The Inhibitory Effect of Canstatin on VEGF-A Induced Lymphangiogenesis in an Oral Squamous Cell Carcinoma SCCVII Animal Model

Tumor lymphangiogenesis, the generation of new lymphatic vessels from the pre-existing lymphatic system, contributes to metastasis by increasing the abundance of lymphatics in and/or nearby a tumor. Tumor lymphangiogenesis is, therefore, an attractive target for therapeutics designed to restrict the metastatic spread of cancer. Here, we describe the effect of recombinant canstatin, a C-terminal globular non-collagenous domain of the α2 chain of type IV collagen, on tumor-induced lymphangiogenesis both in vitro and in vivo. Canstatin inhibited the proliferation and invasion of oral squamous cell carcinoma SCCVII cells. The inhibitory effect of canstatin on tumor growth was further investigated using an orthotopic oral squamous cell carcinoma animal (C3H/He) model. Canstatin reduced the growth of SCCVII-induced tumor. Interestingly, canstatin suppressed lung metastasis caused by lymphangiogenesis and lymph node metastasis of SCCVII. To further determine the effect of canstatin on SCCVII-induced lymphangiogenesis, the expression of lymphangiogenic growth factors was evaluated in hypoxia mimetic agent, CoCl₂- and/or canstatin- treated SCCVII cells. Canstatin reduced the expression of VEGF-A in CoCl₂-treated SCCVII cells. The proliferation, migration and tube formation of lymphatic endothelial cells (LECs) induced by VEGF-A were dramatically inhibited by the treatment of canstatin. In addition, canstatin suppressed the expression of VEGFR-1 and VEGFR-2 in VEGF-A-treated LEC. In immunohistochemical analysis of canstatin-treated tumor sections, the expression of VEGF-A, VEGFR-1, and VEGFR-2 was also suppressed. These results indicate that canstatin reduces the expression of VEGF-A in hypoxia-induced SCCVII cells and inhibits VEGF-A-induced lymphangiogenesis.