Isolation, Purification And Its Antimetastatic Activities In Vitro Of A Novel Protein From Eupolyphaga Sinensis

Eupolyphaga sinensis Walker (Blattaria:Corydiidae), popularly known as’Tubie’, is a well-known edible and medicinal insect in China and now has been artificially propagated on a large scale. In Chinese folk and traditional medicine, it is widely used as a natural healthy product to prevent and treat many diseases, including bone injury, cancer and immune-related diseases, etc. In recent years, it has been reported that E. sinensis has antithrombotic, antitumor, immune-protective and antioxidant activities. The antitumor and thrombolytic activities are the most attractive among these bioactivities. Previous studies on the antitumor activities showed that the crude protein extracts from E. sinensis could inhibit angiogenesis and tumor growth in vivo. However, the components responsible for its activity have not been addressed, and no purified active protein fraction has been obtained from this species.In this study, we purified a72kDa anticancer protein, designated as EPS72, from the fresh female E. sinensis using a line of purification techniques. EPS72showed potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc. To gain insight into the potential antitcancer mechanism of this protein, we further investigated its effects on cellular morphology, proliferation, apoptosis, adhesion, spreading, migration and invasion of A549cells. We found that EPS72showed an antimetastatic activity in vitro. The probable molecular mechanism underlining its effects on A549cancer cells was also studied. All the results of the anticancer protein were summarized as follows:1Isolation, purification and identification of the E. sinensis anticancer proteinThe crude extracts (Fraction I) were obtained by ammonium sulfate precipitation (50%～80%saturation), ultra-filtration (10kDa cut-off) and lyophilization. The anticancer protein fraction(s) was/were purified using three phases Capture, Intermediate Purification and Polishing:(1) Capture:CM-Sepharose FF cation-exchange chromatography (CIEX) and DEAE-Sepharose FF anion-exchange chromatography (AIEX) were used;(2) Intermediate Purification:Q-Sepharose HP anion-exchange chromatography (AIEX) and Butyl Sepharose HP hydrophobic chromatography (HIC) were used;(3) Polishing:Superdex75gel filtration chromatography (GF) was used. During the separation and purification process, the protein elution profile was monitored at280nm and MTT assay was performed to monitor the cytotoxicity of each elution fraction. The final active fraction (Fraction Ⅵ) was lyophilized to obtain a purified anticancer protein. The recovery of the purified anticancer protein was about0.0056%.The Fraction VI appeared as a single band on SDS-PAGE, with an apparent molecular mass of about72kDa. Its accurate molecular mass was71.737kDa as determined by MALDI-TOF mass spectrometry analysis. The results suggested that after performing several steps of purification, a pure72-kDa single-stranded protein with anticancer activity in vitro, named EPS72, was obtained.2Antitumor activities in vitro of EPS72The results of MTT assay indicated that EPS72displayed potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc. We further investigated its effects on cellular morphology, proliferation, apoptosis, adhesion, spreading, migration and invasion using the human lung cancer A549cell line as a tumor model in vitro. The results of microscopic morphological observation, re-culture when withdrawn EPS72, trypan blue staining, JC-1staining to detect mitochondrial membrane potential (ΔΨm) and adridine orange/ethidium bromide(AO/EB) staining of A549cells indicated that EPS72could induce cell detachment in the early stage, which further led to cell apoptosis in the late stage. But the initially detached cell clusters induced by EPS72remained viable, suggesting that EPS72had a direct role on cell adhesion and indirect role on cell apoptosis.5μg/ml of EPS72remarkably inhibited A549cells attachment to fibronectin (FN) and collagen IV (Col IV), but could not inhibit A549cells adhesion to poly-L-lysine (PL) in a cell adhesion assay, could affect spreading ability of A549cells on ECM gel in a time-dependent mode in an spreading assay in vitro, and could inhibit A549cells migration into the wounded areas in a wound healing assay. In a Boyden chamber assay, EPS72was found to inhibit the invasive activities of A549cancer cells in a dose-dependent mode. These results suggested that EPS72exerted its antitumor activities in vitro by affecting adhesion, spreading, migration, invasion, proliferation and survival of cancer cells and EPS72has an antimetastatic activity in vitro.3Possible molecular mechanism underlining its antitumor effects in vitro of EPS72In order to further investigate the possible molecular mechanism underlining the antimetastatic effects in vitro of EPS72, we examined the effect of EPS72on F-actin with an Actin-tracker Green fluorescent probe under a fluorescence microscope and performed flow cytometry and Western blot analysis to detect the expression of β1-integrin and/or integrin-linked kinase (ILK) in A549cells. The results indicated that EPS72could induce depolymerization of F-actin and down-regulate the expression of β1-integrin and ILK in A549cells, suggesting that EPS72exerted the antimetastatic effects in vitro probably by directly targeting β1-integrin, then further influencing the integrin-cytoskeleton signaling pathway.Major achievements and conclusions of the article are as follows:(1) We purified a72-kDa anticancer protein (EPS72) from E. sinensis by using ammonium-sulfate-precipitation, ultra-filtration, CIEX-AIEX-HIC-GF isolation and purification protocol, which is mild, easily operational and easy-to-scaling-up. The purified protein, to our knowledge, is the first purified anticancer protein from this species.(2) In an in vitro study, EPS72was found to display potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc., but the normal cell line human fibroblast cell MRC5was not very sensitive to the treatment of EPS72, suggesting EPS72exhibited relatively broad antitumor activities in vitro and showed some specificity to human cancer cells.(3) We found that EPS72exerted its antitumor effects in vitro by affecting adhesion, spreading, migration, invasion, proliferation and apoptosis of cancer cells, and EPS72has an antimetastatic activity in vitro.(4) We tentativly concluded that EPS72exerted the antimetastatic effects in vitro probably by directly targeting β1-integrin, then further influencing the integrin-cytoskeleton signaling pathway.