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Mar Drugs. 2019 Sep 9;17(9). pii: E528. doi: 10.3390/md17090528.

Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism.

Author information

1
Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, China. joey444@163.com.
2
Center for experimental technology, College of Pharmacy, Jinan University, Guangzhou 510632, China. joey444@163.com.
3
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. sh613050@163.com.
4
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. lcl992@126.com.
5
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. yyluooo@163.com.
6
Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, China. shuimuliunian@163.com.
7
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. tyrm@jnu.edu.cn.
8
Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, MA 02142, USA. Haoran.wang@neolandbio.com.
9
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. lwy5464@stu2017.jnu.edu.cn.
10
Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou 510632, China. tzhujh@jnu.edu.cn.
11
Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, China. huangjuan415@163.com.
12
Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, China. tsly@jnu.edu.cn.

Abstract

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 μg/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.

KEYWORDS:

Arca subcrenata protein; antitumor mechanism; structural characterization

PMID:
31505835
DOI:
10.3390/md17090528
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