Fig 1.
Chemical structures of APLs and edelfosine fluorescent analog PTE-ET used in this manuscript.
The chemical structure of phosphatidylcholine (PC) is also shown. Me, methyl group.
Fig 2.
In vitro and in vivo antileishmanial activity of the antitumor ether phospholipid edelfosine.
Apoptosis-like cell death was quantitated by flow cytometry as percentage of hypodiploid cells (sub-G0/G1) in different Leishmania spp. promastigotes (A) and human cancer cell lines (myeloid leukemia HL-60, multiple myeloma MM144, and cervical carcinoma HeLa) (B), following 24-h incubation with distinct APLs (10 μM). Untreated control cells were run in parallel. ErPC, erucylphosphocholine. (C) Time-course of 10 μM edelfosine-induced apoptosis-like cell death (% hypodiploid cells) in L. panamensis promastigotes. Untreated control parasites were run in parallel. (D) Induction of apoptosis-like cell death in L. panamensis axenic amastigotes treated with the indicated concentrations of edelfosine for 16 h. (E) GFP-L. panamensis (GFP-Lp)-infected J774 macrophages were incubated for 1 h with 10 μM PTE-ET and analyzed by fluorescence microscopy. Incubation of GFP-Lp-infected J774 macrophages with 10 μM edelfosine for 24 h decreased parasite infection (F) and the number of parasites per macrophage (G), as compared to untreated infected cells (Control). (H) Murine BMM were infected with L. panamensis and subsequently treated with distinct APLs (10 μM) or vehicle (Control). Parasite load was measured after 3-day incubation. (I-K) Hamsters were inoculated in the nose with L. panamensis promastigotes, and then treated orally with edelfosine (20 mg/kg, n = 8) or with water vehicle (Control) for 28 days. Evolution index during treatment (I) and parasite load in the nose at the end of the 4-week treatment (J) were determined. Edelfosine treatment dramatically reduced nose inflammation and damage at the end of treatment (K). Data shown are means ± SD or representative of three independent experiments performed. Asterisks indicate that the differences between control and edelfosine-treated groups are statistically significant. (*) P<0.05. (**) P<0.01.
Fig 3.
Edelfosine is taken up by macrophages and inhibits macrophage-derived inflammatory mediators.
(A) 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) 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. (C) 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. (D-F) 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 (D), LPS-induced nitric oxide (E), and IL-12+IL-18-induced IFN-γ (F). 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 P<0.05 (*) and P<0.01 (**).
Fig 4.
Edelfosine induces breakage of kinetoplast DNA prior to nuclear DNA breakdown, and accumulates in mitochondria in Leishmania parasites and cancer cells.
(A) L. panamensis 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) L. panamensis promastigotes and (C) 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.
Fig 5.
Involvement of mitochondria and ROS generation in edelfosine-induced cell death in Leishmania parasites and cancer cells.
(A) L. panamensis promastigotes were untreated (Control) or treated with 10 μM edelfosine at the indicated times, and cells with disrupted ΔΨm (DiOC6(3)low) and ROS production (HE→Eth) were measured by flow cytometry. The numbers in each quadrant refer to the percentages of cells in each population. (B) L. panamensis promastigotes untreated (Control) and treated with edelfosine (EDLF) for 3 h were incubated with 2 μM HE and 10 μg/ml Hoechst 33342, and then analyzed by fluorescence microscopy. (C) L. panamensis promastigotes and (D) Jurkat cells were preincubated with 10 μg/ml CsA for 1 h, or with 10 mM NAC or 10 mM GSH for 2 h, and then incubated in the absence or presence of 10 μM edelfosine for 9 h. After treatment, the percentage of hypodiploid cells was analyzed by flow cytometry. Untreated control cells were run in parallel. (E) L. panamensis promastigotes and (F) Jurkat cells were preincubated with 10 μM rotenone, 5 mM malonate, 10 μM antimycin A, 1.5 mM azide, 50 μM CCCP or with 1 and 10 μM oligomycin (L. panamensis and Jurkat cells, respectively) for 1 h, and then incubated in the absence or presence of 10 μM edelfosine for 9 h. After treatment, cells producing ROS were quantified by flow cytometry. Untreated control cells were run in parallel. Data shown are means ± SD or representative of three independent experiments performed. Asterisks denote that the differences between the indicated groups (C and D) and with control cells (E and F) are statistically significant. (*) P<0.05. (**) P<0.01.
Table 1.
Inhibition of apoptosis-like cell death by ectopic expression of Bcl-XL in L. infantum promastigotes and HeLa tumor cells.
Fig 6.
Involvement of lipid rafts in both antileishmanial and anticancer activities and in edelfosine uptake in Leishmania promastigotes and cancer cells.
(A) L. panamensis 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) L. panamensis 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. (**) P<0.01. (***) P<0.001.
Fig 7.
FOF1-ATPase recruitment into rafts in the antileishmanial activity of edelfosine and oligomycin inhibitory effect on cytotoxicity.
(A) L. panamensis 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) Proteins from lipid rafts of untreated control and edelfosine-treated L. panamensis promastigotes were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial FOF1-ATP synthase β subunit is indicated by an arrow. (C) Mass spectrum of the tryptic peptides of the FOF1-ATP synthase β subunit spot. Mass values (m/z) and putative amino acid position assignments are indicated above peaks. (Inset) Peptide coverage map of Leishmania FOF1-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (D) L. panamensis 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 ΔΨm disruption (Low ΔΨm) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. (*) P<0.05. (**) P<0.01.
Fig 8.
FOF1-ATPase recruitment in lipid rafts of Jurkat cancer cells after edelfosine incubation and oligomycin inhibitory effect on cytotoxicity.
(A) 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) 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 FOF1-ATP synthase β subunit is indicated by an arrow. (C) Mass spectrum of the tryptic peptides of the FOF1-ATP synthase β subunit spot. Mass value (m/z) and putative amino acid position assignments are indicated above peaks. (Inset) Peptide coverage map of human FOF1-ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. (D) 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 ΔΨm disruption (Low ΔΨm) 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. (**) P<0.01.
Fig 9.
Edelfosine resistance of atp7Δ mutant in Saccharomyces cerevisiae yeast.
Growth curves of wild-type (BY4741) (A), ATP7 knock-out mutant (atp7Δ) (B) and the mutant strain harboring the corresponding cognate gene (atp7Δ+pRS416-ATP7) (C) in SDC medium containing different concentrations of edelfosine. The cultures were carried out in duplicate and in at least three independent experiments. Data shown are mean values of three independent experiments. SD values were less than 10% of the mean values.
Fig 10.
Intracellular ATP measurements.
L. panamensis promastigotes were treated with 10 μM edelfosine or 50 μM CCCP for different incubation times and processed for ATP intracellular content. Results are expressed as percentage of the amount of ATP found in untreated control cells. Data shown are means ± SD of three independent experiments.
Fig 11.
Schematic model of mitochondria involvement in the killing activity of edelfosine against Leishmania parasites and tumor cells.
This is a schematic diagram to portray one currently plausible mechanism of how edelfosine induces cell death in Leishmania parasites and tumor cells through its main mitochondrial localization in both biological systems. Protection of mitochondria by Bcl-XL ectopic expression restrains cell death. See text for details.