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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

Characterization of the HCT-116-Irv resistance to elisidepsin.(A) Concentration-response curves showing the activity of elisidepsin after 30 min and 72 h in HCT-116 (⬛) and HCT-116-Irv cells (⬜); results represent the mean±SD of at least three different experiments. (B) Stability of elisidepsin resistance in HCT-116-Irv cells determined by concentration-response curves after 2 (⬛) and 15 (▲) culture passages in absence of the compound. HCT-116 cells (●) are also depicted in the graph. Results represent the mean±SD of three different experiments. (C) Elisidepsin accumulation (pmol/mg protein) in HCT-116 (white bars) and HCT-116-Irv cells (black bars). Both cell lines were treated with elisidepsin 7 μM at different time points and the accumulated compound was quantified by HPLC/MS. Results represent the mean±SD of three different samples. Comparisons between different samples were analyzed by Student’s t test. Differences were considered significant at *P<0.05, **P<0.01 and ***P<0.001. (D) Representative images of HCT-116 and HCT-116-Irv cells exposed to elisidepsin 10 μM for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in the wild-type cells (white arrows). Fluorescence microscopy shows PI stained nuclei only in the parental cells. (E) Electrophysiological effects of elisidepsin 1 μM on HCT-116 and HCT-116-Irv cells. Left panels show original records after applying a ramp pulse protocol from -100 mV to +120 mV during 500 ms. Right panels show the amplitude of the maximum current at the end of the ramp together with the exponential fit of the process. HCT-116-Irv cells are completely insensitive to the elisidepsin effects as shown in the current records. Holding potential was maintained at -80 mV.
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pone.0140782.g001: Characterization of the HCT-116-Irv resistance to elisidepsin.(A) Concentration-response curves showing the activity of elisidepsin after 30 min and 72 h in HCT-116 (⬛) and HCT-116-Irv cells (⬜); results represent the mean±SD of at least three different experiments. (B) Stability of elisidepsin resistance in HCT-116-Irv cells determined by concentration-response curves after 2 (⬛) and 15 (▲) culture passages in absence of the compound. HCT-116 cells (●) are also depicted in the graph. Results represent the mean±SD of three different experiments. (C) Elisidepsin accumulation (pmol/mg protein) in HCT-116 (white bars) and HCT-116-Irv cells (black bars). Both cell lines were treated with elisidepsin 7 μM at different time points and the accumulated compound was quantified by HPLC/MS. Results represent the mean±SD of three different samples. Comparisons between different samples were analyzed by Student’s t test. Differences were considered significant at *P<0.05, **P<0.01 and ***P<0.001. (D) Representative images of HCT-116 and HCT-116-Irv cells exposed to elisidepsin 10 μM for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in the wild-type cells (white arrows). Fluorescence microscopy shows PI stained nuclei only in the parental cells. (E) Electrophysiological effects of elisidepsin 1 μM on HCT-116 and HCT-116-Irv cells. Left panels show original records after applying a ramp pulse protocol from -100 mV to +120 mV during 500 ms. Right panels show the amplitude of the maximum current at the end of the ramp together with the exponential fit of the process. HCT-116-Irv cells are completely insensitive to the elisidepsin effects as shown in the current records. Holding potential was maintained at -80 mV.

Mentions: HCT-116-Irv cells were derived from HCT-116 parental cells by a classical stepwise selection procedure during 18 months. After this period, a pool of resistant cells with a very homogenous behavior in all the analyzed variables was stablished and it was consider as a new cell line named HCT-116-Irv. As shown in Fig 1A and Table 1, cells were 14.7-fold more resistant to elisidepsin than parental HCT-116 cells. The IC50 values after 30 min exposure were 7.7±4.1 μM and >100 μM μM for HCT-116 and HCT-116-Irv cells, respectively (Fig 1A). The IC50 values after 72 h exposure were 5.5±0.8 μM and 81.5±0.8 μM for HCT-116 and HCT-116-Irv cells, respectively (Fig 1A). To determine the stability of the acquired resistance in HCT-116-Irv, cells were cultured for 15 passages in the absence of the drug and concentration-response curves were performed in passage 2 and 15. The levels of resistance at both time points were very similar, indicating that HCT-116-Irv cells had acquired a permanent resistance to the drug (Fig 1B). HCT-116-Irv cell line did not show any cross resistance with other common anticancer agents or other agents interacting with the cell membrane, indicating that the resistance mechanism was specific for elisidepsin (Table 1). Additionally, we evaluated the accumulation of the compound in both parental and resistant cells. Both cell lines were treated with elisidepsin 7 μM at several time points, and the amount of drug retained in cells was quantified by HPLC/MS. While the parental cells showed a rapid and large accumulation of elisidepsin, in the resistant cells the accumulation of the compound was remarkably smaller and slower (Fig 1C).


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)

Characterization of the HCT-116-Irv resistance to elisidepsin.(A) Concentration-response curves showing the activity of elisidepsin after 30 min and 72 h in HCT-116 (⬛) and HCT-116-Irv cells (⬜); results represent the mean±SD of at least three different experiments. (B) Stability of elisidepsin resistance in HCT-116-Irv cells determined by concentration-response curves after 2 (⬛) and 15 (▲) culture passages in absence of the compound. HCT-116 cells (●) are also depicted in the graph. Results represent the mean±SD of three different experiments. (C) Elisidepsin accumulation (pmol/mg protein) in HCT-116 (white bars) and HCT-116-Irv cells (black bars). Both cell lines were treated with elisidepsin 7 μM at different time points and the accumulated compound was quantified by HPLC/MS. Results represent the mean±SD of three different samples. Comparisons between different samples were analyzed by Student’s t test. Differences were considered significant at *P<0.05, **P<0.01 and ***P<0.001. (D) Representative images of HCT-116 and HCT-116-Irv cells exposed to elisidepsin 10 μM for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in the wild-type cells (white arrows). Fluorescence microscopy shows PI stained nuclei only in the parental cells. (E) Electrophysiological effects of elisidepsin 1 μM on HCT-116 and HCT-116-Irv cells. Left panels show original records after applying a ramp pulse protocol from -100 mV to +120 mV during 500 ms. Right panels show the amplitude of the maximum current at the end of the ramp together with the exponential fit of the process. HCT-116-Irv cells are completely insensitive to the elisidepsin effects as shown in the current records. Holding potential was maintained at -80 mV.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140782.g001: Characterization of the HCT-116-Irv resistance to elisidepsin.(A) Concentration-response curves showing the activity of elisidepsin after 30 min and 72 h in HCT-116 (⬛) and HCT-116-Irv cells (⬜); results represent the mean±SD of at least three different experiments. (B) Stability of elisidepsin resistance in HCT-116-Irv cells determined by concentration-response curves after 2 (⬛) and 15 (▲) culture passages in absence of the compound. HCT-116 cells (●) are also depicted in the graph. Results represent the mean±SD of three different experiments. (C) Elisidepsin accumulation (pmol/mg protein) in HCT-116 (white bars) and HCT-116-Irv cells (black bars). Both cell lines were treated with elisidepsin 7 μM at different time points and the accumulated compound was quantified by HPLC/MS. Results represent the mean±SD of three different samples. Comparisons between different samples were analyzed by Student’s t test. Differences were considered significant at *P<0.05, **P<0.01 and ***P<0.001. (D) Representative images of HCT-116 and HCT-116-Irv cells exposed to elisidepsin 10 μM for 5 min. Phase contrast microscopy images show morphological alterations and giant vesicles formation only in the wild-type cells (white arrows). Fluorescence microscopy shows PI stained nuclei only in the parental cells. (E) Electrophysiological effects of elisidepsin 1 μM on HCT-116 and HCT-116-Irv cells. Left panels show original records after applying a ramp pulse protocol from -100 mV to +120 mV during 500 ms. Right panels show the amplitude of the maximum current at the end of the ramp together with the exponential fit of the process. HCT-116-Irv cells are completely insensitive to the elisidepsin effects as shown in the current records. Holding potential was maintained at -80 mV.
Mentions: HCT-116-Irv cells were derived from HCT-116 parental cells by a classical stepwise selection procedure during 18 months. After this period, a pool of resistant cells with a very homogenous behavior in all the analyzed variables was stablished and it was consider as a new cell line named HCT-116-Irv. As shown in Fig 1A and Table 1, cells were 14.7-fold more resistant to elisidepsin than parental HCT-116 cells. The IC50 values after 30 min exposure were 7.7±4.1 μM and >100 μM μM for HCT-116 and HCT-116-Irv cells, respectively (Fig 1A). The IC50 values after 72 h exposure were 5.5±0.8 μM and 81.5±0.8 μM for HCT-116 and HCT-116-Irv cells, respectively (Fig 1A). To determine the stability of the acquired resistance in HCT-116-Irv, cells were cultured for 15 passages in the absence of the drug and concentration-response curves were performed in passage 2 and 15. The levels of resistance at both time points were very similar, indicating that HCT-116-Irv cells had acquired a permanent resistance to the drug (Fig 1B). HCT-116-Irv cell line did not show any cross resistance with other common anticancer agents or other agents interacting with the cell membrane, indicating that the resistance mechanism was specific for elisidepsin (Table 1). Additionally, we evaluated the accumulation of the compound in both parental and resistant cells. Both cell lines were treated with elisidepsin 7 μM at several time points, and the amount of drug retained in cells was quantified by HPLC/MS. While the parental cells showed a rapid and large accumulation of elisidepsin, in the resistant cells the accumulation of the compound was remarkably smaller and slower (Fig 1C).

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