Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics.

Anticancer peptides are promising drug candidates for cancer treatment, but the short circulation time and low delivery efficiency limit their clinical applications. Herein, we designed several lasso-like self-assembling anticancer peptides (LASAPs) integrated with multiple functions by a computer-aided approach. Among these LASAPs, LASAP1 (CRG­DKG­PDC­GKA­FRR­FLG­ALF­KAL­SHLL, 1–9 disulfide bond) was determined to be superior to the others because it can self-assemble into homogeneous nanoparticles and exhibits improved stability in serum. Thus, LASAP1 was chosen for proving the design idea. LASAP1 can self-assemble into nanoparticles displaying iRGD on the surface because of its amphiphilic structure and accumulate to the tumor site after injection because of the EPR effect and iRGD targeting to αVβ3 integrin. The nanoparticles could disassemble in the acidic microenvironment of the solid tumor, and cleaved by the overexpressed hK2, which was secreted by prostate tumor cells, to release the effector peptide PTP-7b (FLG­ALF­KAL­SHLL), which was further activated by the acidic pH. Therefore, LASAP1 could target the orthotopic prostate tumor in the model mice after intraperitoneal injection and specifically inhibit tumor growth, with low systematic toxicity. Combining the multiple targeting functions, LASAP1 represents a promising design of self-delivery of peptide drugs for targeted cancer treatments.

Many lytic peptides contain a heptad sequence with leucine or isoleucine residues at "a" and "d" positions. However, their roles in the peptide-induced cytolytic process remain unclear. We have recently reported an anticancer lytic peptide ZXR-2 (FKIGGFIKKLWRSLLA), which contains a shortened zipper-like sequence with Ile/Leu at "a" and "d" positions. To understand the roles of these Ile/Leu residues, a series of analogs were constructed by sequentially replacing the Ile or Leu residue with alanine (Ala). Significant reduction of the cytolytic activity was observed when the Ile (3rd and 7th) and Leu (10th and 14th) residues at the "a" and "d" positions were substituted, while the replacement of the separate Leu (15th) residue had less effect. Based on the quenching of the intrinsic fluorescence of the peptides and their induced surface pressure changes of lipid monolayer, it was conjectured that the peptide ZXR-2 might insert into cell membranes from the C-terminal and to a depth of the W position. Accordingly, I, I, and L residues which mainly exposed in aqueous solution were more responsible for the peptide self-association on cell membranes, while L, together with L, might help peptide insert and anchor to cell membranes. These results are significant to elucidate the crucial roles of such Ile/Leu residues at "a" and "d" positions in peptide-peptide and peptide-membrane interactions to exert the membrane disruption activity of lytic peptides. With further understanding about the structure-activity relationship of lytic peptides, it would be helpful for designing novel anticancer lytic peptides.

Royal jelly is a nutritious food that has beneficial effects to human health. However, the functional substances remain unclear. Herein, we fractioned the royal jelly proteins of Xinjing black bees according to the Osboren method. Two main proteins from the ethanol-soluble fraction were purified and identified. RJG-1 was determined as glucosylceramidase, and RJG-2 was major royal jelly protein 1 (MRJP1). RJG-1 showed potent cytotoxicity toward various mammalian cells, and caused quick disruption of cell membranes. With glucosylceramidase activity, RJG-1 may degrade the glucosylceramide of the cell membranes and disrupt the membrane structure, thereby resulting in cell necrosis. This study extends our knowledge about the composition and function of royal jelly, and is significant for the application of royal jelly.