Departments of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
BMC Cancer. 2011 Aug 17;11:358. doi: 10.1186/1471-2407-11-358.
The fusion protein VEGF(121)/rGel composed of the growth factor VEGF(121) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF(121)/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.
We investigated the binding, cytotoxicity and internalization profile of VEGF(121)/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.
Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with (125)I-VEGF(121)/rGel demonstrated binding specificity that was competed with unlabeled VEGF(121)/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF(121)/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC(50) levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF(121)/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF(121)/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF(121)/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF(121)/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.
Taken together, these data confirm the selectivity of VEGF(121)/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.
融合蛋白 VEGF(121)/rGel 由生长因子 VEGF(121)和植物毒素 gelonin 组成，靶向肿瘤新生血管，并发挥显著的抗血管作用。我们之前已经表明，VEGF(121)/rGel 对过表达 VEGFR-2 的内皮细胞具有细胞毒性，但对过表达 VEGFR-1 的内皮细胞没有细胞毒性。在这项研究中，我们研究了这种构建物特异性毒性的基础，并评估了其在体外和体内的细胞内效应。
我们研究了 VEGF(121)/rGel 在表达 VEGFR-1 或 VEGFR-2 的内皮细胞上的结合、细胞毒性和内化谱，鉴定了它在体外和体内血管生成模型中的作用，并使用微阵列分析研究了它对许多分子途径的细胞内效应。
用 (125)I-VEGF(121)/rGel 孵育 PAE/VEGFR-2 和 PAE/VEGFR-1 细胞，证明了结合的特异性，这种特异性可以被未标记的 VEGF(121)/rGel 竞争，但不能被未标记的 gelonin 竞争。评估 VEGF(121)/rGel 对阻断体外管形成的影响发现，PAE/VEGFR-2(1 nM)和 PAE/VEGFR-1(100 nM)细胞的 IC50 水平相差 100 倍。VEGF(121)/rGel 在治疗后 1 小时内进入 PAE/VEGFR-2 细胞，但在治疗后 24 小时内未在 PAE/VEGFR-1 细胞中检测到。在用鸡绒毛尿囊膜进行血管生成研究中，1 nM 的 VEGF(121)/rGel 完全抑制了 bFGF 刺激的新生血管生长。VEGF(121)/rGel 的细胞毒性作用不是凋亡，因为处理后的细胞 TUNEL 阴性，没有 PARP 切割或选择凋亡标志物的蛋白水平改变的证据。用 VEGF(121)/rGel 处理的 HUVECs 的微阵列分析显示，控制细胞黏附、凋亡、转录调控、趋化性和炎症反应的 22 个基因的上调形成了独特的“指纹”图谱。
综上所述，这些数据证实了 VEGF(121)/rGel 对过表达 VEGFR-2 的内皮细胞的选择性，并且代表了第一个分析控制哺乳动物内皮细胞被基于 gelonin 的靶向治疗剂中毒的基因的分析。
