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

Comparative interaction study of elisidepsin and the plasma membrane of HCT-116 and HCT-116-Irv cells.Untreated HCT-116 cells: Autofluorescence intensity image (A), FLIM-phasor image (B) and the corresponding phasor plot (C) of representative untreated HCT-116 cells. Grey mask on the FLIM-phasor image, and grey circles on phasor plot correspond to autofluorescence phasors. HCT-116-Irv cells treated with 5μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (D), FLIM-phasor image (E), and the corresponding phasor plot (F) of representative elisidepsin treated HCT-116-Irv cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to FRET donor (Irv-OG488) molecular species outside the cell where donor and acceptor molecules are at distances longer than 70 Å. For these species, the lifetime of Irv-OG488 in the media is not affected by the presence of Irv-A555 molecules, and it was assigned to the donor-only (τD, donor unquenched) phasor in the phasor plot for FLIM-FRET-phasor analysis. The lifetimes inside the cells have not changed substantially with respect to untreated cells, showing the typical autofluorescence behavior (grey mask and cursor). Elisidepsin molecules were not detected inside HCT-116-Irv treated cells. HCT-116 cells treated with 4μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (G), FLIM-phasor image (H), and the corresponding phasor plot (I) of representative elisidepsin treated HCT-116 cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to Irv-OG488 molecular species inside the cell that are not complexed with other elisidepsin molecules, so do not change their lifetime (donor unquenched lifetime). Pink mask and pink cursor correspond to elisidepsin complexes, containing elisidepsin, Irv-OG488 and Irv-A555 molecules at distances shorter than 50 Å, mainly located in the plasma membrane of affected cells. The lifetime of the FRET donor (Irv-OG488) in these complexes (τDA) decreases due to FRET to neighboring FRET acceptor (Irv-A555) molecules, and their corresponding phasor fall in the quenching FRET trajectory. Excitation wavelength: 755 nm. Laser frequency (f): 80x106 s-1. Emission: FF01 520/35. Phasor plot: g and s axis represent the real and imaginary parts of the Fourier transform of the fluorescence impulse response I(t): ; ; ω = 2 π f; M and ϕ are respectively the modulus and the phase of the phasor.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140782.g002: Comparative interaction study of elisidepsin and the plasma membrane of HCT-116 and HCT-116-Irv cells.Untreated HCT-116 cells: Autofluorescence intensity image (A), FLIM-phasor image (B) and the corresponding phasor plot (C) of representative untreated HCT-116 cells. Grey mask on the FLIM-phasor image, and grey circles on phasor plot correspond to autofluorescence phasors. HCT-116-Irv cells treated with 5μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (D), FLIM-phasor image (E), and the corresponding phasor plot (F) of representative elisidepsin treated HCT-116-Irv cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to FRET donor (Irv-OG488) molecular species outside the cell where donor and acceptor molecules are at distances longer than 70 Å. For these species, the lifetime of Irv-OG488 in the media is not affected by the presence of Irv-A555 molecules, and it was assigned to the donor-only (τD, donor unquenched) phasor in the phasor plot for FLIM-FRET-phasor analysis. The lifetimes inside the cells have not changed substantially with respect to untreated cells, showing the typical autofluorescence behavior (grey mask and cursor). Elisidepsin molecules were not detected inside HCT-116-Irv treated cells. HCT-116 cells treated with 4μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (G), FLIM-phasor image (H), and the corresponding phasor plot (I) of representative elisidepsin treated HCT-116 cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to Irv-OG488 molecular species inside the cell that are not complexed with other elisidepsin molecules, so do not change their lifetime (donor unquenched lifetime). Pink mask and pink cursor correspond to elisidepsin complexes, containing elisidepsin, Irv-OG488 and Irv-A555 molecules at distances shorter than 50 Å, mainly located in the plasma membrane of affected cells. The lifetime of the FRET donor (Irv-OG488) in these complexes (τDA) decreases due to FRET to neighboring FRET acceptor (Irv-A555) molecules, and their corresponding phasor fall in the quenching FRET trajectory. Excitation wavelength: 755 nm. Laser frequency (f): 80x106 s-1. Emission: FF01 520/35. Phasor plot: g and s axis represent the real and imaginary parts of the Fourier transform of the fluorescence impulse response I(t): ; ; ω = 2 π f; M and ϕ are respectively the modulus and the phase of the phasor.

Mentions: Fig 2A shows the autofluorescence intensity image of representative untreated HCT-116 cells, measured in the donor channel, in the same experimental conditions as the FRET samples. Fig 2B shows the reciprocal selection of the grey autofluorescence cursors from the phasor plot of the same cells, represented in Fig 2C.


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)

Comparative interaction study of elisidepsin and the plasma membrane of HCT-116 and HCT-116-Irv cells.Untreated HCT-116 cells: Autofluorescence intensity image (A), FLIM-phasor image (B) and the corresponding phasor plot (C) of representative untreated HCT-116 cells. Grey mask on the FLIM-phasor image, and grey circles on phasor plot correspond to autofluorescence phasors. HCT-116-Irv cells treated with 5μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (D), FLIM-phasor image (E), and the corresponding phasor plot (F) of representative elisidepsin treated HCT-116-Irv cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to FRET donor (Irv-OG488) molecular species outside the cell where donor and acceptor molecules are at distances longer than 70 Å. For these species, the lifetime of Irv-OG488 in the media is not affected by the presence of Irv-A555 molecules, and it was assigned to the donor-only (τD, donor unquenched) phasor in the phasor plot for FLIM-FRET-phasor analysis. The lifetimes inside the cells have not changed substantially with respect to untreated cells, showing the typical autofluorescence behavior (grey mask and cursor). Elisidepsin molecules were not detected inside HCT-116-Irv treated cells. HCT-116 cells treated with 4μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (G), FLIM-phasor image (H), and the corresponding phasor plot (I) of representative elisidepsin treated HCT-116 cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to Irv-OG488 molecular species inside the cell that are not complexed with other elisidepsin molecules, so do not change their lifetime (donor unquenched lifetime). Pink mask and pink cursor correspond to elisidepsin complexes, containing elisidepsin, Irv-OG488 and Irv-A555 molecules at distances shorter than 50 Å, mainly located in the plasma membrane of affected cells. The lifetime of the FRET donor (Irv-OG488) in these complexes (τDA) decreases due to FRET to neighboring FRET acceptor (Irv-A555) molecules, and their corresponding phasor fall in the quenching FRET trajectory. Excitation wavelength: 755 nm. Laser frequency (f): 80x106 s-1. Emission: FF01 520/35. Phasor plot: g and s axis represent the real and imaginary parts of the Fourier transform of the fluorescence impulse response I(t): ; ; ω = 2 π f; M and ϕ are respectively the modulus and the phase of the phasor.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140782.g002: Comparative interaction study of elisidepsin and the plasma membrane of HCT-116 and HCT-116-Irv cells.Untreated HCT-116 cells: Autofluorescence intensity image (A), FLIM-phasor image (B) and the corresponding phasor plot (C) of representative untreated HCT-116 cells. Grey mask on the FLIM-phasor image, and grey circles on phasor plot correspond to autofluorescence phasors. HCT-116-Irv cells treated with 5μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (D), FLIM-phasor image (E), and the corresponding phasor plot (F) of representative elisidepsin treated HCT-116-Irv cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to FRET donor (Irv-OG488) molecular species outside the cell where donor and acceptor molecules are at distances longer than 70 Å. For these species, the lifetime of Irv-OG488 in the media is not affected by the presence of Irv-A555 molecules, and it was assigned to the donor-only (τD, donor unquenched) phasor in the phasor plot for FLIM-FRET-phasor analysis. The lifetimes inside the cells have not changed substantially with respect to untreated cells, showing the typical autofluorescence behavior (grey mask and cursor). Elisidepsin molecules were not detected inside HCT-116-Irv treated cells. HCT-116 cells treated with 4μM total elisidepsin (FRET donor: Irv-OG488 200 nM, FRET acceptor: Irv-A555 800 nM, elisidepsin 3 μM, 1:4:15): Fluorescence intensity image (G), FLIM-phasor image (H), and the corresponding phasor plot (I) of representative elisidepsin treated HCT-116 cells. Green mask on FLIM-phasor image and green circles on phasor plot correspond to Irv-OG488 molecular species inside the cell that are not complexed with other elisidepsin molecules, so do not change their lifetime (donor unquenched lifetime). Pink mask and pink cursor correspond to elisidepsin complexes, containing elisidepsin, Irv-OG488 and Irv-A555 molecules at distances shorter than 50 Å, mainly located in the plasma membrane of affected cells. The lifetime of the FRET donor (Irv-OG488) in these complexes (τDA) decreases due to FRET to neighboring FRET acceptor (Irv-A555) molecules, and their corresponding phasor fall in the quenching FRET trajectory. Excitation wavelength: 755 nm. Laser frequency (f): 80x106 s-1. Emission: FF01 520/35. Phasor plot: g and s axis represent the real and imaginary parts of the Fourier transform of the fluorescence impulse response I(t): ; ; ω = 2 π f; M and ϕ are respectively the modulus and the phase of the phasor.
Mentions: Fig 2A shows the autofluorescence intensity image of representative untreated HCT-116 cells, measured in the donor channel, in the same experimental conditions as the FRET samples. Fig 2B shows the reciprocal selection of the grey autofluorescence cursors from the phasor plot of the same cells, represented in Fig 2C.

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