Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells.

Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.

Psd2 pea defensin shows a preference for mimetic membrane rafts enriched with glucosylceramide and ergosterol.

Psd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCerF.solani) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P2, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp42 quantum yield of Psd2 in the presence of GlcCerF.solani and Erg, individually or in simultaneously enriched membranes. The partition coefficient (Kp) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.