Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism
Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism
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一种从Arca subcrenata中纯化的新型蛋白ASP-3的鉴定、表征及其抗肿瘤机制
DOI：
10.3390/md17090528
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发表时间：
2019-09-01
期刊：
影响因子：
5.4
通讯作者：
Song, Liyan
中科院分区：
文献类型：
--
作者：
Guo, Zhongyi;Shi, Hui;Song, Liyan
来源于海洋生物的多种生物活性物质越来越受到人们的关注。除了小分子和多肽外，大量研究表明海洋蛋白也具有抗肿瘤活性。小的抗癌蛋白可以通过病毒载体在体内表达，以发挥局部和长期的抗癌作用。本文从毛蚶中分离纯化了一种具有抗肿瘤活性的新蛋白ASP-3。ASP-3含有179个氨基酸，分子量为20.6 kDa。采用傅里叶变换红外光谱（FTIR）和圆二色谱（CD）对ASP-3的光谱特性进行了解析。ASP-3被鉴定为肌浆钙结合蛋白，通过3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四氮唑（MTT）测定法测定，ASP-3对人肝细胞癌（HepG 2）细胞的增殖表现出强烈的抑制作用，IC 50值为171.18 +/- 18.59 μ g/mL。RNA-seq分析显示ASP-3调控HepG 2细胞中的血管内皮生长因子受体（VEGFR）信号通路。免疫荧光结果表明，ASP-3能有效降低HepG 2细胞中VEGFR 2的磷酸化，并影响VEGF信号通路的下游组分。表面等离子体共振（SPR）分析进一步证明ASP-3与VEGFR 2直接相互作用。更重要的是，ASP-3作为抗血管生成剂的治疗潜力通过使用人脐静脉内皮细胞（HUVEC）的VEGF诱导的管形成测定的体外模型以及使用转基因斑马鱼模型的体内模型进一步证实。总之，ASP-3为开发更有效的抗癌蛋白提供了一个良好的框架，并为使用新方法（如基因治疗）进行癌症治疗提供了重要武器。
Diverse bioactive substances derived from marine organisms have been attracting growing attention. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. Being identified as a sarcoplasmic calcium-binding protein, ASP-3 exhibited strong inhibitory effects on the proliferation of Human hepatocellular carcinoma (HepG2) cells with an IC50 value of 171.18 +/- 18.59 mu g/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. More importantly, the therapeutic potential of ASP-3 as an anti-angiogenesis agent was further confirmed by an in vitro model using VEGF-induced tube formation assay of human umbilical vein endothelial cells (HUVECs), as well as an in vivo model using transgenic zebrafish model. Taken together, the ASP-3 provides a good framework for the development of even more potent anticancer proteins and provides important weapon for cancer treatment using novel approaches such as gene therapy.