Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity

Abstract

b Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ϳ70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture. L eishmaniasis is caused by protozoan parasites of over 20 Leishmania species and is transmitted to humans via the bite of infected female sand flies. There are three main types of leishmaniasis: cutaneous (the most common form), visceral (the most severe clinical manifestation, which is fatal if untreated), and mucocutaneous. According to a recent report from the World Health Organization (WHO) (1), there are an estimated 1.3 million new cases of leishmaniasis worldwide and an estimated 20,000 to 30,000 deaths caused by these parasites annually.Miltefosine (MT) was the first oral drug approved for use for the treatment of visceral and cutaneous leishmaniasis and is currently registered for leishmaniasis treatment in India (2002, as Impavido), Germany (2004), and Colombia (2005) (2). This drug is also used clinically for the topical treatment of skin metastases associated with breast cancer and cutaneous lymphoma (3). MT has shown high cure rates in the treatment of visceral (4), cutaneous (5), and mucocutaneous (6) leishmaniasis. Although many studies have been conducted to identify the mechanisms underlying the action of MT against tumor cells (3) and parasites (7), these mechanisms have not yet been determined. However, due to its chemical structure, which provides MT with a high membrane affinity, the primary molecular targets of MT...

Effects of terpenes on fluidity and lipid extraction in phospholipid membranes

“…The IC 50 values of the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole were 0.008, 0.549, 0.678 and 4.697 mM, respectively [16]. This result demonstrated that the in vitro cytotoxicity of nerolidol was similar to that of miltefosine, which is the first effective oral drug approved for the treatment of visceral and cutaneous leishmaniasis [32]. Overall, these IC 50 values and lysis results partially agree with the results obtained in this work.…”

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

“…3B) indicated that only the 2×10 9 cells/mL concentration was sufficiently concentrated for an accurate calculation. At this cell concentration, the nerolidol IC 50 was 0.32×10 9 terpene molecules/cell, which was similar to that of miltefosine under the same experimental conditions [26]. To obtain the best fit of the IC 50 for nerolidol and (+)-limonene to the theoretical curves given by eqs.…”

“…Tea tree oil and its major active terpene component, terpinen-4-ol, demonstrated antiproliferative activity against two murine tumor cell lines, AE17 mesothelioma and B16 melanoma, with IC 50 values equal to or greater than 0.01% (∼588 µM), and they induced necrotic cell death and cell cycle arrest in both tumor cell lines [38]. Miltefosine is also able to increase membrane fluidity and induce cell lysis in L. amazonensis promastigotes at concentrations close to its IC 50 [26]. Interestingly, miltefosine has been shown to reduce the mitochondrial membrane potential and inhibit cytochrome c oxidase, changes that are linked to the apoptosis-like cell death [39].…”

Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

“…Our data shows that T. brucei has limited tolerance for TbVAMP7B depletion, presumably due to impairment of endosomal-lysosomal fusion (45). Intriguingly, exposure to low concentration miltefosine complements the growth defect seen following TbVAMP7B depletion, suggesting that miltefosine treatment is able to promote vesicle membrane fusion in the endocytic system, a possible consequence of the enhanced membrane fluidity seen upon miltefosine exposure (60). TbVAMP7B has also recently been identified as a putative T. brucei apolipoprotein-L1 sensitivity determinant (61), and other workers have highlighted the importance of the intracellular transit of apoL1-carrying membrane to trypanolysis (62, 63).…”