Brown spider venom phospholipase D as a tool to modulate melanoma cell biology.

Phospholipases D (PLDs) from Loxosceles venoms are enzymes that cleave phospholipids triggering biological effects, as uncontrolled inflammatory response. We treated melanoma cell lines B16-F1 and B16-F10 with recombinant PLD from the venom of the spider Loxosceles intermedia. Toxin binding to cell surface and modulating events were evaluated by specific antibodies, or a chimera PLD-GFP and confocal fluorescence microscopy, flow cytometry, inverted microscopy, and scanning electron microscopy. Cytotoxicity was performed using MTT, Trypan Blue, and CellTiterGlo; and intracellular calcium measured using Fluo-4, spectrofluorimetry, fluorescence confocal microscopy, and flow cytometry. Phosphatidylserine externalization was evaluated using annexin V and flow cytometry. Cellular behaviors as cell growth and proliferation (MTT and Cyquant), colony formation (Clonogenic assay and Soft Agar Assay), migration (Scratch assay), and the expression of related migration and proliferation gene transcripts were studied through Systems Biology and real-timePCR). The PLD binding on the cell surface and the production of ectosomes, followed by nanoclusters, is in a concentration- and time-dependent manner. The treatments did not cause cytotoxicity, but increased intracellular calcium, phosphatidylserine externalization, cell growth, proliferation, colony formation, and migration. The toxin triggers cellular activation, with formation of ectosomes, protrusion-derived ectosomes, tunneling nanotubes, and increased expression of genes related to proliferation and migration. These events are more pronounced in the more aggressive lineage B16-F10. These findings highlight the potential of exploring these toxins as biotools in studies of tumor cell biology.