Screening Of Anticancer Peptides Based On Targeting Skp2 Protein

Objective:Malignant tumors are serious diseases endangering human health. Until now,the commonly used anti-tumor drugs in clinical are of poorly efficacy and toxic side effects. Compared with small-molecule drugs, the anti-tumor peptide and protein drugs have their unique advantages for their high specificity which can specific silence the effects and functions of target protein with light side effects and low research costs. Skp2 is a protein kinase involved in cell cycle through the ubiquitin degradation of varieties of target proteins. The dysfunction of Skp2 is closely associated with the tumor genesis, development and prognosis. So in this study, we intended to screen a peptide targeted to Skp2 from an already constructed human-derived peptide aptamers library with the yeast two hybrid system and find a potential effective anti-tumor peptide drug.Methods and Results:1. The screen of peptide aptamers pool. The LRR region(208aa-390aa) of human Skp2 was used as a bite protein to search its Peptide aptamers. With the yeast two hybrid system, there were found almost 100 peptide aptamers specific binding to skp2. Then the plasmids are extracted and 11 correct coding peptides were identified to carry out the following experiments.2. Identify the anti-tumor effect of the peptide aptamers. The mRNA transcription and protein translation levels of the peptide aptamers were detected by quantitative PCR and western blot in different cancer cells, such as gastric cancer(AGS, MKN28, MKN45, SGC7901),glioma(H4, SH-SY5 Y, SK-N-SH,U251),breast cancer(MCF-7, MDA-MB-231) and lung cancer(HBE, H23, H1299,A549, H292, SPC-A1). The cell lines which expressed a high level of Skp2 with a low level of substrate were chose to evaluate the anti-tumor function. According to the results, the lung cancer cell line A549 was confirmed to meet the requirement and used in the following experiments.3. Identification the interaction between Skp2 and peptide aptamers. The peptide aptamers selected from the yeast two hybrid system were cloned into the expression vector with His-tag and next purified with Nickel-affinity chromatography column. The GST-tag labeled Skp2 expression system was also constructed and purified. Then the interaction between peptide aptamers and Skp2 was determined in vitro by GST pull-down experiment.4. Identify the anti-tumor function of the peptide aptamers. The peptide aptamers were constructed with Eukaryotic expression vectors and transfected into cancer cells. To detect the effect of peptide aptamers on tumor cells proliferation after transfection, the CCK8 assay was involved to detect the survival rate and clone formation experiment was used to determine the non-anchored cellular proliferation ability. To find out the mechanism that the aptamers affect cell cycle and apoptosis of cancer cells, the flow cytometry assay was involved. By the last, the changes of substrates were detected by western blot. The results showed that 3 of the 11 peptide aptamers(6-1,8-1,23-5) could inhibit the degradation of p27 or p21 which is known as the substrate of skp2 and also confirmed to effectively inhibit the growth of tumor cells in the function experiments.Conclusions:The LRR region of human Skp2 was used as a bite protein to screen the Peptide aptamers pool with the yeast two hybrid system. Among all the peptides, three of them(6-1,8-1,23-5) were confirmed to inhibit the degradation of Skp2 substrate p27 or p21 and showed an obviously anti-tumor effect. Thus, the 3 peptides(6-1,8-1,23-5) studied in our job might represent some potential clue for new anti-tumor drugs research.