Abstract
In recent years, the development of anticancer peptides attracts more and more attentions and expectations. Anticancer peptides are suggested to have the selective ability to kill cancer cells and minor side effects on normal cells. However, the use of peptides in clinical treatments is still challenging. For example, unmet needs include reducing the side effects of AMP on normal cells and preventing it from proteolysis during transport in human blood. With our efforts, we screened out the peptide 2163 with antimicrobial activity from the genome of Lactobacillus casei strain ATCC 334, and we proved that it can kill the human colon adenocarcinoma cell SW480. In order to optimize the activity to kill cancer cells, we modified the sequence of peptide 2163 of hydrophobic moments and alpha-helix ratios, resulting in the novel peptide KL15. In this study, we applied the CCK8 assay to evaluate cell viability. In 16 hours treatment, the cell-killing activity of KL15 was significantly improved because of the sequence modifications. According to our observations, the peptide 2163 and KL15 have different cell-killing characteristics. KL15 induced cell membrane disruptions in a shorter time compared with peptide 2163. Through conjugating FITC to KL15, we detected the fluorescent signals by confocal microscopy and found that KL15 can enter the cancer cell SW480 in 10 minutes. Also, the survival rate of SW480 may be reduced by 35% in 2 hours indicated by CCK8 assay, and this effect cannot be rescued by treating the caspase inhibitor Z-VAD-FMK. Furthermore, we investigated the cell-death related protein HMGB1 and PARP1 by western blot. HMGB1, the marker for cell necrosis, can be detected in cell culture media in 6 hours. PARP1, the marker for cell apoptosis, didn't show cleavage in the cells. These results show that KL15 rapidly disrupts cell membranes and induces cell necrosis. We infer that KL15 might cause cell membrane disruptions and induce cell necrosis before being proteolysis by proteases in human blood for clinical use.