Department of Surgical Oncology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Angiogenesis. 2010 Sep;13(3):227-38. doi: 10.1007/s10456-010-9180-2. Epub 2010 Aug 8.
Sulforaphane (SUL), a kind of isothiocyanate, has recently been focused due to its strong pro-apoptotic effect on cancer cells as well as tumor vascular endothelial cells (ECs). And recently, we demonstrated the induction of autophagy by colon cancer cells as a protective mechanism against SUL. In the present study, we aimed to investigate the possible role of autophagy induction by ECs as a defense mechanism against SUL.
Human umbilical vein endothelial cells (HUVECs) were used as the in vitro model of angiogenic ECs. The induction of autophagy was evaluated by the detection of acidic vesicular organelles (AVOs) by flow-cytometry, after the staining with acridine orange, as well as the detection of light chain 3(LC3) by Western blot. Finally, the functional implication of autophagy inhibition and SUL treatment in ECs was investigated by their ability to form vascular-like structures on Matrigel.
Treatment of HUVECs with relatively low concentrations of SUL for 16 h resulted in the evident formation of AVOs and the recruitment of LC3 to autophagosomes, the pathognomonic features of autophagy. Co-treatment of cells with the specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL. And inhibition of autophagy potentiated the inhibitory effect of SUL on the ability of ECs to form capillary-like structures.
Similar to cancer cells, ECs induced autophagy in response to the pro-apoptotic agent, SUL, and the inhibition of autophagy potentiated the pro-apoptotic effect. These findings open premises for the use of autophagy inhibitors in combination with anti-angiogenic agents.
萝卜硫素（SUL）是一种异硫氰酸盐，由于其对癌细胞和肿瘤血管内皮细胞（EC）的强烈促凋亡作用，最近受到了关注。最近，我们证明结肠癌细胞通过自噬诱导作为一种针对 SUL 的保护机制。在本研究中，我们旨在研究 EC 中自噬的诱导作为针对 SUL 的防御机制的可能作用。
用人脐静脉内皮细胞（HUVEC）作为血管生成 EC 的体外模型。通过吖啶橙染色后用流式细胞术检测酸性囊泡细胞器（AVOs）以及用 Western blot 检测轻链 3（LC3）来评估自噬的诱导。最后，通过它们在 Matrigel 上形成血管样结构的能力来研究自噬抑制和 SUL 处理对 EC 的功能影响。
用相对较低浓度的 SUL 处理 HUVEC 16 小时后，明显形成了 AVOs 并募集了 LC3 到自噬体，这是自噬的特征性特征。用特异性自噬抑制剂（3-甲基腺嘌呤）共处理细胞增强了 SUL 的促凋亡作用。并且自噬抑制增强了 SUL 对 EC 形成毛细血管样结构能力的抑制作用。
与癌细胞类似，EC 在受到促凋亡剂 SUL 作用后诱导自噬，并且自噬抑制增强了促凋亡作用。这些发现为在抗血管生成剂中联合使用自噬抑制剂开辟了前提条件。
