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

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.

Figure 1

Isolation, purity, and molecular mass analysis of ASP-3. (A) Separation of proteins by diethyl-aminoethanol (DEAE)-sepharose fast flow anion exchange chromatography; (B) purification of the protein fraction by phenyl sepharose CL-4B hydrophobic chromatography (the blue arrow); (C) molecular mass analysis of ASP-3 by SDS-PAGE (the red box); and (D) RP-HPLC profile of ASP-3.

Figure 2

UV–Vis spectrum of ASP-3.

Figure 3

FTIR spectrum of ASP-3.

Figure 4

Secondary structure determination of ASP-3 by CD spectrum.

Figure 5

Antiproliferative activity of ASP-3 against HepG2 cells.

Figure 6

ASP-3 induced the changes in tumor-related gene expression of HepG2 cells. (A) Distributions of DEGs by volcano diagram; (B) tumor-related gene expression by qRT-PCR. * p < 0.05 versus control; ** p < 0.01 versus control.

Figure 7

ASP-3 reduced VEGFR2 phosphorylation in HepG2 cells. Immunofluorescence staining was used to evaluate the distribution of VEGFR2 phosphorylation. They were stained with DAPI (blue), fluorescent secondary antibody of phospho-VEGFR2 VEGFR2 (green), and phalloidin (red), respectively. The immunofluorescence profile was visualized under a confocal fluoresce (scale bar: 20 µm).

Figure 8

ASP-3 (100 nM) interacted with VEGFR2 based on SPR platform Biacore S200.

Figure 9

The predicted docking model and diagrams of ASP-3 and VEGF/VEGFR2. (A) The predicted structure of ASP-3. (B) The structure of VEGFA/VEGFR2. (C) The functions of ASP-3 and VEGFA/VEGFR2 (orange and blue). (D) Amino acid sequence alignment of ASP-3 and VEGFA/VEGFR2.

Figure 10

ASP-3 inhibits VEGF-induced tube formation of HUVECs in vitro. (A) Representative tubular structure images of HUVECs treated with different concentration of ASP-3 and VEGF (10 ng/mL, magnification 10×). (B) Number of branch points in HUVECs measured. p < 0.05 versus blank control; ** p < 0.01 versus VEGF-treated group.

Figure 11

Anti-angiogenesis activity of ASP-3 in transgenic zebrafish model. (A) Lateral view of fli1a zebrafish embryos at 72 hpf. Live fluorescence microscopy highlights GFP expressing ISVs treated with ASP-3 in the concentration of 0–150 ug/mL (magnification: 4× and 10×). (B) The area of ISVs of zebrafish. (n = 6 for each experimental group). * p < 0.05; ** p < 0.01 versus control.

Figure 12

Proposed mechanism of antitumor effect of ASP-3.