Molecular Mechanisms of the Cytotoxicity of an Anti-Cancer Peptide

The molecular mechanisms of anticancer peptides (ACPs) are largely unknown, which makes their rational design challenging. Here, we investigate the action of an ACP, the peptide NAF-144–67, to elucidate a detailed reaction sequence with cancer cells. NAF-144–67 is a 24-amino-acid ACP derived from the human transmembrane protein NAF-1, which resides in mitochondria and the ER. We investigate the specificity, targeting, and induction of cancer cell death by the peptide using imaging experiments, trypsin assays, and atomistic-level computer simulations. We investigate peptide interactions with and permeation into cancer cells, as well as large (LUVs) and giant unilamellar vesicles (GUVs). The experiments and simulations are conducted on the native peptide and several point mutations. We demonstrate a mechanism with the following sequential steps: 1. The ACPs cross the plasma membrane of cancer cells efficiently without damaging it. 2. Different ACPs target alternate organelles in the cancer cell. The native peptide and two-point mutations (F1W and Q23W) target the mitochondria. Another point mutation (L11W) targets the nucleus. 3. The ACPs initiate apoptosis by damaging the mitochondria. They perturb the mitochondrial membrane and a membrane-embedded protein, NAF-1. We illustrated the critical role of the interaction of the peptide with the protein NAF-1 by examining cells that underexpress or overexpress NAF-1. Overexpressing NAF-1 enhances peptide cytotoxicity, while underexpressing the protein reduces it. The revealed molecular mechanism opens the way for further investigations into ACPs that target different cancer cells and inner organelles and induce apoptosis through multiple pathways.