Deciphering the anti-angiogenic effect of endostatin/cyclophosphamide to normalize tumor micrangium through notch signaling pathway in colon cancer

Background: The invasion of colon cancer is associated with the tumor angiogenesis. Endostatin is an important anti-angiogenic agent, and the additive effect of endostatin with a chemotherapeutic agent, cyclophosphamide, on micrangium has not been established. Methods: Male BALB/c strain nude mice were injected with human colorectal carcinoma cells (HCT-116). The mice were divided into four groups (n = 15, each group) and were treated with different concentrations of endostatin (15, 10, and 5 mg/kg/day), cyclophosphamide (20, 10, and 5 mg/kg/day), and combination of endostatin/cyclophosphamide (15 + 20, 15 + 10, and 15 + 5 mg/kg/day). The tumor inhibition rate was evaluated, followed by the quantification of messenger ribonucleic acid (mRNA) and protein expression of notch signaling components NOTCH-1, NOTCH-3, NOTCH-4, JAG-1, DLL-4, Hes-1, and Hey-1 using quantitative polymerase chain reaction (qPCR). The protein expression of NOTCH-3, JAG-1, and DLL-4 was confirmed using western blotting. Microvessel density (MVD) was evaluated to detect micrangium following the treatment. Results: The endostatin/cyclophosphamide-treated samples exhibited an additive effect on the tumor inhibition rate and the microvessel count. NOTCH-1, NOTCH-3, NOTCH-4, JAG-1, Hes-1, and Hey-1 expression levels were highly correlated and downregulated in the treated samples, whereas DLL-4 expression was upregulated that accounted for its anti-angiogenic property. Conclusions: The combination treatment of colon cancer with endostatin and a chemotherapeutic agent, cyclophosphamide proves to be an efficient therapeutic strategy to inhibit the rapid vasculature formation confirmed by the differential expression of notch signaling components.