Identification Of The Differentiation-Induced Mechanisms Of S5a/angiocidin On THP-1 Monocytic Leukemia Cells

In 1993, Tuszynski's laboratory isolated a protein from lung carcinoma by peptide affinity chromatography using a highly hydrophobic sequence, CSVTCG, from the typeⅠrepeat domain of thrombospondin-1 (TSP-1). They have shown that the recombinant protein, now termed "angiocidin", is able to inhibit critical steps in tumor progression in vitro. A BLAST search of the full length cDNA of angiocidin revealed that the protein sequence has a high degree of homology with two previously described proteins, S5a and antisecretory factor (ASF). Current studies reported that angiocidin was a potent inhibitor of angiogenesis and tumor growth. More recent studies found that angiocidin could induce monocyte-to-macrophage differentiation. Tuszynski and coworkers demonstrated that angiocidin activated monocytes to upregulate cytokine mRNA expression and secretion through pathways mediated by MAPK, NF-κB, and PI3K. It was also found that angiocidin-treatment increased the ability of monocytes to present antigen to T lymphocytes, and that functional macrophages could directly or indirectly elicit tumor cell destruction. But which molecules angiocidin directs toward on the surface of cells remains unclear. The paper discourses the differentiation-inducing effects and potential mechanisms of S5a on THP-1 cell line upon the following aspects.PartⅠ:Expression and purification of MBP-tagged recombinant S5a.For induction of protein production, IPTG was added to LB medium at 0.5 mM final concentration and the bacteria were incubated at 37℃for 6 h. We collect bacteria, break with ultrasound, utilize affinity chromatography column to purify the centrifugal supernatant and make analysis by SDA-PAGE. The results show that there is a new protein band of nearly 88,000D which is consistent with theoretical values of MBP-S5a. After we scan the analysis, the expression of MBP-S5a accounts for about 30% of total bacteria proteins. Results from western blotting suggested successful expression of desired proteins in correct molecular weight.PartⅡ:The apoptosis-inducing effects and differentiation-inducing effects of S5a on THP-1 cells.THP-1 cells were treated with MBP-S5a for 24,48,72 and 96 h, parallel MBP protein is shown as a control. It was found by MTT test that the cell inhibiting rate increased at 24 h and decreased at 48 h significantly. MBP-S5a can promote cells proliferation after 96 h of application. Previous research has shown that S5a can induce monocyte-to-macrophage differentiation. We hypothesized that THP-1 cells differentiate upon S5a-treatment at 48 h. According to the results of the apoptosis rate of THP-1 cells which were analyzed by flow cytometry, there was significantly less apoptosis in MBP-S5a-treated THP-1 cells as compared to MBP-treated THP-1 cells at 48 h. On the other hand, the expression of macrophage markers in MBP-S5a-treated THP-1 cells (CD 14 and CD36) was detected at 48,72 and 96 h by flow cytometry. The result showed the number of CD14/CD36-positive cells treated with MBP-S5a increased with the treatment and increased time, indicating that MBP-S5a induced differentiation of THP-1 cells in a time-dependent manner, as represented by a significant alteration in cell morphology and increased adherence to culture plastic. In conclusion, S5a decides the fate of THP-1 cells to adapt to the environment as follows:THP-1 cells treated with S5a struggle against environmental factors in the first 48 h, cells show resistance to apoptotic stimuli and maintain levels of differentiation during this time.PartⅢ. The interaction of DR6-S5a and the possible mechanisms of its induction on THP-1 cells differentiation.Plasma membrane proteins of THP-1 cells were isolated and the MBP-S5a was expressed as soluble and purified using amylose resin (NEB) according to the manufacturer's instruction. Pulldown experiments were performed to identify proteins associated with MBP-S5a. Among the identified proteins, death receptor-6 (DR6) is a classical membrane protein. Recent studies reported that interactions between beta amyloid former protein (APP) and DR6 could activate caspase enzyme, contributing to caspase-mediated nerve cell apoptosis. However, this claim has not yet been verified in other tissues and cells. There have been no studies reporting typical ligands for DR6, and therefore DR6 remains as an orphan TNF receptor superfamily member. Pulldown-Western blot analyses were performed to confirm the presence of DR6 identified by MS. S5a:DR6 interactions were further tested by far-Western method. The result showed that the 90 kDa and 110 kDa forms of DR6 were consistent with the previous result. The distribution of S5a and DR6 on intact THP-1 membrane was visualized by immunofluorescence confocal microscopy. Western blot analysis revealed that recombinant S5a activated the NF-kB pathway. Our findings provide evidence of interactions between S5a and DR6. We concluded that S5a triggered NF-kB pathways induced by DR6 activation, and contributed to the differentiation of THP-1 cells.As a conclusion, These experimental data fully demonstrate the interactions between S5a and DR6, and assist in identifying ligands of orphan receptors, such as DR6. We conclude S5a induces DR6 activation thereby triggering NF-kB pathways, contributing to the differentiation of THP-1 cells. More importantly, our research provides a novel target that can induce the differentiation of cancer cells especially for biotherapy of leukemia.