A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

Abstract

The high toxicity of most chemotherapeutic drugs and their inactivation by multidrug resistance phenotypes motivated extensive search for drugs with new modes of action. We designed a short cationic diastereomeric peptide composed of D-and L-leucines, lysines, and arginines that has selective toxicity toward cancer cells and significantly inhibits lung metastasis formation in mice (86%) with no detectable side effects. Its ability to depolarize the transmembrane potential of cancer cells at the same rate (within minutes) and concentration (3 M), at which it shows biological activity, suggests a killing mechanism that involves plasma membrane perturbation. Confocal microscopy experiments verified that the cells died as a result of acute injury, swelling, and bursting, suggesting necrosis. Biosensor binding experiments and attenuated total reflectance-Fourier transform infrared spectroscopy using model membranes have substantiated its high selectivity toward cancer cells. Although this is an initial study that looked at tumor formation rather than the ability of the peptides to reduce established tumors, the simple sequence of the peptide, its high solubility, substantial resistance to degradation, and inactivation by serum components might make it a good candidate for future anticancer treatment.Current anticancer chemotherapies that are based on alkylating agents, antimetabolites, and natural products respond incompletely; this is probably related to the development of drug resistance. Most chemotherapeutic agents also affect normal cells and consequently cause severe side effects (1). Thus, there is an urgent need to develop new classes of anticancer drugs with new modes of action that selectively target the cancer cells. A promising group under investigation includes cationic antimicrobial peptides, which are known to play an important role in the innate immunity of a diverse range of organisms, including insects, amphibians, and mammals (2). Most of these peptides have an amphipathic structure, and they preferentially bind and insert into negatively charged cell membranes. Consequently, destabilization of the membrane disturbs the electrolyte balance and induces the intracellular contents to leak, leading to cell death. In contrast to normal eukaryotic cells, which generally have low membrane potentials and whose outer leaflet almost exclusively consists of zwitterionic phospholipids, the prokaryotic and cancer cell membranes maintain large transmembrane potentials and have a higher content of anionic phospholipids on their outer leaflet. Many antimicrobial peptides therefore preferentially disrupt prokaryotic and cancer cell membranes rather than eukaryotic membranes (3).Several in vitro studies were conducted with native all L-amino acid antimicrobial peptides with defined ␣-helical or ␤-sheet secondary structures (3). However, the use of native antimicrobial peptides in vivo is mainly limited due to the loss of their function in serum, partially because of enzymatic degradation and binding to serum compone...

Suppression of Human Prostate Tumor Growth in Mice by a Cytolytic d-, l-Amino Acid Peptide

“…This binding is governed mainly by electrostatic interactions (45). Membrane binding forces the peptide to adopt a functional structure, which allows destabilization of the membrane (31). In agreement with this, many cancer cells have approximately (46)] they create the additional negative charge.…”

“…A plausible explanation for this selective activity could be as follows: A common feature found in amphipathic-L and all of the other noncellselective peptides is their stable amphipathic ␣-helical structure in membranes as well as their similar affinity to negatively charged and zwitterionic membranes. Structural studies revealed that both amphipathic-L and amphipathic-D have unordered structures in solution (31). However, whereas amphipathic-L strongly binds and becomes fully helical in any type of membrane, amphipathic-D binds ϳ15-fold better negatively charged membranes compared with zwitterionic membranes (45).…”

“…The following day, the media were replaced with 90 l of fresh media and 10 l of a solution containing different concentrations of the peptides. The plate was then incubated for 24 h before adding to each well 50 l of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) reaction solution (Biological Industries); viability was determined as described previously (31). The LC 50 (concentration at which 50% of the cells die) for each peptide was obtained from the dose-dependent cell viability curves.…”

“…35), using a ABI 433A automatic peptide synthesizer, and purified as described previously in detail (31). Labeling of the NH 2 terminus of the peptides with rhodamine was done on the resin-bound peptide (36).…”

“…Most importantly, this family of peptides preserved their activity in serum, and their enzymatic degradation could be controlled. One peptide, a 15-amino acids diastereomer composed of leucines, arginines, and lysines, was shown recently to act against the mouse melanoma and lung carcinoma cell lines and to significantly inhibit lung metastasis in mice with no detectable side effects (31). Here, we show that a 15-mer all L-amino acids lytic peptide and its diastereomer, both of which are composed of only lysines and leucines, are highly active preferentially toward both androgen-dependent and androgen-independent human prostate carcinoma cells.…”