Department of Medical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
Int J Cancer. 2010 Dec 1;127(11):2685-98. doi: 10.1002/ijc.25276.
Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF-induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF-induced endothelial cell proliferation and VEGF-induced phosphorylation of extracellular-regulated kinase 1/2, phospholipase C-γ, and VEGF receptor-2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF-activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL-modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF-activated HUVECs as compared to peptide-unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL-17 carcinoma cell-bearing mice. In summary, BiP may regulate VEGF-induced endothelial cell proliferation through VEGFR-2-mediated signaling and be an effective target molecule for cancer antineovascular therapy.
血管生成对于肿瘤生长和血行转移至关重要。在血管生成的内皮细胞中，特别是在质膜上，特异性表达和功能的蛋白分子可能是癌症治疗中抗血管生成药物和药物输送系统（DDS）的分子靶标。为了发现这种靶标分子，我们使用二维差异凝胶电泳（2D-DIGE）和基质辅助激光解吸/电离串联飞行时间质谱（MALDI-TOF/TOF-MS）对用或不用血管内皮生长因子（VEGF）处理的人脐静脉内皮细胞（HUVEC）进行了亚细胞蛋白质组分析。在鉴定出的蛋白质中，分子伴侣 BiP/GRP78 在 VEGF 处理后 HUVEC 的膜/细胞器部分高表达。通过 RNA 干扰研究了 BiP 在 VEGF 诱导的血管生成中的作用。BiP 敲低显著抑制了 VEGF 诱导的内皮细胞增殖和 VEGF 诱导的细胞外调节激酶 1/2、磷脂酶 C-γ 和 VEGF 受体-2 的磷酸化。细胞表面生物素化分析显示，在 VEGF 激活的 HUVEC 中 BiP 的细胞表面表达增加。为了将 BiP 应用于脂质体 DDS 的靶标分子，我们开发了用已显示与 BiP 结合的 WIFPWIQL 肽修饰的脂质体，并研究了其在癌症治疗中的潜力。与肽未修饰的脂质体相比，WIFPWIQL 肽修饰的脂质体（WIFPWIQL 脂质体）被 VEGF 激活的 HUVEC 显著摄取。WIFPWIQL 脂质体似乎在体内积聚在肿瘤内皮细胞中。载有多柔比星的 WIFPWIQL 脂质体显著抑制肿瘤生长并延长携带结肠 26 NL-17 癌的小鼠的存活时间。总之，BiP 可能通过 VEGFR-2 介导的信号通路调节 VEGF 诱导的内皮细胞增殖，并且是癌症抗血管生成治疗的有效靶标分子。
