Department of Oncology and Southwest Cancer Center, Southwest Hospital, The Third Military Medical University, Chongqing, P. R. China.
J Cell Biochem. 2011 Aug;112(8):2106-14. doi: 10.1002/jcb.23130.
Endostatin is a natural occurring anti-angiogenic peptide and has been shown to inhibit tumor lymphangiogenesis by suppressing the expression of tumor-stimulating growth factors. We have previously shown that fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of colorectal tumors. Since it is known that EDA interacts with integrin α9 in the lymphatic endothelial cells (LECs), we hypothesized that endostatin may target EDA-integrin α9 pathway to inhibit colorectal tumor-induced lymphangiogenesis. To test this hypothesis, we examined the effect of endostatin on EDA secreted by SW480 colorectal cancer cells and treated human LECs with different doses of endostatin in the presence of conditional medium from SW480 cells. We found that endostatin significantly reduced EDA secretion by SW480 cells and the expression of integrin α9 in LECs. Immunofluorescence studies showed that EDA and integrin α9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by endostatin. Co-immunoprecipitation studies demonstrated that EDA interacted with integrin α9 in LECs, and showed that endostatin treatment inhibited the formation of EDA-integrin α9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by endostatin substantially, leading to a reduced formation of tube-like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and integrin α9 expressions as well as lymphangiogenesis were significantly suppressed by endostatin in colorectal cancer xenografts. In conclusion, our results suggest that endostatin reduces colorectal tumor-induced lymphangiogenesis, at least in part, by inhibiting EDA-integrin α9 pathway.
内皮抑素是一种天然存在的抗血管生成肽，已被证明通过抑制肿瘤刺激生长因子的表达来抑制肿瘤淋巴管生成。我们之前已经表明，纤连蛋白替代外显子 A（EDA）促进结直肠肿瘤的淋巴管生成。由于已知 EDA 在淋巴管内皮细胞（LEC）中与整合素α9 相互作用，我们假设内皮抑素可能靶向 EDA-整合素α9 途径抑制结直肠肿瘤诱导的淋巴管生成。为了验证这一假设，我们研究了内皮抑素对 SW480 结直肠癌细胞分泌的 EDA 的影响，并在 SW480 细胞的条件培养基存在下，用不同剂量的内皮抑素处理人 LEC。我们发现内皮抑素显著降低了 SW480 细胞的 EDA 分泌和 LEC 中整合素α9 的表达。免疫荧光研究表明，EDA 和整合素α9 在 LEC 的细胞膜上共定位，这些共定位通过内皮抑素显著减少。共免疫沉淀研究表明，EDA 在 LEC 中与整合素α9 相互作用，并表明内皮抑素处理抑制了 LEC 中 EDA-整合素α9 复合物的形成。此外，我们发现内皮抑素基本上破坏了 LEC 细胞骨架的排列和极性，导致 LEC 管样结构的形成减少，并抑制了 LEC 向 SW480 细胞的趋化性。一致地，内皮抑素在结直肠癌异种移植中显著抑制了 EDA 和整合素α9 的表达以及淋巴管生成。总之，我们的结果表明，内皮抑素通过抑制 EDA-整合素α9 途径减少结直肠肿瘤诱导的淋巴管生成，至少在部分程度上是这样。
