Perifosine Mechanisms of Action in Leishmania Species

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Leishmania species are obligate intracellular parasites that are transmitted to the mammalian host by the bites of infected sand flies. Cutaneous leishmaniasis (CL), the most common form of leishmaniasis, is a group of diseases with a varied spectrum of clinical manifestations that range from small cutaneous nodules to wide mucosal tissue destruction. Visceral leishmaniasis (VL), characterized by prolonged fever and splenomegaly, is the most severe form, and it is often fatal if untreated (1).

The World Health Organization (WHO) estimates that 1.5 million cases of CL and 500,000 cases of VL occur every year in 82 countries. CL is endemic in more than 70 countries worldwide, and 90% of cases occur in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi Arabia, and Syria. VL presents in 65 countries, and the majority (90%) of these cases occur in five countries, Bangladesh, India, Nepal, Sudan, and Brazil. Estimates indicate that there are approximately 350 million people at risk for acquiring leishmaniasis, with 12 million currently infected worldwide (1).

The current available leishmanicidal treatments include pentavalent antimonials, amphotericin B, miltefosine, paromomycin, and pentamidine. These drugs can be administered alone or in combination with each other. However, these drugs are normally highly toxic, even at low doses. Furthermore, most of these treatments require several days of hospitalization because of their intravenous or parenteral manner of administration. The appearance of strains resistant to these active compounds presents a major problem for the current therapeutic measures against these parasites (2). Moreover, since there is no immediate prospect of a vaccine for leishmaniasis (3), there is an urgent need to develop novel leishmanicidal agents.

Several typical markers of mammalian apoptosis have been found in Leishmania, suggesting the existence of an apoptosis-like death in these organisms. The markers include cell shrinkage, nuclear chromatin condensation, DNA fragmentation, membrane blebbing, mitochondrial transmembrane potential loss, and phosphatidylserine exposure (4).

Perifosine was purchased from Cayman Chemical, and miltefosine, used as reference drug, was provided by Æterna Zentaris. In this study, strains of Leishmania amazonensis (MHOM/BR/77/LTB0016) and Leishmania donovani (MHOM/IN/90/GE1F8R) were used. The murine macrophage J774A.1 (ATCC TIB-67) cell line was also used to evaluate host cell toxicity.

For the activity assays against the promastigote stage of Leishmania spp., a colorimetric assay based on alamarBlue reagent (Invitrogen, Life Technologies) was used as previously described (5).

Activity assays against intracellular amastigotes were performed as previously described with minor modifications (6). For the toxicity assay, an LDH cytotoxicity detection kit (Roche Applied Science) was used (7). The results against the promastigote and intracellular amastigote stages are shown in Table 1, as are the cytotoxicities and selectivity indexes (SIs). The 50% inhibitory concentrations (IC₅₀s) are consistent with data obtained in previous studies (5, 8). A study by Glaser et al. (9) showed similar cytotoxicity data for miltefosine.

For measuring changes in the mitochondrial membrane potential (ΔΨm) of L. amazonensis and L. donovani, the JC-1 mitochondrial membrane potential assay kit (Cayman Chemical) was used. The ratio of the reading at 595 nm to the reading at 535 nm was considered the relative ΔΨm value (10). The two molecules induced decreases in the ΔΨm in both parasitic strains; the decrease was greater in the case of L. amazonensis (Fig. 1).

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FIG 1

(A) ATP levels of L. amazonensis and L. donovani after 24 h of incubation with the IC₉₀s of perifosine and miltefosine. (B) Changes in the mitochondrial membrane potential (ΔΨm) of L. amazonensis and L. donovani after 24 h of incubation with the IC₉₀ of perifosine and miltefosine. Error bars represent the standard deviation (SD). Each data point indicates the mean of three measurements. (C) Images of L. amazonensis incubated with JC-1 dye after 24 h of treatment with the IC₉₀ of perifosine; images of negative controls of parasites without treatment are shown on the upper left and upper right.

ATP levels were measured using the Cell Titer-Glo luminescent cell viability assay (Promega). This assay proved the strong effects of miltefosine and perifosine in decreasing ATP levels (Fig. 1). The apoptosis-like process in Leishmania spp. requires ATP to carry out all the involved procedures; however, it seems that ATP levels are maintained by either glycolysis and/or minimal mitochondrial activity to support the parasite metabolism until the conclusion of the apoptosis-like process (11).

The Tali apoptosis kit (annexin V Alexa Fluor 488 and propidium iodide) (Life Technologies) double-staining assay was performed according to manufacturer instructions using a Tali image-based cytometer. Figure 2 shows all the obtained results. Moreover, the differences in the amounts of annexin V observed between the two strains of Leishmania (amounts were greater in L. amazonensis) might have been due to differences in the amount of polysaccharides (PSs) present in the membrane of the parasites (12).

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FIG 2

(A) Results of phosphatidylserine exposure after 48 h of incubation with the IC₉₀s of perifosine and miltefosine. Error bars represent the standard deviation (SD). Each data point indicates the mean of three measurements. Images of L. amazonensis (B) and L. donovani (C) captured using a Tali image-based cytometer.

Plasma membrane permeability was measured using SYTOX Green (Molecular Probes). The results confirmed that the plasma membrane integrity was not altered in the treated parasites, at least not in the first 6 h of treatment (Fig. 3). This provided us with important data with which to distinguish between apoptosis and necrosis, considering the rupture of the plasma membrane, which is a morphological feature of necrosis (13). Also, this assay might indicate that the target of these molecules is not likely to be primarily the plasma membrane of the parasites (14).

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FIG 3

(A) Plasma membrane permeability assay results for L. amazonensis and L. donovani. Triton X-100 was added to obtain 100% permeabilized cells, and amphotericin B (10 μM) was used as positive control. (B) Changes in the relative expression of phosphorylated kinases on L. donovani after 24 h of incubation with the IC₉₀ of perifosine. Error bars represent the standard deviation (SD). Each data point indicates the mean of three measurements.

The Human Phospho-Kinase Array 1 proteome profiler (R&D Systems) was used for the phosphorylation analysis of different kinases during the promastigote stage and was developed by chemiluminescence using ChemiDoc XRS+ image capture (Bio-Rad). The Q-View software (Quansys Biosciences) was used to measure pixel density. All statistical analyses were performed with Sigma Plot 12.0 (Systat Software). After incubation of L. donovani with perifosine, one protein presented a difference in its relative expression, a decrease in the phosphorylation levels of Akt (phosphorylation of S473) necessary for its activation (Fig. 3). Miltefosine is an inhibitor of Akt in Leishmania (15), and perifosine also inhibits Akt protein in human cancer cells (16). These facts lead us to believe that perifosine has the same Akt-inhibiting capacity in Leishmania, which leads to the induction of an apoptosis-like cell death that is known to occur with miltefosine in L. amazonensis (17) and L. donovani (18).

Previous results obtained in our laboratory on alkylphospholipids showed that perifosine was superior to miltefosine and edelfosine in killing Leishmania promastigotes in vitro (5), and we also showed that perifosine was superior to other alkylphospholipids in an in vivo mouse model (19). We now conclude that perifosine induces an apoptosis-like process in L. amazonensis and L. donovani at very low doses, which makes perifosine a promising candidate for further study and possible therapeutic use in humans.