Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis

Edelfosine induces breakage of kinetoplast DNA prior to nuclear DNA breakdown, and accumulates in mitochondria in <i>Leishmania</i> parasites and cancer cells.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes were untreated (Control) or treated with 10 μM edelfosine (EDLF) for 6 and 9 h, and then analyzed by confocal microscopy for propidium iodide (PI) staining and TUNEL assay. The positions of the nucleus (N) and kinetoplast (K) are indicated by arrows. Merging of PI and TUNEL panels (Merge) shows the DNA-containing organelles with DNA disruption in yellow. The corresponding differential interference contrast (DIC) images were included in the Merge panels to highlight parasite morphology and facilitate kinetoplast identification. (<b>B</b>) <i>L</i>. <i>panamensis</i> promastigotes and (<b>C</b>) HeLa cancer cells were incubated with 10 μM PTE-ET (blue fluorescence) for 1 h, 100 nM MitoTracker (red fluorescence) for 20 min to localize mitochondria, and then analyzed by fluorescence microscopy. Areas of colocalization between mitochondria and PTE-ET in merge panels are purple. The corresponding differential interference contrast (DIC) images are also shown. Images are representative of three independent experiments. Bar, 20 μm.</p

F_OF₁-ATPase recruitment into rafts in the antileishmanial activity of edelfosine and oligomycin inhibitory effect on cytotoxicity.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes untreated (Control) and treated with 10 μM edelfosine for 9 h were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Individual fractions were electrophoresed, and location of GM1 was determined. (<b>B</b>) Proteins from lipid rafts of untreated control and edelfosine-treated <i>L</i>. <i>panamensis</i> promastigotes were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial F_OF₁-ATP synthase β subunit is indicated by an arrow. (<b>C</b>) Mass spectrum of the tryptic peptides of the F_OF₁-ATP synthase β subunit spot. Mass values (m/z) and putative amino acid position assignments are indicated above peaks. (<i>Inset</i>) Peptide coverage map of <i>Leishmania</i> F_OF₁-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (<b>D</b>) <i>L</i>. <i>panamensis</i> were untreated (Control) or preincubated with 1 μM oligomycin for 1 h and then incubated in the absence or presence of 10 μM edelfosine for 9 h, and ΔΨ_m disruption (Low ΔΨ_m) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. (*) <i>P</i><0.05. (**) <i>P</i><0.01.</p

Schematic model of mitochondria involvement in the killing activity of edelfosine against <i>Leishmania</i> parasites and tumor cells.

<p>This is a schematic diagram to portray one currently plausible mechanism of how edelfosine induces cell death in <i>Leishmania</i> parasites and tumor cells through its main mitochondrial localization in both biological systems. Protection of mitochondria by Bcl-X_L ectopic expression restrains cell death. See text for details.</p

Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.

<p>Inhibition of apoptosis-like cell death by ectopic expression of Bcl-X_L in <i>L</i>. <i>infantum</i> promastigotes and HeLa tumor cells.</p

Edelfosine is taken up by macrophages and inhibits macrophage-derived inflammatory mediators.

<p>(<b>A</b>) Incorporation of edelfosine (EDLF) in mouse bone marrow-derived macrophages (BMM) and mouse RAW 309 Cr.1 tumor macrophage cell line. 10⁶ cells were incubated with 10 μM edelfosine (containing 0.05 μCi [³H]edelfosine) for the indicated times to measure drug uptake. (<b>B</b>) Edelfosine is cytotoxic for transformed macrophages but spares BMM. 2 x 10⁶ cells were incubated for 24 h in the absence or presence of the indicated concentrations of edelfosine (EDLF), and cytotoxicity was determined by the WST-1 reduction method. (<b>C</b>) Edelfosine inhibits superoxide anion generation in BMM. Superoxide anion was measured as lucigenin-dependent chemiluminescence (relative light units, RLU) in untreated control (C) or edelfosine (EDLF)-treated BMM that were incubated with medium alone or zymosan to induce the respiratory burst. (<b>D-F</b>) BMM from edelfosine-fed mice show a decreased generation of inflammatory mediators. BMM from untreated control mice (C) and from mice given orally edelfosine (EDLF) for two weeks were analyzed for their capacity to generate zymosan-induced superoxide anion (<b>D</b>), LPS-induced nitric oxide (<b>E</b>), and IL-12+IL-18-induced IFN-γ (<b>F</b>). Cells incubated with medium alone were run in parallel as a negative control of each assay. Data shown are means ± SD of five independent determinations. Asterisks indicate values that are significantly different from those of control mice (comparison between the black histograms of control and edelfosine-treated groups) at <i>P</i><0.05 (*) and <i>P</i><0.01 (**).</p

Involvement of lipid rafts in both antileishmanial and anticancer activities and in edelfosine uptake in <i>Leishmania</i> promastigotes and cancer cells.

<p>(<b>A</b>) <i>L</i>. <i>panamensis</i> promastigotes and T-cell leukemia Jurkat cells were untreated (Control) or pretreated with MCD, and then incubated in the absence or presence of 10 μM edelfosine for 24 h. Percentage of hypodiploid cells were measured by flow cytometry. (<b>B</b>) <i>L</i>. <i>panamensis</i> promastigotes and T-cell leukemia Jurkat cells were untreated (Control) or pretreated with MCD and then incubated with 10 μM [³H]edelfosine for 1 h. Drug uptake was determined as shown in the Materials and Methods section. Data shown are means ± SD of three independent experiments performed. Asterisks denote that the differences between the indicated groups are statistically significant. (**) <i>P</i><0.01. (***) <i>P</i><0.001.</p

Chemical structures of APLs and edelfosine fluorescent analog PTE-ET used in this manuscript.

<p>The chemical structure of phosphatidylcholine (PC) is also shown. Me, methyl group.</p

F_OF₁-ATPase recruitment in lipid rafts of Jurkat cancer cells after edelfosine incubation and oligomycin inhibitory effect on cytotoxicity.

<p>(<b>A</b>) Jurkat cells untreated (Control) and treated with 10 μM edelfosine for 9 h were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Individual fractions were subjected to SDS-PAGE, and location of GM1 was determined using CTx B subunit conjugated with horseradish peroxidase. (<b>B</b>) Proteins from lipid rafts of untreated control and edelfosine-treated Jurkat cells were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial F_OF₁-ATP synthase β subunit is indicated by an arrow. (<b>C</b>) Mass spectrum of the tryptic peptides of the F_OF₁-ATP synthase β subunit spot. Mass value (m/z) and putative amino acid position assignments are indicated above peaks. (<i>Inset</i>) Peptide coverage map of human F_OF₁-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (<b>D</b>) Jurkat cells were untreated (Control) or preincubated with 10 μM oligomycin for 1 h and then incubated in the absence or presence of 10 μM edelfosine for 9 h, and ΔΨ_m disruption (Low ΔΨ_m) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. Asterisks denote that the differences between the indicated groups are statistically significant. (**) <i>P</i><0.01.</p