C7 Peptide Inhibits Hepatocellular Carcinoma Metastasis By Targeting The HGF/c-Met Signaling Pathway

[Backgrounds] Hepatocellular carcinoma(HCC),characterized by a high rate of metastasis and recurrence after surgery,is caused by malignant proliferation of hepatocytes with epigenetic and/or genetic mutations.Studies have found abnormal expression of c-mesenchymal-epithelial transforming receptor(c-Met)in liver cancer.In particular,abnormal activation of the hepatocyte growth factor(HGF)/ c-Met axis is closely associated with HCC metastasis.At present,effective treatments or drugs that target the HGF/c-Met signaling pathway are still in the research pipeline.Such as Rilotumumab that is the first monoclonal antibody that selectively targets HGF,and tyrosine kinase inhibitors(TKIs)targeting c-Met(i.e.Cabozantinib).However,the effects of these drugs in clinical trials are not ideal,and there are also many side effects.Therefore,the development of new targeted drugs and rational combination therapeutic strategies are necessary to maintain a pool of effective treatment at all times.At present,more and more drug researches are beginning to turn to targeting molecular peptides,because targeting peptides are characterized by biomolecules with lower molecular weights,specific sequences and good organizational penetration abilities,emerging as attractive or important therapeutic agents.To screen for peptide sequences that specifically bind to c-Met,we screened the T7 select Human Liver Tumor c DNA Library and the Ph.D-C7 CTM phage display peptide library in our laboratory.After four rounds of screening,the results showed that M13 phage was significantly enriched.The phage peptide sequence was analyzed by sequencing the phage plasmid ss DNA and named C7 peptide.Does the C7 peptide actually bind specifically to c-Met and block HGF/c-Met signaling to inhibitor cancer development? We conducted a series of experiments to test this hypothesis.The mechanism of possible existence was verified by Western blot.At the same time,the anti-cancer effect of C7 peptide was demonstrated in vivo,which provided new insight into the mechanism of HCC and future clinical treatments.[Research methods] 1.Exploring the appropriate concentration of the C7 peptide,HGF and EGF using Hep G2 cell line.2.Scatter assay was used to detect the effects of the C7 peptide on cell scatter induced by HGF and EGF.3.MTT assay was used to detect the growth effects of C7 peptide,HGF and EGF in HCC and vascular endothelial cells.4.Scratch and migration assays were used to detecte the effects of the C7 peptide on cell migration induced by HGF and EGF.5.Transwell assay was used to detect the effects of the C7 peptide on cell invasion induced by HGF and EGF.6.The effects of the C7 peptide on phosphorylation of c-Met,Akt and Erk induced by HGF and EGF were detected by Western blot.7.The effects of the C7 peptide on the metastasis of HCCLM3 cell line in vivo was examined by liver orthotopic xenograft models.8.H&E test detected the effect of C7 peptide on the size and number of lung metastasis nodules in nude mice.9.Detecting the effects of C7 peptide on the expression of Met,p-Met,Erk and p-Erk in hepatic tumors by IHC.[Experiment results] 1.The appropriate concentration of the C7 peptide,HGF and EGF was 100 ?g/ml,20 ng/ml and 10 ng/ml respectively.2.The C7 peptide inhibited HGF-induced cell scatter but can not inhibit EGF-induced cell scatter.3.HGF does not promote the proliferation in hepatoma cell lines,and the C7 peptide can inhibit HGF-induced proliferation in vascular endothelial cells.4.The C7 peptide can inhibit HGF-induced cell migration but can not inhibit EGF-induced cell migration.5.The C7 peptide can inhibit HGF-induced cell invasion but did not inhibit EGF-induced cell invasion.6.HGF stimulates phosphorylation of c-Met receptors,whereas EGF does not phosphorylate c-Met receptors.However,the C7 peptide was able to inhibit HGF-induced c-Met receptor phosphorylation.Both HGF and EGF can phosphorylate Akt and Erk1/2 proteins,however the C7 peptide only inhibited HGF-induced downstream protein phosphorylation.7.The lung metastasis nodules in the C7 group were significantly lower than that in control group.8.H&E results showed that the number and size of tumor nodules in the C7 group were lower than that in control group.9.IHC staining of liver tumor showed that phospho-Met,phospho-Erk level in C7 group were significantly lower than that in control group.[Conclusion] C7 peptide inhibits metastasis of hepatocellular carcinoma by targeting the HGF/c-Met signaling pathway.