The International Journal of Biochemistry & Cell Biology
Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth
Graphical abstract
Introduction
Sphingolipids are an essential class of membrane lipids and signaling molecules in eukaryotic cells. Ceramides are the basic structural elements of all sphingolipids. They are composed of sphingosine and a fatty acid with a variable chain length. Ceramides and other intermediates of sphingolipid metabolism are key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation (Pettus et al., 2002). The precise ratio of pro-proliferative and anti-proliferative sphingolipid metabolites seems to play a crucial role in the fate of a given cell. After treatment of cells with diverse apoptosis-inducing agents – including ionizing radiation, UV light, TNF-alpha and chemotherapeutic agents – accumulation of ceramides is detectable. Because of their proven apoptosis-inducing effects in cancer cells, ceramides have been termed “tumor suppressor lipids”, but their selective role in the mechanisms of apoptosis remains unclear (Taha et al., 2006). Besides their apoptotic actions, ceramides are assumed to play a pivotal role in other cellular death pathways, such as autophagy, necrosis or mitochondrial stress (Hannun and Luberto, 2000, Mimeault, 2002, Hou et al., 2011).
There are two main pathways for ceramide synthesis. (1) De novo synthesis takes place at the endoplasmic reticulum and starts with the condensation of l-serine and palmitoyl-CoA by l-serine palmitoyl transferase (SPT), generating 3-oxosphinganine. The 3-oxosphinganine reductase subsequently converts this intermediate to sphinganine, which is then acetylated by ceramide synthase (CerS) to produce dihydroceramide. At the surface of the endoplasmic reticulum, dihydroceramide desaturase converts dihydroceramide to ceramide. Ceramides are then transported to the Golgi apparatus (Futerman and Riezman, 2005), either by vesicular trafficking or by a specific ceramide transfer protein called CERT (Hanada et al., 2003). (2) The salvage pathway, generated either at the plasma membrane or in the late endosomes and lysosomes, leads to a continuous degradation of complex sphingolipids, such as glycosphingolipids and sphingomyelin, to sphingosine. Free sphingosine, released from the lysosome, is also accepted by ceramide synthases as a substrate, leading to the synthesis of ceramides at the surface of the endoplasmic reticulum or in endoplasmic reticulum-associated membranes (Kitatani et al., 2008). The ceramide-synthesizing ceramide synthases (CerS) were formerly known as LASS (Longevity Assurance) genes, because they are the mammalian homologs of the yeast longevity assurance gene LAG1 (Venkataraman and Futerman, 2002). So far, six different mammalian CerS (CerS1–6) have been described. They differ in their tissue specific expression pattern and in their substrate specificity resulting in the production of ceramides with different N-acyl side chains (C14:0-Cer–C26:0-Cer). All six CerS isoenzymes have similar Km values with sphinganine (ranging from 2 to 5 μM) as substrate (Lahiri et al., 2007) but vary in their specificity toward acyl-CoAs (Laviad et al., 2008, Mizutani et al., 2008, Riebeling et al., 2003). Their differential substrate specificities are: CerS1 (C18:0-Cer); CerS2 (C20:0-Cer, C22:0-Cer, C24:0-Cer); CerS3 (C18:0-Cer, C20:0-Cer); CerS4 (C18:0-Cer, C20:0-Cer, C22:0-Cer, C24:0-Cer); CerS5 (C16:0-Cer, C18:0-Cer, C18:1-Cer); and CerS6 (C16:0-Cer, C18:0-Cer). These data point out a remarkable degree of overlap in substrate specificity among the different CerS isoforms, precluding any assignment of a specific ceramide to a specific CerS isoform. It should be emphasized that these reported CerS specificities were determined in different organisms, cells and tissues by various groups (Min et al., 2007, Riebeling et al., 2003, Mizutani et al., 2006).
In previous studies, we demonstrated that ceramide production and their biological activities are highly dependent on the length of their covalently linked fatty acid. Especially in human breast tumor tissue, the levels of C16:0-Cer, C24:0-Cer, and C24:1-Cer are upregulated in dependence on tumor dignity (Schiffmann et al., 2009a). Furthermore, we showed that the selective COX-2 inhibitor, celecoxib, mediates its anti-proliferative effects, in part, by selectively activating CerS6 in human colon carcinoma cells, leading to an increase in C16:0-Cer (Schiffmann et al., 2010). These data indicated that ceramides, particularly in relation to the length of their side chain, are associated with various effects in tumorigenesis. To further clarify the biological relevance of these findings, we overexpressed CerS2, CerS4 and CerS6 in human breast and colon carcinoma cell lines and investigated their effects on ceramide synthesis, cell survival and apoptosis.
Section snippets
Cells and reagents
The human breast cancer cell line MCF-7 and the human colon cancer cell line HCT-116 were ordered from “Deutsche Sammlung für Mikroorganismen und Zellkulturen” (DSMZ, Braunschweig, Germany). HCT-116 cells were incubated in McCoy's 5A (Invitrogen, Darmstadt, Germany) supplemented with 100 units/ml penicillin G, 100 μg/ml streptomycin and 10% FCS (fetal calf serum). MCF-7 cells were incubated in DMEM + Glutamax™ (Invitrogen, Darmstadt, Germany) supplemented with 100 units/ml penicillin G, 100 μg/ml
Expression levels of ceramide synthases in transfected MCF-7 and HCT-116 cells
Mock-transfected MCF-7 and HCT-116 cells were used to determine the basal level of CerS2, CerS4 and CerS6 by quantitative real-time-PCR. The most predominantly expressed mRNA was ceramide synthase 2 (CerS2) in MCF-7- and in HCT-116 cells (Fig. 1A and B). In comparison, CerS4 and CerS6 were barely expressed in either cell line, when normalized to GAPDH. Transfection of cells with pTA-CerS2, pTA-CerS4, and pTA-CerS6 was accompanied by a selective increase in CerS2, CerS4, or CerS6, respectively,
Discussion
Previous data indicate that especially CerS2, 4, and 6 are overexpressed in human breast cancer tissue depending on tumor dignity (Schiffmann et al., 2009a, Erez-Roman et al., 2010). Here, we investigated the molecular effects of CerS2, 4 and 6 upregulation in order to clarify the impact of ceramide synthases and ceramides with various side chain lengths on breast and colon cancer cells. Our data suggest that overexpression of CerS4 and 6 has a toxic effect mediated by mitochondrial damage,
Acknowledgement
We thank Dr. M. Parnham for critically reading the manuscript.
Grant support: This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Forschergruppe FOG 784/TP5 (GR2011/2-1) and DFG project GR2011/3-1 and the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF).
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These authors contributed equally to this work.