Golgi Fragmentation Is Associated with Ceramide-induced Cellular Effects

Ceramide has been shown to cause anoikis, a subtype of apoptosis due to inadequate cell adhesion. However, the underlying mechanism is unclear. Herein, we report that D-e-C(6)-ceramide (D-e-Cer), via generating sphingosine, disrupts the Golgi complex (GC), which is associated with various cellular effects, including anoikis. Treatment of HeLa cells with D-e-Cer caused cell elongation, spreading inhibition, rounding, and detachment before apoptosis (anoikis). In D-e-Cer–treated cells, glycosylation of β1 integrin in the GC was inhibited, thus its associated integrin receptors failed to translocate to the cell surface. Ceramide treatment also inhibited the reorganization of both microtubule and F-actin cytoskeletons, focal adhesions, and filopodia. These cellular effects were preceded by fragmentation of the Golgi complex. In contrast, L-e-C(6)-ceramide (L-e-Cer), the enantiomer of D-e-Cer, failed to induce these cellular effects. Mass spectrometric analysis revealed that treatment HeLa cells with D-e-Cer but not L-e-Cer caused a >50-fold increase in the levels of sphingosine, a product of hydrolysis of ceramide. Treatment with D-e-sphingosine and its enantiomer, L-e-sphingosine, caused massive perinuclear vacuolization, Golgi fragmentation, and cell rounding. Together, these results suggest that sphingosine generated from hydrolysis of ceramide causes the GC disruption, leading to various cellular effects

Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase

<div><p>Acid ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid ceramidase inhibitor <i>in vitro</i>, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid ceramidase is an efficient and valuable therapeutic strategy.</p></div

Upregulation of the Human Alkaline Ceramidase 1 and Acid Ceramidase Mediates Calcium-Induced Differentiation of Epidermal Keratinocytes

Extracellular calcium (Cao2+) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Cao2+ markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Cao2+-induced growth arrest and differentiation of HEKs. haCER1 is the human ortholog of mouse alkaline ceramidase 1 that we previously identified. haCER1 catalyzed the hydrolysis of very long-chain ceramides to generate sphingosine (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER1 in HEKs decreased the levels of D-e-C24:1-ceramide and D-e-C24:0-ceramide but elevated the levels of both SPH and its phosphate (S1P), whereas RNA interference-mediated knockdown of haCER1 caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER1 overexpression, Cao2+ increased the levels of SPH and S1P, and this was attenuated by haCER1 knockdown. haCER1 knockdown also inhibited the Cao2+-induced growth arrest of HEKs and the Cao2+-induced expression of keratin 1 and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Cao2+; and its knockdown attenuated the Cao2+-induced expression of keratin 1 and involucrin in HEKs. These results strongly suggest that upregulation of haCER1 and AC mediates the Cao2+-induced growth arrest and differentiation of HEKs by generating SPH and S1P

LCL521 represents an acute and potent inhibitor of ACDase.

<p><b>(A)</b> MCF7 cells were treated with vehicle, or with 0.1, 0.25, 0.5, 1, 1.5, 2.5, 5, and 10μM LCL521 for 1h. Cer, Sph and S1P were then extracted and quantified by LC-MS/MS. (n = 4, three times experiments with one time duplicates and two times single experiment). The actual amounts of Cer, Sph and S1P after treatment with LCL521 are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177805#pone.0177805.s001" target="_blank">S1B–S1D Fig</a> (<b>B</b>). MCF7 cells were treated with vehicle or 1μM LCL521 for 15min, 30min, 1, 2, and 5h. Sph was then extracted and quantified by LC-MS/MS. (*<i>p</i><0.05, n = 7, 3 times experiments with 2 times duplicates and 1 time triplicates).</p

Synergistic effect of LCL521 and single dose of ionizing radiation.

<p>MCF7 cells were irradiated with 0 or 2Gy using 137 Cesium irradiator. 1h after IR, each replicated treatment were further treated with vehicle or 5x 1μM LCL521. For the 5-time treatment, media were replaced every 24h with fresh media that contained either vehicle or 1μM LCL521. After that, cells were cultured for 4 weeks and then stained with crystal violet (1g/500ml formalin). Representative crystal staining is shown. Colonies count is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177805#pone.0177805.s002" target="_blank">S2 Fig</a>.</p

2′-Hydroxy C16-Ceramide Induces Apoptosis-Associated Proteomic Changes in C6 Glioma Cells

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as <i>N</i>-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (<i>R</i>)-2′-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (<i>S</i>) isomer or non-hydroxylated C16-ceramide. Upon exposure to (<i>R</i>)-2′-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (<i>R</i>)-2′-hydroxy-ceramide. Experimental validation confirmed that 2′-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3β, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2′-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (<i>R</i>)-2′-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer