Investigation of possible effects of a new antimicrobial peptide on A549 cell line

Antimicrobial peptides (AMP) are molecules that play important roles in the innate immune system from insects to mammals. It is known that these peptides regulate the immune system in inflammation and infections, neutralize lipopolysaccharides, and are effective against gram positive and gram negative bacteria by regulating adaptive immune responses. Lung cancer has a prevalence of 14% worldwide and has two subtypes as small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). In recent years, antimicrobial peptides have gained attention as potential anticancer drugs. The aim of this study was to investigate the possible anticancer effects of cryptonin in A549 cells. This AMP is firstly defined in hemolymph of Cicada, Cryptotympana dubia and has 24 amino acids long, 50% hydrophobicity and +8 net charge. This peptide is also effective on gram-negative and gram-positive bacteria and has antibacterial activity. In order to determine the anticancer activity of cryptonin, A549 cells grown in cell culture were exposed to this AMP at different concentrations. Cell viability, LDH release, ROS levels and morphological changes after peptide treatment were investigated. Morphological changes were examined by inverted microscope and acridine orange/ethidium bromide staining method. Cryptonin reduced cell viability and incresead LDH release in dose dependent manner. Cryptonin also rised the reactive oxygen species in high doses and led swollen cell morphology. Increased number of necrotic cells having nuclei stained with ethidium bromide was observed parallel to increasing peptide concentration for 24h. Western blot analysis used to detect the expression of Caspase-3, Bax, Atg5, Beclin1, LC3B, HMGB1 in A549 cells revealed that all these proteins were dose-dependently decreased in A549 cells by cryptonin, and cell death occurred by necrosis, not apoptosis and autophagy. The possible synergistic effects with doxorubicin were also investigated. Doxorubicin administration for 24, 48 and 72 hours decreased cell viability in a dose-dependent manner. In addition, the combined application of 1, 2.5, 5 and 10 μg/ml cryptonin and 1μM doxorubicin for 48 hours caused a significant decrease in cell viability compared to doxorubicin alone, while the synergistic effect as a result of the combined application of 5μM doxorubicin and cryptonin at the same low doses for 48 hours was only observed at the combined application of 5μg/ml cryptonin and 5μM doxorubicin. With the administration of 1 μM doxoribosin for 48 hours, the level of active caspase 3 protein increased significantly compared to the control, while the level of LC3B protein decreased. The combined application of 1 μM doxorubicin and 5 μg/ml cryptonin significantly decreased caspase-3 levels and significantly increased LC3B levels compared to the application of 1 μM doxorubicin alone. Application of 5 μM doxorubicin and 5 μg/ml cryptonin significantly decreased caspase-3 and LC3B levels compared to the control and LC3B levels compared to the application of 5 μM doxorubicin. In conclusion, these findings showed that cryptonin has an anticarcinogenic effect on lung cancer cells and has the potential for chemotherapeutic effects, also showed that the combined application of cryptonin and doxorubicin was effective at lower doses by showing a synergistic anticancer effect.