Effects of ceramide-1-phosphate on cultured cells: dependence on dodecane in the vehicle

Loic Tauzin, Christine Graf, Mei Sun, Philipp Rovina, Nicolas Bouveyron, Markus Jaritz, Anthony Winiski, Nicole Hartmann, Frank Staedtler, Andreas Billich, Thomas Baumruker, Mei Zhang, and Frederic Bornancin

Novartis Institutes for BioMedical Research, Vienna

Corresponding Author: frederic.bornancin{at}novartis.com

Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A₂ $alpha$ . Much of the present knowledge has relied on the use of C1P of various acyl chain length, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here we show that, addition of C1P in ethanol/dodecane to culture medium, leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation and cell death. Not only the toxicity of C1P, but also its ability to trigger PGE₂ release, are fully dependent upon addition of a pre-made C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and in turn, help unraveling C1P signaling, which is still poorly understood.

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