Protocols for studying antimicrobial peptides (AMPs) as anticancer agents

Antimicrobial peptides (AMPs) are a class of small cationic peptides that are important for host defense. In a manner that is similar to AMP-mediated destruction of microbial pathogens, certain AMPs can physically associate with the anionic lipid membrane components of cancer cells, resulting in destabilization of the lipid membrane and subsequent peptide binding to intracellular targets, which ultimately leads to the death of the cancer cell. In comparison, normal healthy cells possess a neutral membrane charge and are therefore less affected by AMPs. Based on the selective cytotoxicity of certain AMPs for cancer cells, these peptides represent a potential reservoir of novel anticancer therapeutic agents. The development and improvement of AMPs as anticancer agents requires appropriate methods for determining the effects of these peptides on the viability and function of cancer cells. In this chapter, we describe methods to assess the ability of AMPs to cause cell membrane damage (measured by propidium iodide uptake), apoptosis and/or necrosis (measured by annexin V-FLUOS/propidium iodide staining), and mitochondrial membrane destabilization (measured by 3,3′-dihexyloxacarbocyanine iodide staining), as well as reduced motility (measured by a migration and invasion assay) of cancer cells growing in suspension or as monolayers. We also describe a tubule-forming assay that can be used to assess the effect of AMPs on angiogenesis.