The antimicrobial peptide cecropin A induces caspase-independent cell death in human promyelocytic leukemia cells
Introduction
Over the past decades, cationic peptides have been isolated and characterized from a wide variety of insects, amphibians and mammals, including humans. These peptides that possess antimicrobial activities are known to be important components of innate immunity and they constitute a primitive immune defense system in a broad range of species [6]. Nowadays, cationic peptides are considered as a potential alternative for the treatment of emerging drug-resistant infections. However, some of these peptides display a wide range of biological activities and at least three classes of antimicrobial peptides have been shown to have antitumoral activity [51]. Antimicrobial peptides act rapidly to combat the invasion of potential pathogens, and thus serve to limit the extent of infection prior to activation of the adaptive immune response. Cecropins constitute one of the most extensively studied antimicrobial polypeptides among the many that are synthesized by insects as components of their host defense systems against bacterial infection [19], [20], however, cecropin also exhibits cytotoxic effects for a number of tumor derived cell lines [31].
Cecropin A is a cationic 37-residue antimicrobial peptide composed entirely of ordinary l-amino acids [45], which was initially isolated from Hyalaphora cecropia pupae [5]. This antimicrobial peptide belongs to cecropin family, which was originally discovered to have a broad spectrum against protozoa and metazoan parasites in addition to fungi and bacteria [53]. Antibacterial activity of cecropins includes Escherichia coli, Pseudomonas aeruginosa, Bacillus megaterium or Micrococcus luteus [3]. These antimicrobial peptides have been identified in other insects (e.g., bactericidin, moricin, and sarcotoxin) as well as in pig intestines (cecropin P) and tunicates [6], which contribute to the innate immunity of the animal. They are largely alpha-helical and consist of an amphipathic N-terminal helix and a hydrophobic C-terminal helix and lack hemolytic activity [23], [38]. The mode of action for this class of peptides is based on the formation of pores or channels across the bacterial membrane causing permeabilization of this structure [43]. They develop ion-permeable channels subsequently resulting in cell depolarization, irreversible cytolysis and finally death [4].
However, differences between cell membrane organization and composition may modify membrane fluidity, and interfering with the membrane insertion of lytic peptides in eukaryotic cells [28]. Irrespective of the fundamental role as antimicrobial agents, several investigation have described to these peptides as natural cytolytic agents capable of destroying tumoral cells without exhibiting any cytotoxic and hemolytic effects for peripheral blood lymphocytes [9], [22] and erythrocytes [41], respectively. Nevertheless, cecropins showed selective cytotoxicity and antiproliferative activities in bladder cancer cells [47], leukemia cells [11], colon adenocarcinoma cells [31] and gastric cancer cells [10].
An important aspect to elucidate is the mechanism whereby some defense peptides exert cytotoxic effects on tumoral cells. The aim of the present study was to analyze the cytotoxic action of cecropin A as well as the potential role of this cationic peptide as apoptotic inducer on a promyelocytic HL-60 cell line. Therefore, data from this study may serve to consider the potential value of cecropin A as a chemotherapeutic option and may offer a new strategy for overcoming multi-drug-resistant proteins, which represent a major problem in cancer therapy.
Section snippets
Cell line, culture conditions and treatment
The human promyelocytic leukemia HL-60 cell line was obtained from American Type Culture Collection (ATCC, CCL-240) and cells were maintained in RPMI 1640 (PAA Laboratories GmbH, Austria) supplemented with 0.2% sodium bicarbonate (Sigma–Aldrich, St. Louis, MO, USA), 10% heat-inactivated fetal calf serum (FCS, PAA Laboratories GmbH), l-glutamine, penicillin 50 U/ml and 50 mg/ml streptomycin (PAA Laboratories GmbH). Cells were maintained under a fully humidified atmosphere of 95% room air and 5% CO2
Cecropin A induces cytotoxicity on leukemic cells
The induction of cytotoxicity or inhibition of cell viability in tumoral cells is an important property for the application of cationic peptides as chemotherapeutic agents. The MTT assay constitutes an important indicator of mitochondrial activity and it has been applied to the measurement of cell viability. Fig. 1A indicates that the reduction of cell viability after the treatment of cecropin A occurs in a dose-dependent manner. The loss of cell viability is maximal at final concentrations of
Discussion
Antimicrobial peptides isolated from insects, including melittin [39], [40], cecropin related peptides [24], [31] and the magainins [13], [14] have been shown to exhibit antitumoral activity for cells derived from mammalian tumors (reviewed in [21]), although the detailed signaling pathways involved in antitumoral activities are not completely established. Currently, there is a considerable interest in developing antitumoral agents with a new mode of action because of the acquisition of
Acknowledgements
This research was supported by Consejería de Innovación y Universidades, Junta de Andalucia, Spain (group PAI, CTS105). JCM receives a predoctoral fellowship from the University of Jaen. We would like to thank Ms Nieves de la Casa (Servicios Técnicos de Investigación, Universidad de Jaén) for her assistance with the confocal microscopy.
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Both the authors contributed equally to this work.