ABSTRACT
The alkyl-lysophospholipids edelfosine and miltefosine induce apoptosis in Leishmania infantum promastigotes. The finding that edelfosine-induced cell death can be regulated by the ectopic expression of the antiapoptotic and proapoptotic members of the Bcl-2 family of proteins Bcl-XL and Hrk suggests that this process is similar to apoptosis in eukaryotic cells.
Miltefosine, edelfosine, and several other alkyl-lysophospholipids originally developed as anticancer agents have proved to be effective antileishmanial drugs (4, 7). An increasing number of reports have shown that culture saturation (3), heat shock treatment (1, 5), and exposure to chemotherapeutic agents such as glucantime (8) and miltefosine (6, 9) induce features of programmed cell death (PCD) on Leishmania parasites. To gain further insight into this process, we have investigated whether proteins that regulate apoptosis in higher eukaryotes have any effect on edelfosine-induced PCD in Leishmania infantum.
Exponentially growing (2 × 106 parasites/ml) L. infantum (M/CAN/ES/96/BCN150 MON-1) promastigotes were subjected to incubation with increasing concentrations of either edelfosine or miltefosine for 24 h, and the percentage of dead parasites was evaluated by flow cytometry after the parasites were stained with 5 μM propidium iodide (PI) (1). The increase in drug concentrations correlated with the percentages of PI staining-positive parasites, which was an indication of the cytotoxic effects of the drugs (Fig. 1A and B). The estimated 50% lethal doses were 27 μM (R2 > 0.99) for edelfosine and 47 μM (R2 > 0.99) for miltefosine. The DNA content in the drug-treated parasites was analyzed by flow cytometry (1). Both drugs induced a concentration-dependent process of DNA degradation, as shown by the progressive increase in the percentages of hypodiploid cells (Fig. 1C and D). The results also reveal that edelfosine has greater potency than miltefosine against L. infantum promastigotes.
Changes in the mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry after staining of the parasites with tetramethylrhodamine methyl ester (TMRM) (2). As already shown for heat-induced cell death (1), edelfosine or miltefosine treatment causes a nonhomogeneous effect on the ΔΨm of the parasites (Fig. 1E and F). After 24 h, the parasites can be divided into two populations according to their mitochondrial status: the first one is composed of parasites with a reduced ΔΨm, and the second one is composed of a population in which the parasites show a clear increase in ΔΨm compared to that for the untreated controls. This increase in ΔΨm can be observed as soon as 30 min after drug treatment (data not shown) and may be interpreted as a strategy that the cell uses to obtain enough energy to develop the apoptotic process. The observed decrease in ΔΨm, together with the presence of a sub-G1 peak in the cell cycle analysis, is suggestive of a death process similar to that of apoptosis in response to edelfosine or miltefosine. The induction of apoptosis following miltefosine treatment has already been reported in Leishmania donovani (6, 9, 10).
Apoptosis in higher eukaryotes is regulated by members of the Bcl-2 family of proteins (11). When parasites transfected with a pX63-Neo vector containing the bcl-XL-coding sequence were treated with edelfosine, significant decreases in the number of hypodiploid cells (Fig. 2A) and the number of cells present in the population with a low ΔΨm (Fig. 2B) were observed. Our results clearly indicate that both apoptotic processes are partially reverted by Bcl-XL expression.
To check whether a proapoptotic member of the Bcl-2 family could also modify the response of the cell population to edelfosine, L. infantum promastigotes were transfected with a pX63-Neo vector containing the hrk-coding sequence. Significant increases in the percentage of hypodiploid parasites (Fig. 2A) and the number of promastigotes with low ΔΨm values (Fig. 2B) were observed when these transfected promastigotes were treated with 40 μM edelfosine for 24 h.
The effect of Bcl-XL or Hrk expression was further confirmed by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick-end-labeling (TUNEL) method (Fig. 2C). Statistical analysis of the images (Table 1) revealed that the percentages of fluorescent cells in nontransfected parasites and in parasites transfected with an empty vector (pX63) were similar (42.5% and 47.4%, respectively), whereas only 26.7% of the parasites expressing the antiapoptotic Bcl-XL was TUNEL assay positive. On the other hand, 92.5% of the parasites expressing the proapoptotic Hrk were positive for staining by the TUNEL assay, which confirms the proapoptotic effect of the expression of this protein in Leishmania parasites.
We have previously shown that Bcl-XL is able to partially revert heat shock-induced cell death in L. infantum promastigotes (1). The prosurvival activity of this protein was also confirmed in the present work. On the other hand, the expression of Hrk severely impairs the ability of L. infantum promastigotes to survive in the presence of edelfosine. Conservation of the antiapoptotic and proapoptotic activities of Bcl-XL and Hrk, respectively, in Leishmania promastigotes may be considered an indication of the presence of BH3-bearing proteins, which have been implicated in the regulation of cell death, in the Leishmania proteome.
Leishmanicidal effects of edelfosine and miltefosine. (A and B) Percentages of PI-positive L. infantum promastigotes after 24 h of treatment with increasing concentrations of edelfosine (A) or miltefosine (B); (C and D) percentages of hypodiploid promastigotes after 24 h of treatment with increasing concentrations of edelfosine (C) or miltefosine (D); (E and F) monoparametric histograms comparing the relative TMRM-derived fluorescence of control and 45 μM edelfosine-treated (E) or 45 μM miltefosine-treated (F) promastigotes. LD50, 50% lethal dose.
Proteins from the Bcl-2 family modulate edelfosine-induced cell death in L. infantum promastigotes. Transfected L. infantum strains carrying the empty vector (pX63) or the genes encoding the antiapoptotic Bcl-XL (BclXL) or proapoptotic Hrk were exposed to 40 μM edelfosine for 24 h. Cell death was measured in terms of DNA degradation (A) and the decrease in the mitochondrial membrane potential (B). Light gray bars, untreated parasites; black bars, edelfosine (Edf)-treated parasites; lines above the bars, standard deviations (n = 3); *, P < 0.05. (C) Confocal microscopy images of L. infantum strains transfected with empty pX63 (L. infantum), pX63-bcl-XL (L. infantum-Bcl-XL), or pX63-hrk (L. infantum-Hrk) and incubated in the absence of edelfosine (control) or exposed to 40 μM edelfosine for 24 h and then analyzed by the TUNEL assay, PI staining, and differential interference contrast (DIC). (D) Western blot analysis of the Leishmania strains expressing either Bcl-XL or HrK. Detection of the ectopic proteins expressed was carried out with anti-HA (Bcl-XL) or anti-FLAG (Hrk) antibodies. Antibodies against the Kmp-11 protein were used to confirm that the same amount of protein was loaded in each lane.
Measurement of apoptosis by TUNEL techniquea
ACKNOWLEDGMENTS
This work was supported by grants FIS-PI021052, FIS-FEDER 04/0843, SAF 2006-12713-CO2, and CAM-UAH2005/035; by the Fundación de Investigación Médica Mutua Madrileña; by the Fundación la Caixa (grant BM05-30-0); and by a research grant from the Vicerectoría de Investigación, Universidad de Antioquia.
FOOTNOTES
- Received 25 December 2007.
- Returned for modification 25 March 2008.
- Accepted 7 July 2008.
↵▿ Published ahead of print on 21 July 2008.
- American Society for Microbiology
