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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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Exogenous ceramide mimics cytoskeletal effects of cisplatin. MCF-7 cells were treated with D-e-C16-ceramide (5 μM) or vehicle (2% dodecane/98% ethanol) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel). Nuclei were visualized using DRAQ5 nuclear dye. (B) Cells were homogenized and fractionated into a cytosolic and membrane fractions by centrifugation at 100,000 g. Amount of ezrin in total, cytosolic, and membrane fractions (concentrated eightfold) was detected by Western blotting. (C) Cells were treated with D-e-C16-ceramide (5 μM), dh-C16-ceramide (5 μM), or vehicle (2% dodecane/98% ethanol) up to 3 h. Levels of p-ezrin and total ezrin were detected by Western blotting. (D) Cells treated with bSMase (100 mU/ml) for 0, 15, 30, and 60 min were subjected to Western blot analysis for ezrin and p-ezrin levels. (E) Subcellular distribution of ezrin in control cells and cells treated with bSMase (100 mU/ml) for 60 min. (F) Cells treated as in D were stained for p-ezrin (green channel) and phalloidin (red channel). Results are representative of three independent experiments. Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Bars, 20 μm.
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fig6: Exogenous ceramide mimics cytoskeletal effects of cisplatin. MCF-7 cells were treated with D-e-C16-ceramide (5 μM) or vehicle (2% dodecane/98% ethanol) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel). Nuclei were visualized using DRAQ5 nuclear dye. (B) Cells were homogenized and fractionated into a cytosolic and membrane fractions by centrifugation at 100,000 g. Amount of ezrin in total, cytosolic, and membrane fractions (concentrated eightfold) was detected by Western blotting. (C) Cells were treated with D-e-C16-ceramide (5 μM), dh-C16-ceramide (5 μM), or vehicle (2% dodecane/98% ethanol) up to 3 h. Levels of p-ezrin and total ezrin were detected by Western blotting. (D) Cells treated with bSMase (100 mU/ml) for 0, 15, 30, and 60 min were subjected to Western blot analysis for ezrin and p-ezrin levels. (E) Subcellular distribution of ezrin in control cells and cells treated with bSMase (100 mU/ml) for 60 min. (F) Cells treated as in D were stained for p-ezrin (green channel) and phalloidin (red channel). Results are representative of three independent experiments. Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Bars, 20 μm.

Mentions: Because the above results suggested that ceramide production is necessary for cytoskeletal changes induced by cisplatin, we next evaluated whether ceramide is sufficient for these changes. C16-ceramide was chosen for these experiments, as it was abundant in MCF-7 cells and almost doubled in response to cisplatin (Fig. 3 C). Microscopically, treatment with C16-ceramide recapitulated the effects of cisplatin, including loss of filopodia and redistribution of ezrin to the cytosol (Fig. 6 A). After separation of cytosolic and membrane pools by cell fractionation, Western blot analysis revealed reduction of membrane-associated ezrin after ceramide treatment (Fig. 6 B). In line with these results, treatment of HL-60 cells with short chain C2-ceramide was earlier reported to decrease the levels of ERM proteins in the membrane fraction (Kondo et al., 1997), suggesting that the above findings are neither specific to a particular cell line nor a ceramide species. Additionally, similar to cisplatin, administration of exogenous C16-ceramide caused a time-dependent dephosphorylation of ezrin, although dephosphorylation occurred in a more acute manner (30 min) (Fig. 6 C). Of note, treatment with the ceramide analogue, dihydro-C16-ceramide, failed to influence the levels of p-ezrin, suggesting a ceramide-specific response. Thus, ceramide is not only necessary but also sufficient for stress-induced cytoskeletal responses.


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

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

Exogenous ceramide mimics cytoskeletal effects of cisplatin. MCF-7 cells were treated with D-e-C16-ceramide (5 μM) or vehicle (2% dodecane/98% ethanol) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel). Nuclei were visualized using DRAQ5 nuclear dye. (B) Cells were homogenized and fractionated into a cytosolic and membrane fractions by centrifugation at 100,000 g. Amount of ezrin in total, cytosolic, and membrane fractions (concentrated eightfold) was detected by Western blotting. (C) Cells were treated with D-e-C16-ceramide (5 μM), dh-C16-ceramide (5 μM), or vehicle (2% dodecane/98% ethanol) up to 3 h. Levels of p-ezrin and total ezrin were detected by Western blotting. (D) Cells treated with bSMase (100 mU/ml) for 0, 15, 30, and 60 min were subjected to Western blot analysis for ezrin and p-ezrin levels. (E) Subcellular distribution of ezrin in control cells and cells treated with bSMase (100 mU/ml) for 60 min. (F) Cells treated as in D were stained for p-ezrin (green channel) and phalloidin (red channel). Results are representative of three independent experiments. Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Bars, 20 μm.
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fig6: Exogenous ceramide mimics cytoskeletal effects of cisplatin. MCF-7 cells were treated with D-e-C16-ceramide (5 μM) or vehicle (2% dodecane/98% ethanol) for 2 h. (A) Cells were fixed and stained with ezrin-specific polyclonal antibody (green channel). Nuclei were visualized using DRAQ5 nuclear dye. (B) Cells were homogenized and fractionated into a cytosolic and membrane fractions by centrifugation at 100,000 g. Amount of ezrin in total, cytosolic, and membrane fractions (concentrated eightfold) was detected by Western blotting. (C) Cells were treated with D-e-C16-ceramide (5 μM), dh-C16-ceramide (5 μM), or vehicle (2% dodecane/98% ethanol) up to 3 h. Levels of p-ezrin and total ezrin were detected by Western blotting. (D) Cells treated with bSMase (100 mU/ml) for 0, 15, 30, and 60 min were subjected to Western blot analysis for ezrin and p-ezrin levels. (E) Subcellular distribution of ezrin in control cells and cells treated with bSMase (100 mU/ml) for 60 min. (F) Cells treated as in D were stained for p-ezrin (green channel) and phalloidin (red channel). Results are representative of three independent experiments. Blots from three independent experiments were quantitated using NIH ImageJ (*, P < 0.05). Bars, 20 μm.
Mentions: Because the above results suggested that ceramide production is necessary for cytoskeletal changes induced by cisplatin, we next evaluated whether ceramide is sufficient for these changes. C16-ceramide was chosen for these experiments, as it was abundant in MCF-7 cells and almost doubled in response to cisplatin (Fig. 3 C). Microscopically, treatment with C16-ceramide recapitulated the effects of cisplatin, including loss of filopodia and redistribution of ezrin to the cytosol (Fig. 6 A). After separation of cytosolic and membrane pools by cell fractionation, Western blot analysis revealed reduction of membrane-associated ezrin after ceramide treatment (Fig. 6 B). In line with these results, treatment of HL-60 cells with short chain C2-ceramide was earlier reported to decrease the levels of ERM proteins in the membrane fraction (Kondo et al., 1997), suggesting that the above findings are neither specific to a particular cell line nor a ceramide species. Additionally, similar to cisplatin, administration of exogenous C16-ceramide caused a time-dependent dephosphorylation of ezrin, although dephosphorylation occurred in a more acute manner (30 min) (Fig. 6 C). Of note, treatment with the ceramide analogue, dihydro-C16-ceramide, failed to influence the levels of p-ezrin, suggesting a ceramide-specific response. Thus, ceramide is not only necessary but also sufficient for stress-induced cytoskeletal responses.

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