Jing Yue, Wang Gang, Xiao Qi, Zhou Yachun, Wei Yingjie, Gong Zhunan
Center for New Drug Research and Development, College of Life Science, Nanjing Normal University, Nanjing, China.
Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
Onco Targets Ther. 2018 Apr 4;11:1871-1884. doi: 10.2147/OTT.S157747. eCollection 2018.
Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angio-genesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear.
In this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. expression in whole embryos following AA-PMe treatment was also determined.
We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling.
For the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research.
血管生成在许多生理和病理过程中起着至关重要的作用，一些疾病与其调节异常有关。齐墩果酸（AA）已显示出抗癌特性，我们怀疑这可能归因于其对血管生成的影响。AA的一种修饰衍生物，N-(2α,3β,23-乙酰氧基乌苏-12-烯-28-酰基)-L-脯氨酸甲酯（AA-PMe），比其母体化合物具有更高的疗效，但其对血管发育的影响仍不清楚。
在本研究中，我们研究了AA和AA-PMe在斑马鱼胚胎和人脐静脉内皮细胞（HUVECs）中的抗血管生成活性。首先，我们用不同浓度的AA-PMe或AA处理HUVECs，有无血管内皮生长因子（VEGF）存在，并评估细胞活力、管形成以及细胞迁移和侵袭。随后使用定量实时聚合酶链反应和蛋白质印迹分析来确定血管内皮生长因子受体2（VEGFR2）介导的信号传导在AA-PMe抑制血管生成中的作用。我们将这些研究扩展到使用转基因斑马鱼胚胎跟踪血管生成。对于这些实验，在受精后24至72小时用不同浓度的AA-PMe或AA处理胚胎，然后进行形态学观察、血管生成评估和内源性碱性磷酸酶测定。还测定了AA-PMe处理后整个胚胎中的表达。
我们发现AA-PMe以剂量依赖性方式降低HUVECs的细胞活力并抑制迁移和管形成。同样，AA-PMe破坏了斑马鱼胚胎中节间血管、背主动脉和后主静脉的形成。在体外和体内，AA-PMe通过抑制VEGF诱导的VEGFR2磷酸化以及破坏下游细胞外调节蛋白激酶和AKT信号传导，在阻断血管生成方面都超过了AA。
本研究首次揭示AA-PMe作为一种有效的VEGFR2激酶抑制剂发挥作用，并具有强大的抗血管生成活性，表明它是进一步研究中一个有前景的治疗候选物。
