A lytic mechanism based on soluble phospholypases A2 (sPLA2) and b-galactosidespecific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractilehemocytes against K562 cell line and mammalian erythrocytes

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca2þ-dependent cytotoxic activity towardmammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations wereseparated (B1eB6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxicactivity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In additionthe separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture.Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLScontains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. TheB5 activity was blocked by D-Galactose, a-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), L-Fucose, DMannose,D-Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain,quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca2þ-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggestedthat sPLA2 could be responsible for changes and large alterations of the target cell membrane. Anapoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very dilutedHLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cellsrespectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.