell viability inhibition assessment
for both es-ss and es-zn treatments (different doses for 24 hours), average ct26 cell viability rates did not show any significant differences from those of the control treatments (p>0.05).
In conclusion, results of this study show that replacement of the n-terminal zn binding site of the endostatin-derived peptide with an engineered disulfide bond considerably improves its antiangiogenic and antitumor activities. besides, the endostatin-derived peptides exert their antiangiogenic and ant-tumor activities exclusively through their inhibitory effect on growth and proliferation of vascular endothelial cells of tumors, whereas they do not show any effect on tumor epithelial cells.
Tumor, colon, antiangiogenic, endostatin, peptide