The VEGF/VEGFR axis has also become an ideal target for the development of molecularly targeted agents. Therefore, the discovery of new antitumor drugs with high selectivity and low toxic effects from natural products has become a key area of investment by researchers worldwide. Both VEGF and VEGFR are expressed in a variety of HCC cell lines, including HepG2, BEL-7402, SMMC-7721, Huh7, and MHCC-97H. Several studies have focused on the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) axis, which can trigger multiple downstream signaling pathways, causing tumor cell proliferation, migration, and angiogenesis. Although several signaling pathways have been consistently found dysregulated in hepatocellular carcinoma (HCC) (e.g., WNT-β-catenin, PI3K/AKT/MTOR, RAS/MAPK, IGF, HGF/MET, VEGF, EGFR, and PDGF), the incomplete understanding of the molecular mechanism of HCC contributes poor overall prognosis in HCC patients. Liver cancer is recognized as the fourth leading cause of cancer mortality in the world. Meanwhile, most types of cancers have the potential for high invasiveness and subsequent recurrence and metastasis, which is especially common in liver cancer. However, while killing tumour cells, chemical drugs often have severe toxicity on normal cells, which impairs patient immunity, increases recurrence rates, and reduces survival rates.
Many small molecule antitumor drugs were developed over the past decades.
Surgical resection combined with radiotherapy and chemotherapy can reduce cancer mortality, but surgical treatment may effectively remove only macroscopic tumors. Therefore, it is extremely urgent to find safe and effective antitumor drugs. Globally, cancer is still a critical health threat that remains to be solved. According to a report, in 2015, the number of new malignant tumor cases in China was about 42.92 million, and the number of deaths caused by this kind of tumor was about 2.814 million. Malignant tumors pose a serious threat to human health and result in high global morbidity and mortality. 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. 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. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. 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 IC 50 value of 171.18 ± 18.59 μg/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa.
Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Diverse bioactive substances derived from marine organisms have been attracting growing attention.
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