Effects Of HP-10 On Proliferation Of Human Colon Adenocarcinoma LOVO Cells In Vitro And In Vivo

nuclear factor-κB (NF-κB) is an importent transcription facter, which play a crucial role in the control of inflamation, ageing, cancer and other physiological and pathological progresses. It is constitutively activated in most human cancers ,and the inhibitor of NF-κB can at least partly prevent cancer growth and metastasis. So the anti-tumor strategy antogonizing NF-κB activity became the centre researchers concerned. We have got p65 cell-penetrating-binding peptides through coupling the cell-penetrating peptide with some polypeptides which was screened out by yeast two-hybrid technology and bioinformatic technique and can competitively bind with DNA domain of NF-κB p65 subunit. Finaly, we reconstructed and got the hexa-peptide HP-10. In this study, we investigated the effect of HP-10 on proliferation of human colon adenocarcinoma LoVo cells and the penetration ability of HP-10 into LoVo cells in vitro and in vivo.Major research contents and results1. The invitro experiments of HP-10 on human colon adenocarcinoma LoVo cells. MTT assay was used to test the effect of HP-10 on proliferation of LoVo cells at 75μmol/L,37.5μmol/L,18.75μmol/L. The repeated results showed that HP-10 can markedly inhibit proliferation of LoVo cells in a dose-dependent manner(P<0.05).Confocal laser fuuorescence microscopy was used to observe the penetration ability of HP-10 into LoVo cells, after coculturing LoVo cell with FITC labeled HP-10 2 hours in vitro. The results demonstrated that HP-10 can penetrate into cell after coculture 2 hours.Furtherly, we test cell cycle and apoptosis of LoVo cell with flow cytometry advancely. We found HP-10 can not affect cell cycle, but can induce cell apoptosis.2. The invivo experiments of HP-10 in colon adenocarcinoma LoVo cells bearing nude mice.The model of colon cancer of nude mice was established with implantation of LoVo cells. The therapeutic effect of HP-10 was observed at 750μmol/L , 375μmol/L , 75μmol/L, after intratumor injection of HP-10 for 14 days, once every other day. HP-10 can signifciantly prevent the growth of tumor than PBS control and untreated control, but no obvious dosage-effect relation(P<0.05).confocal laser fuuorescence microscopy was used to observe the penetration ability of HP-10 into colon adenocarcinoma tumor cells, after intratumor injection of FITC labeled HP-10 2 hours in vivo. It was demonstrated that HP-10 can transduce into tumor cells in vivo.The DNA-binding activity of NF-κB p65 was measured using an enzyme-linked immunosorbent assay (ELISA) to observe the effect of HP-10 on NF-κB activity in tumor cell nucleus. The results showed that HP-10 can inhibit the NF-κB activity in tumor cell nucleus(P<0.05).Terminal deoxynucleotidyl transferase–mediated nick end labeling staining of the tumor sections was conducted to determine the effect of HP-10 on the apoptosis of tumor cells in vivo. Compared with PBS control, HP-10 markly enhanced the apoptosis of tumor cells .Western blot was used to test the expression changes of Bcl-2, Bax, pro-Caspase 9, pro-Caspase 8 and pro-Caspase 3 which all are the improtant apoptosis related proteins. The results revealed that the expression of pro-Caspase 9, pro-Caspase 8, pro-Caspase 3 and Bax were reduced,whereas Bcl-2 increased. It was supposed that HP-10 can induced apoptosis of tumor cells through extrinsic and intrinsic pathways simultaneously.ConclusionHP-10 is an oligopeptide able to directly transduce into cells without the coupling cell-penetrating peptide. It can inhibit the growth of LoVo cells in vitro and in vivo through inducing cell apoptosis,which at least partly induced by the reduction of NF-κB activity.