Finally, we studied the impact of recombinant brown spider phospholipase-D on the proliferation of B16-F10 cells because it has been demonstrated that exogenous autotaxin is a powerful inducer of cell proliferation. To this end, B16-F10 cells (5 × 103 cells/well) were treated with recombinant GW 572016 brown spider
phospholipase-D (10 and 25 μg/mL for 48 h), and their cell proliferation was evaluated using the CyQUANT method and spectrofluorimetry. As shown in Fig. 7A, exogenous treatment of B16-F10 cells with the recombinant phospholipase-D led to an increase in cell growth in a concentration-dependent manner. Additionally, cells (5 × 103 cells/well) were treated with recombinant phospholipase-D (10 μg/mL) for 24, 48 or 72 h, and their proliferation was examined under conditions identical to those described above. It was observed that Selumetinib exogenous treatment with recombinant brown spider phospholipase-D induced proliferation
in a time-dependent manner (Fig. 7B), strengthening the idea that the lipid-modulating and other activities of this molecule in cells stimulate increases in proliferation. The putative lipid substrates that are targeted Cobimetinib supplier following brown spider phospholipase-D exposure include sphingomyelin, which produces ceramide 1-phosphate following phospholipase-D treatment, and other interconvertible bioactive molecules, such as ceramide and sphingosine 1-phosphate (both of which are bioactive lipids involved in increasing cell proliferation) (Chalfant
and Spiegel, 2005). Therefore, we repeated the proliferation assays (5 × 103 cells/well), but using exogenous sphingomyelin (5 and 10 mM) in the culture medium together with the recombinant phospholipase-D LiRecDT1 at a concentration of 10 μg/mL for 48 h. As depicted in Fig. 7C, cells incubated with exogenous sphingomyelin showed a higher proliferation index, indicating that brown spider phospholipase-D can act as an exogenous factor that stimulates proliferation. Phospholipase-D proteins have been described as important regulators of several critical physiological processes (Exton, 2002). These enzymes catalyze the hydrolysis of various phospholipids, generating bioactive molecules that play a role in distinct events in intracellular signaling cascades. Phospholipase-D proteins have also been shown to regulate the cell cycle, cell proliferation and apoptosis (Foster and Xu, 2003).