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

Activity of elisidepsin in B16, GM95 and GM95-UGCG cell lines.(A) HPTLC analysis of the total lipid extracts from the three cell lines. Lipids were separated in chloroform:methanol (8:2) and stained with thioflavine S. (B) Orcinol staining of glycolipids present in the lipid extracts from the three cell lines. Lipids were separated as mentioned before. In the last lane, C16-β-D-glucosyl ceramide was included as control. In (A) and (B): 1, lipid extract from B16 (200 μg); 2, lipid extract from GM95 (200 μg); 3, lipid extract from GM95-UGCG (200 μg); 4, C16-β-D-glucosyl ceramide (6 μg). Arrows indicate the lipid fractions that are present or completely absent in the cell lines (C) Concentration-response curves for elisidepsin obtained after 72 h treatment in B16 (▲), GM95 (⬛) and GM95-UGCG cells (⬜); results represent the mean±SD of at least three different experiments. (D) Representative images of B16, GM95 and GM95-UGCG cells exposed to different concentrations of elisidepsin (10, 25 and 50 μM) for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in B16 and GM95-UGCG cells (white arrows). PI stained nuclei are shown in the fluorescence microscopy images from both cell lines. GM95 cells do not show any response to the treatment with elisidepsin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140782.g004: Activity of elisidepsin in B16, GM95 and GM95-UGCG cell lines.(A) HPTLC analysis of the total lipid extracts from the three cell lines. Lipids were separated in chloroform:methanol (8:2) and stained with thioflavine S. (B) Orcinol staining of glycolipids present in the lipid extracts from the three cell lines. Lipids were separated as mentioned before. In the last lane, C16-β-D-glucosyl ceramide was included as control. In (A) and (B): 1, lipid extract from B16 (200 μg); 2, lipid extract from GM95 (200 μg); 3, lipid extract from GM95-UGCG (200 μg); 4, C16-β-D-glucosyl ceramide (6 μg). Arrows indicate the lipid fractions that are present or completely absent in the cell lines (C) Concentration-response curves for elisidepsin obtained after 72 h treatment in B16 (▲), GM95 (⬛) and GM95-UGCG cells (⬜); results represent the mean±SD of at least three different experiments. (D) Representative images of B16, GM95 and GM95-UGCG cells exposed to different concentrations of elisidepsin (10, 25 and 50 μM) for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in B16 and GM95-UGCG cells (white arrows). PI stained nuclei are shown in the fluorescence microscopy images from both cell lines. GM95 cells do not show any response to the treatment with elisidepsin.

Mentions: We assessed if glycosylceramides were needed for elisidepsin-induced disruption of the plasma membrane and necrosis. For this purpose we used the mouse melanoma B16 cell line and its mutant derivative GM95, described to lack UDP-glucose:ceramide glucosyltransferase (UGCG; EC 2.4.1.80) activity [19]. HPTLC analysis of lipid extracts from both cell lines confirmed the absence in GM95 cells of lipids in the region of the identified glycosylceramides (Fig 4A and 4B). B16 cells were sensitive to elisidepsin, displaying the typical membrane permeabilization and formation of large vesicles observed in other tumor cell lines (Fig 4C and 4D; Table 2). In contrast, UGCG-deficient GM95 cells were resistant to elisidepsin (Fig 4C and Table 2) showing a behavior similar to that of HCT-116-Irv cells (Fig 4D). Interestingly, over-expression of UGCG gene in GM95 cells (GM95-UGCG cells) rendered them sensitive to elisidepsin, at a similar level of that of B16 cells (mean IC50 values of 9.0 and 6.9 μM, respectively; Fig 4C). Furthermore, as observed in Fig 4D and S2 Movie, GM95-UGCG showed typical elisidepsin-induced morphological changes and membrane permeabilization by contrast phase or fluorescence microscopy. Similarly, an increase in cell resistance was observed in HCT-116 parental cells after pretreatment with the UGCG inhibitors D,L-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and n-Butyldeoxynojyrimicin (NB-DNJ, Miglustat hydrochloride) (Fig 5).


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)

Activity of elisidepsin in B16, GM95 and GM95-UGCG cell lines.(A) HPTLC analysis of the total lipid extracts from the three cell lines. Lipids were separated in chloroform:methanol (8:2) and stained with thioflavine S. (B) Orcinol staining of glycolipids present in the lipid extracts from the three cell lines. Lipids were separated as mentioned before. In the last lane, C16-β-D-glucosyl ceramide was included as control. In (A) and (B): 1, lipid extract from B16 (200 μg); 2, lipid extract from GM95 (200 μg); 3, lipid extract from GM95-UGCG (200 μg); 4, C16-β-D-glucosyl ceramide (6 μg). Arrows indicate the lipid fractions that are present or completely absent in the cell lines (C) Concentration-response curves for elisidepsin obtained after 72 h treatment in B16 (▲), GM95 (⬛) and GM95-UGCG cells (⬜); results represent the mean±SD of at least three different experiments. (D) Representative images of B16, GM95 and GM95-UGCG cells exposed to different concentrations of elisidepsin (10, 25 and 50 μM) for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in B16 and GM95-UGCG cells (white arrows). PI stained nuclei are shown in the fluorescence microscopy images from both cell lines. GM95 cells do not show any response to the treatment with elisidepsin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140782.g004: Activity of elisidepsin in B16, GM95 and GM95-UGCG cell lines.(A) HPTLC analysis of the total lipid extracts from the three cell lines. Lipids were separated in chloroform:methanol (8:2) and stained with thioflavine S. (B) Orcinol staining of glycolipids present in the lipid extracts from the three cell lines. Lipids were separated as mentioned before. In the last lane, C16-β-D-glucosyl ceramide was included as control. In (A) and (B): 1, lipid extract from B16 (200 μg); 2, lipid extract from GM95 (200 μg); 3, lipid extract from GM95-UGCG (200 μg); 4, C16-β-D-glucosyl ceramide (6 μg). Arrows indicate the lipid fractions that are present or completely absent in the cell lines (C) Concentration-response curves for elisidepsin obtained after 72 h treatment in B16 (▲), GM95 (⬛) and GM95-UGCG cells (⬜); results represent the mean±SD of at least three different experiments. (D) Representative images of B16, GM95 and GM95-UGCG cells exposed to different concentrations of elisidepsin (10, 25 and 50 μM) for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in B16 and GM95-UGCG cells (white arrows). PI stained nuclei are shown in the fluorescence microscopy images from both cell lines. GM95 cells do not show any response to the treatment with elisidepsin.
Mentions: We assessed if glycosylceramides were needed for elisidepsin-induced disruption of the plasma membrane and necrosis. For this purpose we used the mouse melanoma B16 cell line and its mutant derivative GM95, described to lack UDP-glucose:ceramide glucosyltransferase (UGCG; EC 2.4.1.80) activity [19]. HPTLC analysis of lipid extracts from both cell lines confirmed the absence in GM95 cells of lipids in the region of the identified glycosylceramides (Fig 4A and 4B). B16 cells were sensitive to elisidepsin, displaying the typical membrane permeabilization and formation of large vesicles observed in other tumor cell lines (Fig 4C and 4D; Table 2). In contrast, UGCG-deficient GM95 cells were resistant to elisidepsin (Fig 4C and Table 2) showing a behavior similar to that of HCT-116-Irv cells (Fig 4D). Interestingly, over-expression of UGCG gene in GM95 cells (GM95-UGCG cells) rendered them sensitive to elisidepsin, at a similar level of that of B16 cells (mean IC50 values of 9.0 and 6.9 μM, respectively; Fig 4C). Furthermore, as observed in Fig 4D and S2 Movie, GM95-UGCG showed typical elisidepsin-induced morphological changes and membrane permeabilization by contrast phase or fluorescence microscopy. Similarly, an increase in cell resistance was observed in HCT-116 parental cells after pretreatment with the UGCG inhibitors D,L-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and n-Butyldeoxynojyrimicin (NB-DNJ, Miglustat hydrochloride) (Fig 5).

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