Purification Of Ancylostoma Caninum Anticoagulantpeptide C2 And Its Effects On The Growth And Apoptosis Of Human Non-small Cell Lung Cancer Cell Line NCI-H460

Ancylostoma caninum anticoagulantpeptide c2(AcAPc2) was extracted from Ancylostoma caninum, and it has been found to inhibit the activity of factor VIIa in complex with membrane-bound tissue factor (TF) and prothrombinase; formation of this complex is a crucial stage in the thrombotic response as the factor VIIa/TF complex initiates the production of thrombin. Due to its distinct spectrum of anticoagulant activity and its novel mechanism, rAcAPc2 is currently being developed as a therapeutic agent for the potential treatment and prevention of thrombosis in humans. However, with traditional expression and purification systems, it is difficult to yield recombinant AcAPc2 (rAcAPc2). Expression of recombinant protein in E. coli usually contains a permanent methionine or other tag that may interfere with the bioactivity of recombinant molecules. Expression of recombinant protein in yeast and other eukaryotic cells commonly results in a low yield of products. Inteins are intervening protein sequences that are capable of catalyzing their excision from a precursor protein with the concomitant joining of the flanking sequences, termed exteins, through a native peptide bond. The intein-mediated self-cleaving mechanism has recently been exploited to purify recombinant proteins from bacterial cultures. This opens the way for using protein splicing as a tool that allows expression of inactive precursor proteins that can be activated by protein splicing. The IMPACT^TM-TWIN protein fusion and purification system utilizes the inducible self-cleavage activity of engineered protein splicing elements (termed inteins) for protein purification and manipulation. This system has been used for the generation of recombinant native proteins. In the present study, we employed this simple system to generate high-purity rAcAPc2 with higher yield using the overlapping PCR with synthesized oligonucleotides. The gene for rAcAPc2 was fused with the intein and chitin-binding domain (CBD), which functions as an affinity tag for expression in E. coli and purification of rAcAPc2 by pH-dependent cleavage. Importantly, the generated rAcAPc2 has antitrypsin protease activity and potent anticoagulant activities as evidenced by significantly prolonging the prothrombin time (PT) and activated partial thromboplastin time (aPTT) of human plasma in vitro in a dose-dependent manner.Serine protease inhibitors are involved in the control of many enzymme activity, they play a very important role in life. Previous studies have shown that the serine protease inhibitors can down-regulate tumor growth and metastasis of several types of cancers. As analysis of the amino acid sequences of the hookworm anticoagulant proteins reveals that they are members of a family related to serine protease inhibitors, AcAPc2 might inhibit the growth of cancer cells. By using the SRB method, we found that rAcAPc2 inhibited the proliferation of NCI-H460 in a dose manner. Flow cytometric analysis of Annexin V and propidium iodide staining, TUNEL and DAPI staining showed that rAcAPc2 induced substantial apoptosis. The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in processes leading to cancer cell invasion and metastasis. It is reported that the overexpression of uPA/uPAR is correlated with the progression of cancers. The prognostic value of uPA and its receptor uPAR are well established, plasma levels of uPA and uPAR were elevated in patients with bladder carcinoma and were associated with features of biologically aggressive disease. Subramanian et al. reported that when uPA and uPAR were downregulated simultaneously, the apoptotic cascade was triggered. By using the method of RT-PCR, we found that levels of mRNA of uPA and uPAR were obviously decresed in rAcAPc2⁺ than in rAcAPc2^- cells. To further define the role of rAcAPc2 in inducing apoptosis, we examined NF-κB of the ribonucleoprotein by Western blot using anti- NF-κB polyclone antibody. The results showed that level of NF-κB in ribonuleoprotein decreased in the rAcAPc2⁺ cells. Nuclear extracts from NCI-H460 cell lines were analyzed by EMSA.The results showed that nuclear translocation of NF-κB decreased in the rAcAPc2⁺ cells. Evidence of NF-κB nuclear translocation in NCI-H460 by immunohistochemical and immuno-fluorescence method also showed that nuclear translocation of NF-κB decreased in the rAcAPc2⁺ cells.NF-κB is a transcription activator that controls expression of genes associated with inflammation and cell proliferation and survival, such as uPA and uPAR gene. NF-κB is a heterodimer composed of p50 and p65/RelA, which is retained in the cytoplasm by the IκB group of inhibitory proteins. In response to stressful stimuli, such as cancer chemotherapy, IκB is phosphorylated, ubiquitinated, and subsequently degraded by the 26S proteasome.κB degradation results in the nuclear translocation of NF-κB and the activation of NF-κB-dependent gene transcription. The 26S proteasome is a large multisubunit protein complex found in the cytoplasms and nuclei of all eukaryotic cells; its principle function is to degrade proteins via the ubiquitin pathway. It is composed of a 20S core cylinder, with tryptic and chymotryptic activity, and is capped at each end by a 19S regulatory particle. As AcAPc2 is the member of a serine protease inhibitors family, AcAPc2 might inhibit the proteolytic activity of 26S proteasome and reflected the activation of NF-κB. Inhibition of NF-κB could induce cell apoptosis and also decrease the expression of the uPA and the uPAR. To investigate a possible relationship between rAcAPc2 and proteasome function, we examined its effects on the tryptic cleavage activity of the 26s proteasome in NCI-H460 cells. The results showed that rAcAPc2 inhibited the tryptic cleavage activity of the 26s proteasome. We also examined IκBαof the cytoplasmprotein by Western blot using anti- IκBαpolyclone antibody. The results showed that incubation with rAcAPc2 caused an increase of IκBαprotein level.In conlusion, we used a simple system to generate a bacterial clone that expressed high levels of rAcAPc2-CBD-intein fusion proteins. With a spontaneous pH-dependent splicing, a high yield of rAcAPc2 with 98% purity was obtained from bacterial lysate with a one-step procedure. Importantly, the purified rAcAPc2 appeared to have the native sequence and strongly prolonged the PT and aPTT clotting time of freshly isolated human plasma and antitrypsin protease activity in a dose-dependent manner. Futher study showed that rAcAPc2 could inhibit the proliferation and induce apoptosis of NCI-H460 in a dose manner. rAcAPc2 also inhibited the level of mRNA of the uPA and the uPAR gene.The reason might be that rAcAPc2 inhibit the proteolytic activity of 26S proteasome and reflected the activation of NF-κB, which could induce cell apoptosis and also decrease the expression of uPA and uPAR. Our experiments supplied a new insight for how rAcAPc2 reflected the growth and apoptosis of non-small cell lung cancer cell line NCI-H460.