Design And Synthesis Of ERC(N~5) Peptide And Its Effect On The Proliferation And Invasion Behaviors Of Human Hepatocellular Carcinoma Cell Line HepG2

Objectives1. To design and synthesize RGD peptide ERC(N~5).2. To analyze the binding condition of ERC(N~5) and hepatocellular cancer cell line HepG2.3. To investigate the effects of ERC(N~5) suppression on the proliferation and invasionbehaviors of HepG2.Methods1. Design and synthesis of ERC(N~5) peptideThe RGD motif and C-terminal amino acid sequences of Echistatin (Ech) were conjugatedtogether to present a small peptide designated as ERC(N~5). The sequence of ERC(N~5) isARGDNMPRNPHKGPAT. It was synthesized by CL.(Xi'an)Bio-Scientific CO.LTD.2. Analysis of binding condition between ERC(N~5) and HepG2The expression of integrinαvβ3 on cell surface was detected by flow cytometry. Threekinds of cell lines were investigated including HepG2, Hela and SW620. The content ofαvβ3were also detected under conditions of paraformaldehyde fixation or not and typsin or EDTAdigestion. The mean fluorescence intensity and positively staining cells were examined afterERC(N~5) or Ech, FITC-conjugatedαvβ3 monoclonal antibody (LM609) and HepG2 wereincubated together. They were used to analyze the competitive binding of ERC(N~5)/Ech andLM609.3. Effects of ERC(N~5) on proliferation and invasion behaviors of HepG2CCK-8 method was used to draw cell growth curve. The curve was used to analyzeproliferation effects of ERC(N~5). The effects of ERC(N~5) on the adhesion were investigated byCCK-8 assay. The effects of ERC(N~5) on migration and invasion behaviors of HepG2 wereobserved by Transwell migration assay, wound healing assay and Matrigel invasion assay; Theapoptotic effect of ERC(N~5) on HepG2 was examined by Annexin V-FITC/PI double stainingmethod combined with fiow cytometry. Cell morpHology change was observed by microscope.Results1. The amino acid sequence is correct after analyzed by mass spectrum. The purity of ERC(N~5)was more than 95% detected by HPLC chromatography. 2. Theαvβ3 positively staining cells of HepG2 cell, Hela cell and SW620 cell were 47%, 35.4%and 5.8% separately.3. The mean fluorescence intensity and positively staining cells of paraformaldehyde treatedgroup were 2.23 and 4.6%, whereas 6.97 and 30.1% were observed in paraformaldehydeuntreated group. Theαvβ3 positively staining cells in tryspin digested group was 3.6%, whichwas much lower than EDTA group with 47% positive cells.4. The positively staining cells of FITC-LM609 treated group was 57.6%, which was muchhigher than FITC-LM609 and ERC(N~5) treated group. The positively staining cells decreasedwith the increasing concentration of ERC(N~5). At the concentration of 64μM, the positivelystaining cells were 0.3% and 30.1%. This indicates that ERC(N~5) could bind toαvβ3 strongerthan Ech.5. CCK-8 assay results showed that the OD values decreased with the increasing concentrationof ERC(N~5). All the dates were significant in statistical analysis(P<0.05).The OD values wereincreased along with the elongation of culture time when ERC(N~5) was 16μM, but the valueswere lower than the control group.6. The results of FCM showed that the apoptosis percentage increased 1-fold after HepG2 cellwere treated by ERC(N~5).7. The cell adhesion ratio of 16μM ERC(N~5) was decreased by 46.2% on fibronectin.8. The migration and invasion ability of HepG2 were decreased by 76.4% and 79.2%respectively. Wound healing assay showed that the negative control cells moved rapidly andfilled the denuded surface after 24 h,while ERC(N~5)-treated cells could not.ConclusionConclusions1. Human Hepatocellular carcinoma HepG2 cell could express integrinαvβ3, ERC(N~5) couldbind to HepG2 cell through competitive binding toαvβ3 with LM609.2. ERC(N~5) could suppress HepG2 cell proliferation and induce apoptosis. It could inhibitHepG2 cell adhesion to ECM, as well as tumor cell migration and invasion .