Sceening Of High-affinity SAPRIL-binding Peptides And Their In Vitro And In Vivo Inhibitory Effects On Proliferation In Colorectal Cancer Cells

A proliferation-inducing ligand(APRIL) is a TNF-like cytokine modulate a variety of biological processes,including initiation of proliferation, prolonged cellular survival and accelerated tumor formation.therefore,try to effective inhibition of APRIL functional activity,has the potential to provide a novel treatment strategy for high expression of APRIL associated tumors.APRIL is synthesized as a 32kDa typeⅡtransmembrane protein which is cleaved by furin in the Golgi to release a 17 kDa soluble molecule.Phage display describes a selection technique in which a peptide or protein is expressed as a fusion with a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the virion, while the DNA encoding the fusion resides within the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called panning.Our studies is founded on cloning and expression of human soluble APRIL. Proposed to selection and identification of high affinity APRIL binding peptides from phage random 12-mer peptide libraray. The peptide sequences were deduced from DNA sequences.then Synthesized the peptides. and biologic activity of the peptide was texted by using ELISA.Then to explore the inhibitory effect of APRIL-binding peptide on colotectal cancer or in vivo by observing the growth and the growth inhibition ratio of tumor.Materials and MethodsReagentsAPRIL, PhD Phage Display Peptide Library Kit, HRP-M13 antibody, Monoclonal antibody to APRIL(sacha-2).Phage DisplayPhage libraries were amplified, purified, and titered according to the manufacturer's protocols. First, we used a direct screening approach and biopanned APRIL for 4 rounds. APRIL were coated into 96-well plates and incubated overnight at 4℃until 2 days after confluence, washed plate rapidly 6 times with TBST(TBS+Tween-20). then dilute 4×1010(10ul of original library) with 100ul of TBST, pipet onto coated plate and rock gently for confluence.discard nonbinding phage then wash plates 6 times with TBST.Weakly associated phages were eluted in 100ul 0.2 mol/L glycine (pH 2.2) for 10 minutes at room temperature, followed by neutralization with 200 mL Tris-HCl (pH 8.0). High-affinity phages (tightly bound phages) were isolated.Phages were amplified and titered between each round to ensure that 109pfu of input phages was used at the start of each successive round. Carry out a second,tird,forth round of panning repeat above steps using.After 4 rounds of biopanning to phage random 12-mer peptide library,phage clones binding to APRIL were identified.They were selected and amplified.The binding activity measureed by ELISA of these phage clone were performed. The DNA of these phage clones were extracted for sequencing using-96 primer as described in manual. The peptide sequences were deduced from DNA sequences.Those peptides were analyesd by bioinformatics.Through Blast,the peptide sequences were compared for homology analysis.The peptide was synthesized by the solid-phage method,and biologic activity of the peptide was texted by using ELISA.Cell proliferation assaysthe cells were plates in 96-well plates,cells were treated with APRIL binding phage or peptid for variouse concentration and various time periods. The single-cell suspension were seeded at concentrations of 2×103 per well in RPMI 1640 medium per well in 96-well plates. After drugtreatment, Add 20μl of the CCK-8 solution to each well, attached cells were incubated with cell counting kit-8(CCK-8) according to the manufacturer's instructions. The absorbency at 450nm was then the amount of light with spectrophotometer at the different time. repeat trials 3 times.Analysis of Apoptosis and Cell cycleAdjust LOVO cell concentration to 1×105/ml, treated with 20nm or 40nm of APRIL-binding peptides, Control Group without peptides. Flow cytometry uses the principles of light excitation, The wavelength was 488 nm. used to detect in any phase of the LOVO cell cycle and apoptosis. Apoptosis was measured simultaneously by staining cells with Annexin-V-PE and 7AAD according to the manufacturer's protocol.Western Blot AnalysisTotal protein extraction from cultured cells, determination of protein concentration with BCA, the preparation of 10% separating gel,5% of the laminated plastic, line SDS PAGE. take samples:M 4ul,SW620 10ul. LOVO 10ul,HCT116 8ul,HT-298ul,SW480 6ul, constant voltage 80V 50min, then the gel was run at 120v constant voltage for at least 1 hour and 20 min until the tracking dye (bromophenol blue) reaches bottom of the gel. electrophoresis after the use of semi-dry transfer apparatus to transfer constant 60 min,5% nonfat dry milk The TTBS at room temperature closed 1 h, respectively, APRIL mouse monoclonal antibody working concentration 1:1000, HRP-labelled Rabbit Anti-Rat IgG working concentration 1:5000 overnight reaction,at last add chromogenic substrate.Tumor cell xenotransplantationHuman colorectal cancer LOVO cells were inoculated into the lower flanks hypodermis of NCr nu/nu mice at a dose of 2×107/ml viable tumor cells.We established the nude mice model with colorectal cancer successfully.The nude mice with colorectal cancer were randomly divided into three groups,Low concentration group,High concentration group and control group.Every group had 5 nude mice.The number of nude mice model with colorectal cancer was 15. APRIL-binding peptide 2000nm (100ul), APRIL-binding peptide 4000nm(100ul) and PBS(100ul) were injected into the tumor directly,respectively.Those drugs were injected every two days and seven times totally. Mouse tumor growth was measured with digital caliper and calculated by using the formula,where V is volume, is the longest tumor axis,and is the perpendicular shorter tumor axis.The longest and shortest diameter was measured every two days befor injection. Nude mice were executed by breaking their cervical vertebra after therapy one weeks.Tumor was separated and weighted.The growth inhibition ratio of tumor was calculated.ResultsSelected peptides that bind to APRIL from phage peptide libraryUsing APRIL as target,4 rounds of biopanning were performed as described in methods.The bound phages were eluted by the glycine solution.The phage clones that display APRIL binding peptide were concentrated from the NEB Ph.D.-12TM phage-displayed peptide library.After four rounds of biopannung,the eluted phage were characterized by ELISA and DNA sequencing.20 eluted clones were examined and most of them could bind APRIL specifically,and amino acid sequence of random peptide of 8 positive clones were identified. The core DNA sequence of 12-mer peptide library is A A A P L A Q P H M W A, S S T T T S D K Y L S A, SNLHDN N T E K N V.The peptide sequences were deduced from DNA sequences from these phage clones,we found the core peptide sequence of 8 phage clones was A A A P L A Q P H M W A.then Synthesized the peptides.The synthesized peptide purity exceed 99%,MWt assayed by mass-spectrum is 1263.5.Effect of APRIL binding peptide on proliferation and apoptosis of relatively colorectal carcinoma cellsWe investigated the degree of expressions of APRIL in relatively colorectal carcinoma cell. After the Western-Blot we selected relatively high expression LOVO cell and low expression SW620 cell in vitro experiments, For CCK-8 assay,the cells were plates in 96-well plates,cells were treated with APRIL binding phage or peptid for variouse concentration and various time periods. phage or peptide inhibited the proliferation of LOVO cell After 48h and 72h(P<0.05). and SW620 cells no significant inhibition.The percentage of LOVO cells in G0/G1 phase of the cell cycle after exposed to 20,40nm APRIL-bingding peptide versus untreated were significant decrease(P<0.05) after 24h and 48h.in G2/M phase of the cell cycle were increase(P<0.05). APRIL binding peptide increased the percentage of G0/G1 phase and decreased G2/M phase of LOVO cells.The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death,compared with the control group,after exposed to 20,40nm peptide for 24h and 48h,LOVO cells no significant decrease(P=0.059).APRIL binding peptide decreased proliferation and tumorigenicity in vivoAPRIL-binding peptide was injected into nude mice with tumor,which were induced by colorectal cancer LOVO cell.The LOVO cell of the growth of tumor was two weeks.The average volumes was about 110.34 mm3 in the fourteenth day.The percentage of forming tumor was 100%.No animals died after four weeks.There was significant after the eighth day between APRIL-binding peptid group versus control group(D8:F=6.608, P=0.012; D10:F=6.571, P=0.012; D12:F=8.469, P=0.005; D14: F=21.24,P=0.000).ConclusionThe selected peptides that bind to APRIL from NEB Ph.D.-12TM phage-displayed peptide library were screened by using APRIL as a ligand.After four rounds of biopanning,the core amino acid sequence of random peptide was A A A P L A Q P H M W A.APRIL binding peptide could inhibit the LOVO cells significantly in proliferation.The cell cycle also support these poimts. But apoptosis have no signifcant change.The peptide also could inhibit the growth of tumor in vivo.The results show that peptides binding APRIL could be selected from phage-displayed peptide library. It provide new ideas of direct-evolution for understanding APRIL relatively tumor and may fimd new treatment fo colorectal carcinoma.