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受体酪氨酸激酶抑制剂对在表达人血管内皮生长因子受体2（VEGFR2）的人胚肾293（HEK-293）细胞中血管内皮生长因子165a（VEGF165a）和血管内皮生长因子165b（VEGF165b）刺激的基因转录的影响。

Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.

作者信息

Carter Joanne J, Wheal Amanda J, Hill Stephen J, Woolard Jeanette

机构信息

Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.

出版信息

Br J Pharmacol. 2015 Jun;172(12):3141-50. doi: 10.1111/bph.13116. Epub 2015 Apr 10.

DOI:10.1111/bph.13116
PMID:25684635
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4459029/
Abstract

BACKGROUND AND PURPOSE

Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF(165)a and VEGF(165)b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells.

EXPERIMENTAL APPROACH

HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF(165)a- and VEGF(165)b-stimulated luciferase gene expression.

KEY RESULTS

VEGF(165)a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF(165)b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF(165)a. Analysis of the concentration-response data using the operational model of agonism indicated that both VEGF(165) isoforms had similar affinity for VEGFR2.

CONCLUSIONS AND IMPLICATIONS

Quantitative pharmacological analysis of the interaction of VEGF(165) isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF(165)a and VEGF(165)b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.

摘要

背景与目的

基于其减少血管生成的能力，靶向血管内皮生长因子受体2（VEGFR2）的受体酪氨酸激酶抑制剂（RTKIs）已被证明是一种有吸引力的癌症治疗方法。在此，我们通过研究活的人胚肾293（HEK-293）细胞中活化T细胞核因子（NFAT）报告基因的活性，对RTKIs与两种血管内皮生长因子剪接变体VEGF(165)a和VEGF(165)b与VEGFR2的相互作用进行了定量分析。

实验方法

使用表达人VEGFR2和由NFAT反应元件调控的萤火虫荧光素酶报告基因的HEK-293细胞，对RTKIs对VEGF(165)a和VEGF(165)b刺激的荧光素酶基因表达的影响进行定量分析。

关键结果

VEGF(165)a在活细胞中产生浓度依赖性的NFAT-荧光素酶报告基因激活，四种不同的RTKIs（西地尼布、帕唑帕尼、索拉非尼和凡德他尼）以非竞争性方式抑制这种激活。通过该分析获得的每种RTKIs的效力与使用纯化的VEGFR2激酶结构域进行的结合研究中获得的效力相似。与VEGF(165)a相比，VEGF(165)b是NFAT-荧光素酶反应的低效能激动剂。使用激动作用的操作模型对浓度-反应数据进行分析表明，两种VEGF(165)异构体对VEGFR2具有相似的亲和力。

结论与意义

在完整的活细胞中对VEGF(165)异构体和RTKIs与VEGFR2的相互作用进行定量药理学分析，为VEGF(165)a和VEGF(165)a对该酪氨酸激酶受体激活钙调神经磷酸酶-NFAT信号通路的相对亲和力和效力提供了重要见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/d7bcef6d7036/bph0172-3141-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/9486caf71a02/bph0172-3141-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/32c2237c4c82/bph0172-3141-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/df0079d07fd5/bph0172-3141-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/d7bcef6d7036/bph0172-3141-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/9486caf71a02/bph0172-3141-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/32c2237c4c82/bph0172-3141-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/df0079d07fd5/bph0172-3141-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fcc/4459029/d7bcef6d7036/bph0172-3141-f4.jpg

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