Inhibitory Effects And Preliminary Mechanisms Of Peptide 9R-P201 To Hepatocellular Carcinoma HepG2 Cells

Cancer is a fatal disease to human health,and the search for more effective targets associated with cancer and the development of anti-cancer drugs are urgently needed.FoxMl was an activating transcription factor and was overexpressed in various cancer cells which was closely related to the proliferation,metastasis,chemo-resistance and poor prognosis.FoxMl was regarded as a potential target for anti-cancer drug discovery.We previously obtained one high affinity peptide,namely P201,from the phage random library against the DNA binding domain of FoxM1c?FoxM1c-DBD?protein.Here in this study,we used the modified peptide 9R-P201 as the anti-cancer drug and take the HepG2 cells as the main model to elucidate the anti-cancer effects and its preliminary mechanisms.By cell viability,we found that 9R-P201 showed stronger inhibition to HepG2 cancer cells than those of DU 145,HUVEC,L-02,HL-7702 and mouse spleen cells with an IC₅₀ of 43.6 ?g/mL?13.1 ?M?in a dose-dependent manner.The peptide was highly effective to liver cancer cells with an IC₅₀ for L-02 and HL-7702 cells of 2855.9,93.5 ?g/mL,respectively.Subsequently,HepG2 cells were taken for further investigation of molecular mechanisms.We confirmed that 9R-P201 aggregated in the cell nucleus and the expression of FoxM1 was significantly down-regulated at both transcriptional and translational levels in HepG2 cells.Then,by colony formation,scratch wound migration and Transwell migration assays,we revealed that the proliferation and migration of HepG2 cells were significantly inhibited by 9R-P201.The results were further confirmed by qRT-PCR and Western blot assays:9R-P201 could significantly down-regulate the expression of matrix metalloproteinase?mmp2,mmp9?,Vimentin and vegf,leading to the suppression of proliferation,migration and angiogenesis of HepG2 cells.Moreover,by AO-EB staining assay,we found that 9R-P201 could induce apoptosis in HepG2 cells,which was further supported by qRT-PCR and Western blot assays:the apoptosis induction was associated with the up-regulated expression of p53,caspases,and down-regulated expression of E-cadherin,bcl-2/bax and Surviving.In addition,the nude mice HepG2 cells xenograft models were established to evaluate the effect of 9R-P201 on tumors in vivo.The results revealed that tumor growth in mice treated with 9R-P201 or 5-Fu was significantly suppressed compared with that of the blank and vehicle control?p<0.001?,while no statistical significance was found between 9R-P201 and 5-Fu treated mice?p>0.05?.Notably,the mRNA expression of foxM1 was markedly down-regulated by 9R-P201 as well.The results were further supported both in histopathological HE staining and protein immunohistochemistry with the significant down-regulation of Ki67 and up-regulation of cleaved Caspase3 expression in tumors.All the results indicate that 9R-P201 could suppress the growth and induce cell death of HepG2 xenograft tumors in nude mice.In conclusion,our findings suggested that 9R-P201 could strongly and effectively inhibit the viability,proliferation,migration and invasion of liver cancer HepG2 cells and induce apoptosis by down-regulation of FoxMl and regulation of related gene expression in signal transduction passways.Thus,9R-P201 holds great potential as a lead anti-cancer drug directly targeting FoxM1.