Effects Of The Terminal Amino Acids Of (AABB) Peptides On Its Antitumor Activity And Drug Resistance Research

The spread of cancer and its high fatality rate makes it a kind of aeipathia in human civilization development period. A lot of nature anticancer extractives and new type drugs modified and synthesized artificially were applied to carcinoma therapy after clinical validation in recent years, which bring hopes of human health life. While up to now, we can’t suppress its swift and violate momentum completely. Reports of tremendous side-effects produced by chemotherapeutics and emergence of drug-resistant strains were frequently heard. We are urgent to find a new drug which is efficient to kill cancer cells and keeps low toxicity to normal tissue while hard to cause drug resistance. A series of α-helical peptides G(IIKK)nI-NH2(n=1-4) were designed by our lab before. We confirmed successfully that both anticancer activities and toxicities to normal cells increase with the augment of n. G(IIKK)3I-NH2 is the best among them on account of its high anticancer activities and low toxicities to normal cells. Based on that result, we excogitate three new amphipathic cationic peptides by deleting its N- or C-terminal amino acid whose sequences were(IIKK)3I-NH2, G(IIKK)3-NH2,(IIKK)3-NH2, in order to investigate its anticancer mechanism and effects of the terminal amino acids on its antitumor activity. First of all we discover that these four peptides show random coil in water while change toα-helical in hydrophobic environment with the help of circular dichroism spectrometer. We choose cancer cells such as Hepg2, Hela, A375, B16, HL60 and normal cells such as HDFa, COS7, human red blood cells to test activities of these peptides by MTT assay. The results demonstrate that the antitumor ability and toxicity to normal cells of these peptides are in the order: G(IIKK)3I-NH2 ≈(IIKK)3I-NH2 >G(IIKK)3-NH2 >(IIKK)3-NH2, suggesting that isoleucine in C-terminal plays a more significant role in killing tumor cells than glycine in the N-terminal. Then we use Calcein-AM, DAPI and Rhodamine-Phalloidin to stain cells separately, which corroborate that adding drugs will lead to cytomembrane rupture, cytoplasm deconcentration, cytoskeleton collapse and deformation. For the sake of analysingpeptides location in cells, we synthesize FITC-G(IIKK)3I-NH2, Rhodamine-(IIKK)3-NH2. We conclude that both of them combine with membrane through electrostatic attraction and hydrophobic effect. There is no specific biding site. The discrepancy in anticancer activity depend on the pressure they make to membrane surface. At last we select nowadays widely-used DOX as a reference substance. We cultivate cells in an environment containing G(IIKK)3I-NH2 or DOX, hoping to screen a kind of cell strain which be able to endure DOX while don’t have drug resistance to helical peptides.