Limits...
Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death.

Molina-Guijarro JM, García C, Macías Á, García-Fernández LF, Moreno C, Reyes F, Martínez-Leal JF, Fernández R, Martínez V, Valenzuela C, Lillo MP, Galmarini CM - PLoS ONE (2015)

Bottom Line: Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane.Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane.These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Investigación y Desarrollo, PharmaMar S.A., Colmenar Viejo, Madrid, Spain.

ABSTRACT
Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy.

No MeSH data available.


Related in: MedlinePlus

Glycosylceramides pattern and elisidepsin interaction with glycosylceramides in HCT-116 and HCT-116-Irv cells.(A) Monodimensional silica-gel HPTLC of the lipid extract from HCT-116 and HCT-116-Irv. The plate was stained with thioflavine S. Arrows show a group of nearby fractions with different presence in both cell lines. The total extract was divided in 8 fractions (a to h) that were purified for subsequent experiments. (B) Glycolipid detection in lipid extracts from HCT-116 and HCT-116-Irv. Total lipid extracts from HCT-116 and HCT-116-Irv were developed on HPTLC silica plates and visualized with orcinol-sulphuric acid staining to reveal the presence of glycosylated lipids. Control samples were added to confirm the results; left panel: control HPTLC incubated with sulphuric acid alone; right panel: HPTLC stained with orcinol-sulphuric acid. The samples were: 1, HCT-116-Irv lipid extract (200 μg); 2, HCT-116 lipid extract (200 μg); 3, neutral glycosphingolipid mixture (cerebrosides, lactosylceramides, ceramide trihexosides, globosides –Gb4-) (10 μg); 4, C16-β-D-glucosyl ceramide (6 μg); 5, glucose (5 μg). The lipid fractions that are related to elisidepsin binding or resistance are pointed out inside the dot line square. (C) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the lipid fractions from the wt and the resistant cells. Arrows indicate the fraction with specific binding to elisidepsin-biotin only present in HCT-116 cells. (D) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the selected lipid fractions from the parental and the resistant cells. Fraction 4 from HCT-116 is the only fraction that interacts with elisidepsin-biotin. (E) Competitive binding assay with elisidepsin. Nitrocellulose membranes with spots of the lipid fraction 4 from HCT-116 were incubated with different proportions of elisidepsin-biotin and elisidepsin (1:1, left; 1:15, right). A lower signal was detected when elisidepsin quantity was increased. (F) NMR analysis of a purified lipid fraction of HCT-116 cells. NMR spectra from C16-β-D-glucosyl ceramide and purified F4 lipid fraction from HCT-116 are shown. Letters and arrows indicate the assignation of signals from F4 spectra in a model glycosylceramide molecule.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608773&req=5

pone.0140782.g003: Glycosylceramides pattern and elisidepsin interaction with glycosylceramides in HCT-116 and HCT-116-Irv cells.(A) Monodimensional silica-gel HPTLC of the lipid extract from HCT-116 and HCT-116-Irv. The plate was stained with thioflavine S. Arrows show a group of nearby fractions with different presence in both cell lines. The total extract was divided in 8 fractions (a to h) that were purified for subsequent experiments. (B) Glycolipid detection in lipid extracts from HCT-116 and HCT-116-Irv. Total lipid extracts from HCT-116 and HCT-116-Irv were developed on HPTLC silica plates and visualized with orcinol-sulphuric acid staining to reveal the presence of glycosylated lipids. Control samples were added to confirm the results; left panel: control HPTLC incubated with sulphuric acid alone; right panel: HPTLC stained with orcinol-sulphuric acid. The samples were: 1, HCT-116-Irv lipid extract (200 μg); 2, HCT-116 lipid extract (200 μg); 3, neutral glycosphingolipid mixture (cerebrosides, lactosylceramides, ceramide trihexosides, globosides –Gb4-) (10 μg); 4, C16-β-D-glucosyl ceramide (6 μg); 5, glucose (5 μg). The lipid fractions that are related to elisidepsin binding or resistance are pointed out inside the dot line square. (C) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the lipid fractions from the wt and the resistant cells. Arrows indicate the fraction with specific binding to elisidepsin-biotin only present in HCT-116 cells. (D) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the selected lipid fractions from the parental and the resistant cells. Fraction 4 from HCT-116 is the only fraction that interacts with elisidepsin-biotin. (E) Competitive binding assay with elisidepsin. Nitrocellulose membranes with spots of the lipid fraction 4 from HCT-116 were incubated with different proportions of elisidepsin-biotin and elisidepsin (1:1, left; 1:15, right). A lower signal was detected when elisidepsin quantity was increased. (F) NMR analysis of a purified lipid fraction of HCT-116 cells. NMR spectra from C16-β-D-glucosyl ceramide and purified F4 lipid fraction from HCT-116 are shown. Letters and arrows indicate the assignation of signals from F4 spectra in a model glycosylceramide molecule.

Mentions: Since HCT-116-Irv resistant cells accumulated less elisidepsin and the compound was detected at lower levels in their cell membrane, we investigated whether this was due to an altered lipid composition. To this end, we extracted total lipids from HCT-116 and HCT-116-Irv cell pellets using standard methods based on chloroform and methanol combinations. Lipid extracts were then fractionated by HPTLC (high performance thin layer chromatography) and the plates were stained with specific dyes for lipids (Fig 3A). Interestingly, we identified two subfractions in HCT-116 cells that were almost absent in their resistant counterpart (white arrows 1 and 4 in Fig 3A). Staining HPTLC plates with orcinol, a classic sugar staining method, demonstrated that the two differential lipid species identified contained glycosylated lipids (Fig 3B).


Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death.

Molina-Guijarro JM, García C, Macías Á, García-Fernández LF, Moreno C, Reyes F, Martínez-Leal JF, Fernández R, Martínez V, Valenzuela C, Lillo MP, Galmarini CM - PLoS ONE (2015)

Glycosylceramides pattern and elisidepsin interaction with glycosylceramides in HCT-116 and HCT-116-Irv cells.(A) Monodimensional silica-gel HPTLC of the lipid extract from HCT-116 and HCT-116-Irv. The plate was stained with thioflavine S. Arrows show a group of nearby fractions with different presence in both cell lines. The total extract was divided in 8 fractions (a to h) that were purified for subsequent experiments. (B) Glycolipid detection in lipid extracts from HCT-116 and HCT-116-Irv. Total lipid extracts from HCT-116 and HCT-116-Irv were developed on HPTLC silica plates and visualized with orcinol-sulphuric acid staining to reveal the presence of glycosylated lipids. Control samples were added to confirm the results; left panel: control HPTLC incubated with sulphuric acid alone; right panel: HPTLC stained with orcinol-sulphuric acid. The samples were: 1, HCT-116-Irv lipid extract (200 μg); 2, HCT-116 lipid extract (200 μg); 3, neutral glycosphingolipid mixture (cerebrosides, lactosylceramides, ceramide trihexosides, globosides –Gb4-) (10 μg); 4, C16-β-D-glucosyl ceramide (6 μg); 5, glucose (5 μg). The lipid fractions that are related to elisidepsin binding or resistance are pointed out inside the dot line square. (C) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the lipid fractions from the wt and the resistant cells. Arrows indicate the fraction with specific binding to elisidepsin-biotin only present in HCT-116 cells. (D) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the selected lipid fractions from the parental and the resistant cells. Fraction 4 from HCT-116 is the only fraction that interacts with elisidepsin-biotin. (E) Competitive binding assay with elisidepsin. Nitrocellulose membranes with spots of the lipid fraction 4 from HCT-116 were incubated with different proportions of elisidepsin-biotin and elisidepsin (1:1, left; 1:15, right). A lower signal was detected when elisidepsin quantity was increased. (F) NMR analysis of a purified lipid fraction of HCT-116 cells. NMR spectra from C16-β-D-glucosyl ceramide and purified F4 lipid fraction from HCT-116 are shown. Letters and arrows indicate the assignation of signals from F4 spectra in a model glycosylceramide molecule.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608773&req=5

pone.0140782.g003: Glycosylceramides pattern and elisidepsin interaction with glycosylceramides in HCT-116 and HCT-116-Irv cells.(A) Monodimensional silica-gel HPTLC of the lipid extract from HCT-116 and HCT-116-Irv. The plate was stained with thioflavine S. Arrows show a group of nearby fractions with different presence in both cell lines. The total extract was divided in 8 fractions (a to h) that were purified for subsequent experiments. (B) Glycolipid detection in lipid extracts from HCT-116 and HCT-116-Irv. Total lipid extracts from HCT-116 and HCT-116-Irv were developed on HPTLC silica plates and visualized with orcinol-sulphuric acid staining to reveal the presence of glycosylated lipids. Control samples were added to confirm the results; left panel: control HPTLC incubated with sulphuric acid alone; right panel: HPTLC stained with orcinol-sulphuric acid. The samples were: 1, HCT-116-Irv lipid extract (200 μg); 2, HCT-116 lipid extract (200 μg); 3, neutral glycosphingolipid mixture (cerebrosides, lactosylceramides, ceramide trihexosides, globosides –Gb4-) (10 μg); 4, C16-β-D-glucosyl ceramide (6 μg); 5, glucose (5 μg). The lipid fractions that are related to elisidepsin binding or resistance are pointed out inside the dot line square. (C) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the lipid fractions from the wt and the resistant cells. Arrows indicate the fraction with specific binding to elisidepsin-biotin only present in HCT-116 cells. (D) Dot-blot assay for the interaction of biotin or elisidepsin-biotin with the selected lipid fractions from the parental and the resistant cells. Fraction 4 from HCT-116 is the only fraction that interacts with elisidepsin-biotin. (E) Competitive binding assay with elisidepsin. Nitrocellulose membranes with spots of the lipid fraction 4 from HCT-116 were incubated with different proportions of elisidepsin-biotin and elisidepsin (1:1, left; 1:15, right). A lower signal was detected when elisidepsin quantity was increased. (F) NMR analysis of a purified lipid fraction of HCT-116 cells. NMR spectra from C16-β-D-glucosyl ceramide and purified F4 lipid fraction from HCT-116 are shown. Letters and arrows indicate the assignation of signals from F4 spectra in a model glycosylceramide molecule.
Mentions: Since HCT-116-Irv resistant cells accumulated less elisidepsin and the compound was detected at lower levels in their cell membrane, we investigated whether this was due to an altered lipid composition. To this end, we extracted total lipids from HCT-116 and HCT-116-Irv cell pellets using standard methods based on chloroform and methanol combinations. Lipid extracts were then fractionated by HPTLC (high performance thin layer chromatography) and the plates were stained with specific dyes for lipids (Fig 3A). Interestingly, we identified two subfractions in HCT-116 cells that were almost absent in their resistant counterpart (white arrows 1 and 4 in Fig 3A). Staining HPTLC plates with orcinol, a classic sugar staining method, demonstrated that the two differential lipid species identified contained glycosylated lipids (Fig 3B).

Bottom Line: Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane.Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane.These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Investigación y Desarrollo, PharmaMar S.A., Colmenar Viejo, Madrid, Spain.

ABSTRACT
Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy.

No MeSH data available.


Related in: MedlinePlus