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Remodeling of cellular cytoskeleton by the acid sphingomyelinase/ceramide pathway.

Zeidan YH, Jenkins RW, Hannun YA - J. Cell Biol. (2008)

Bottom Line: This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta.Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation.Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
The chemotherapeutic agent cisplatin is widely used in treatment of solid tumors. In breast cancer cells, cisplatin produces early and marked changes in cell morphology and the actin cytoskeleton. These changes manifest as loss of lamellipodia/filopodia and appearance of membrane ruffles. Furthermore, cisplatin induces dephosphorylation of the actin-binding protein ezrin, and its relocation from membrane protrusions to the cytosol. Because cisplatin activates acid sphingomyelinase (ASMase), we investigate here the role of the ASMase/ceramide (Cer) pathway in mediating these morphological changes. We find that cisplatin induces a transient elevation in ASMase activity and its redistribution to the plasma membrane. This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta. Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation. Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin. Collectively, these results highlight a novel tumor suppressor property for Cer and a function for ASMase in cisplatin-induced cytoskeletal remodeling.

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LC/MS analysis of cisplatin effects on ceramide and sphingomyelin. Cells were treated with cisplatin (5 μg/ml) for 15, 30, 60, or 180 min. After Bligh Dyer extraction of lipids, the different treatment groups were subjected to mass spectrometric analysis as indicated in Materials and methods. Sphingolipids measurements were normalized to total phospholipids. Shown are the normalized results for (A) total ceramide, (B) total SM, (C) C16 and C24:1-ceramide, and (D) C16 and C24:1-SM. Ceramide and SM results represent averages ± S.E. from three independent experiments.
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fig3: LC/MS analysis of cisplatin effects on ceramide and sphingomyelin. Cells were treated with cisplatin (5 μg/ml) for 15, 30, 60, or 180 min. After Bligh Dyer extraction of lipids, the different treatment groups were subjected to mass spectrometric analysis as indicated in Materials and methods. Sphingolipids measurements were normalized to total phospholipids. Shown are the normalized results for (A) total ceramide, (B) total SM, (C) C16 and C24:1-ceramide, and (D) C16 and C24:1-SM. Ceramide and SM results represent averages ± S.E. from three independent experiments.

Mentions: The effects of cisplatin on dephosphorylation of ezrin raised the possibility of activation of a serine/threonine phosphatase. Importantly, previous studies have shown that the bioactive lipid ceramide activates phosphatases in vitro and in cells (Pettus et al., 2002). Moreover, the ASMase/ceramide pathway has been previously shown to be activated by cisplatin (Levade and Jaffrezou, 1999; Lacour et al., 2004). Therefore, it became important to evaluate whether the observed changes with cisplatin are due to modulation of sphingolipid metabolism. To that end, MCF-7 cells were treated with cisplatin (5 μg/ml) over a time course of 3 h, and sphingolipids were extracted by the Bligh Dyer technique. Mass spectrometric measurements revealed a transient elevation in total ceramide (twofold) within 30 min of cisplatin treatment (Fig. 3 A). Further analysis indicated that this change was mainly accounted for by an increase in ceramide species carrying C16 and C24:1 acyl chains (Fig. 3 C). Importantly, the observed changes in ceramide levels were paralleled by a transient drop in sphingomyelin (SM) levels (Fig. 3 B). In particular, a major change in C16 SM and a minor one in C24:1 SM were detected by mass spectrometry after 15 min of cisplatin treatment (Fig. 3 D). Together, this sphingolipidomic profile suggests the operation of a sphingomyelinase.


Remodeling of cellular cytoskeleton by the acid sphingomyelinase/ceramide pathway.

Zeidan YH, Jenkins RW, Hannun YA - J. Cell Biol. (2008)

LC/MS analysis of cisplatin effects on ceramide and sphingomyelin. Cells were treated with cisplatin (5 μg/ml) for 15, 30, 60, or 180 min. After Bligh Dyer extraction of lipids, the different treatment groups were subjected to mass spectrometric analysis as indicated in Materials and methods. Sphingolipids measurements were normalized to total phospholipids. Shown are the normalized results for (A) total ceramide, (B) total SM, (C) C16 and C24:1-ceramide, and (D) C16 and C24:1-SM. Ceramide and SM results represent averages ± S.E. from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315679&req=5

fig3: LC/MS analysis of cisplatin effects on ceramide and sphingomyelin. Cells were treated with cisplatin (5 μg/ml) for 15, 30, 60, or 180 min. After Bligh Dyer extraction of lipids, the different treatment groups were subjected to mass spectrometric analysis as indicated in Materials and methods. Sphingolipids measurements were normalized to total phospholipids. Shown are the normalized results for (A) total ceramide, (B) total SM, (C) C16 and C24:1-ceramide, and (D) C16 and C24:1-SM. Ceramide and SM results represent averages ± S.E. from three independent experiments.
Mentions: The effects of cisplatin on dephosphorylation of ezrin raised the possibility of activation of a serine/threonine phosphatase. Importantly, previous studies have shown that the bioactive lipid ceramide activates phosphatases in vitro and in cells (Pettus et al., 2002). Moreover, the ASMase/ceramide pathway has been previously shown to be activated by cisplatin (Levade and Jaffrezou, 1999; Lacour et al., 2004). Therefore, it became important to evaluate whether the observed changes with cisplatin are due to modulation of sphingolipid metabolism. To that end, MCF-7 cells were treated with cisplatin (5 μg/ml) over a time course of 3 h, and sphingolipids were extracted by the Bligh Dyer technique. Mass spectrometric measurements revealed a transient elevation in total ceramide (twofold) within 30 min of cisplatin treatment (Fig. 3 A). Further analysis indicated that this change was mainly accounted for by an increase in ceramide species carrying C16 and C24:1 acyl chains (Fig. 3 C). Importantly, the observed changes in ceramide levels were paralleled by a transient drop in sphingomyelin (SM) levels (Fig. 3 B). In particular, a major change in C16 SM and a minor one in C24:1 SM were detected by mass spectrometry after 15 min of cisplatin treatment (Fig. 3 D). Together, this sphingolipidomic profile suggests the operation of a sphingomyelinase.

Bottom Line: This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta.Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation.Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
The chemotherapeutic agent cisplatin is widely used in treatment of solid tumors. In breast cancer cells, cisplatin produces early and marked changes in cell morphology and the actin cytoskeleton. These changes manifest as loss of lamellipodia/filopodia and appearance of membrane ruffles. Furthermore, cisplatin induces dephosphorylation of the actin-binding protein ezrin, and its relocation from membrane protrusions to the cytosol. Because cisplatin activates acid sphingomyelinase (ASMase), we investigate here the role of the ASMase/ceramide (Cer) pathway in mediating these morphological changes. We find that cisplatin induces a transient elevation in ASMase activity and its redistribution to the plasma membrane. This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta. Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation. Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin. Collectively, these results highlight a novel tumor suppressor property for Cer and a function for ASMase in cisplatin-induced cytoskeletal remodeling.

Show MeSH
Related in: MedlinePlus