Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100S, S9—S16

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Mechanisms of action of lysophospholipid analoguesagainst trypanosomatid parasites

Julio A. Urbina ∗

Laboratorio de Quımica Biologica, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientificas,Apartado 21827, Caracas 1020A, VenezuelaAvailable online 22 August 2006

KEYWORDSLeishmaniasis;Lysophospholipidanalogues;Antiparasitic activity;Trypanosomatid;Phosphatidylcholine;Apoptosis

Summary Lysophospholipid analogues (LPAs) comprise a class of metabolically stable com-pounds that have been developed as anticancer agents for over two decades, but which havealso potent and selective antiparasitic activity, particularly against trypanosomatid parasitessuch as Leishmania and Trypanosoma cruzi, both in vitro and in vivo. The in vivo activitiesof LPAs result from direct effects on their target cells and are not dependent on a functionalimmune system. Because of their chemical nature, LPAs have a potential for interaction with avariety of subcellular structures and biochemical pathways. However, in mammalian cells LPA-induced growth inhibition and programmed cell death is usually associated with a blockade ofphosphatidylcholine (PC) biosynthesis at the level of CTP: phosphocholine citidyltransferase,probably through an increase of cellular ceramide levels due to depressed sphingomyelin syn-thesis. Although in trypanosomatid parasites much less information is available, inhibition of PCbiosynthesis by LPA has also been documented but at the level of phosphatidylethanolamine N-methyl-transferase, as well as LPA-induced classical apoptotic phenomena. The higher activityof LPAs as inhibitors of PC biosynthesis in parasites than in mammalian cells, probably due to

different biochemical pathways involved in the two types of cells, could explain their selectiveantiparasitic action in vivo.

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1. Introduction

Lysophospholipid analogues (LPAs) comprise a broad classof metabolically stable compounds, which include alkyl-phosphocholines, such as hexadecylphosphocholine (milte-fosine), and alkyl-glycerophosphocholines, such as ET-18-

OCH3 (edelfosine) and BM 41 440 (ilmofosine) (see Figure 1).There has been a significant effort over the past 25 years toresearch and develop LPAs as anticancer agents (Kuhlencordet al., 1992; Unger et al., 1992; Wieder et al., 1999).

∗ Tel.: +58 212 5041660; fax: +58 212 5041093.E-mail address: jaurbina@ivic.ve.

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0035-9203/$ — see front matter © 2006 Royal Society of Tropical Medicindoi:10.1016/j.trstmh.2006.03.010

l Medicine and Hygiene. Published by Elsevier Ltd. All rights

It has been shown that these compounds are potent initro growth inhibitors of transformed cell lines, as well aseing selective antitumour agents in several animal mod-ls (Unger et al., 1992). Clinical studies were performed forany years in cancer patients, but with limited results (deastro et al., 2004; Wieder et al., 1999). Thus, althoughdelfosine is effective as a purging agent in autologous bonearrow transplantation (Ruiter et al., 2001), the anticancer

ctivity of miltefosine was shown to be limited due to unac-

eptable gastrointestinal toxicity and limited activity wheniven by the oral route (Planting et al., 1993; Verweij etl., 1992). However, this compound has been shown to beffective as a topical formulation against cutaneous lym-homas and has been licensed in Europe for the treatment

e and Hygiene. Published by Elsevier Ltd. All rights reserved.

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Anttecompounds retain their antileishmanial activities in sev-eral immunodeficient mouse models (Escobar et al., 2001;

igure 1 Chemical structure of anticancer and antiparasiticysophospholipid analogues. Reproduced from Lira et al. (2001),ith permission.

f skin metastases due to breast cancer (Smorenburg etl., 2000). Similar findings have been obtained with otherPAs and it has been concluded that as anticancer agentsphospholipid analogs have a therapeutic benefit only asopical formulations or in ex-vivo applications’ (Wieder etl., 1999). Nevertheless, clinical investigations continue onhe potential safety and efficacy of LPAs, such as ilmo-osine, as systemic anticancer agents (Giantonio et al.,004).

In a parallel development, LPAs were shown to haveotent and selective antiparasitic activity, particularlygainst trypanosomatid parasites such as Leishmania spp.nd Trypanosoma cruzi (reviewed in de Castro et al., 2004).uch results led to clinical studies of orally administerediltefosine in patients with visceral leishmaniasis, includ-

ng those infected with antimony-resistant strains of L.onovani. The very high response rate and relatively mildide-effects have led many investigators to consider thisompound as the long-awaited oral treatment for this life-hreatening condition and its registration in India and Ger-any for this indication as well as in Colombia for cutaneous

eishmaniasis (Bhattacharya et al., 2004; Croft et al., 2003;e Castro et al., 2004; Jha et al., 1999a, 1999b; Murray,001, 2004a, 2004b; Sundar et al., 1998, 1999a, 1999b, 2000,002, 2003; Thakur et al., 2000).

Although the molecular mechanisms underlying theelective anticancer activities of LPAs have been the sub-

ect of many studies and controversies (Arthur and Bittman,998; de Castro et al., 2004; Wieder et al., 1999), theasis of their even more remarkable antiparasitic activityas attracted much less interest, despite their obvious

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iological and clinical significance. In this review I willresent a summary of the available information and indi-ate how the combination of the results obtained in bothammalian and trypanosomatid parasitic cells suggests

n explanation for the observed therapeutic activities ofPAs.

. Mechanisms of action

iven their chemical nature and physicochemical proper-ies, LPAs are expected to interact with a variety of sub-ellular structures and enzymes, particularly those asso-iated with cellular membranes. Despite this fact, whichas been recognized by many authors (Wieder et al.,999; Wright et al., 2004), the results of many indepen-ent studies have concluded that interference with a rel-tively small number of targets seems to underlie theirnown biological effects. These targets are summarizedelow. However, before considering these specific mech-nisms, some considerations on physicochemical and bio-ogical actions of LPAs that have been shown to have noelevance for their specific antiproliferative effects seemsppropriate.

.1. Surfactant effects

ue to their amphiphilic nature LPAs naturally can haveon-specific cytotoxic activity at concentrations equal to orbove their critical micelle concentration (cmc, in the orderf 10 �M for pharmacologically active LPAs, see Stafford etl., 1989). The exact threshold concentrations for these non-pecific effects depend on the particular cell type and theresence of lipid-binding serum components, which reducehe free monomer concentration and elevate the effectivemc. Investigating an immortalized human keratinocyte celline (HaCaT), Wieder et al. (1998) found that the thresholdor non-specific cytotoxic effects of miltefosine was around5 �M. Santa-Rita et al. (2000), investigating the effects ofeveral LPAs against T. cruzi trypomastigotes in Dulbecco’sodified Eagle medium, found that the ED50/24 h values

or lytic effects at 37 ◦C were in the range 30—50 �M andhat addition of 5% blood increased these values 2—3-fold.t is thus clear that any effects of LPAs at concentrationsbove 30—50 �M in conventional cell culture media are mostrobably non-specific actions due to their detergent-likeroperties.

.2. Immunomodulatory effects

lthough early studies showed that LPAs were capable ofon-specific activation of macrophages, which suggestedhat their anticancer activities could be linked to modula-ion of the immune system (Ngwenya et al., 1992; Yamamotot al., 1987), more recent work has shown that these

urray and Delph-Etienne, 2000). These findings demon-trated that the antiparasitic effects of LPAs result fromirect effects on the parasite and do not require host T cellr macrophage-dependent activation.

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In trypanosomatid parasites the first cellular/molecularstudies on the cytotoxicity of LPAs were carried out inL. donovani (Achterberg and Gercken, 1987a, 1987b): itwas shown that edelfosine was 10-fold more toxic thannatural ester and ether lysophospholipids, with IC50 val-ues in the submicromolar range in a lipid- and protein-free blood dialysate medium. Also it was found that etherlysophospholipids were not degraded by intact cells for over15 h, indicating the absence of O-alkenyl cleaving enzymes(Achterberg and Gercken, 1987b). The metabolism of mil-tefosine and edelfosine was investigated in L. mexicanaby Lux et al. (2000), and it was found that these com-pounds inhibit alkyl-specific acyl-CoA-acyltransferase, a keyenzyme in ether-lipid remodelling. However, the correla-tion between ether-lipid remodelling and inhibition of cellgrowth was not directly established as the IC50 for the formereffect was about 4-fold higher than that for growth inhibi-tion (Lux et al., 2000).

Lira et al. (2001) carried out a detailed study of theeffects of miltefosine, edelfosine and ilmofosine on thephospholipid and sterol composition of T. cruzi epimastig-otes. It was found that PC biosynthesis in these cells pro-ceeds mostly through the Brenmer-Greenberg (transmethy-lation) pathway, which is also the main pathway in fungi(Robson et al., 1990) but is only used in mammals undercholine deprivation conditions (Walkey et al., 1997, 1998).LPA treatment, at concentrations close to the IC50 for growthinhibition (1—3 �M), led to a modification of the ratio of PCto phosphatidylethanolamine (PE) from 1.5 in control cellsto ca. 0.67 in treated cells (Figure 2), and edelfosine inhib-ited the incorporation of L-methyl-[14C]-methionine into PCwith an IC50 of 2 �M. These observations suggested that inhi-bition of the de novo synthesis of PC through the Greenberg’spathway, specifically at the level of PE N-methyl-transferase(PEMT; EC 2.1.1.17; Figure 3), is a primary effect underlying

Figure 2 Effects of lysophospholipid analogues on the phos-

Mechanisms of action of lysophospholipid analogues

3. Mechanisms of action: inhibition ofmembrane lipid metabolism

Given their molecular structure, LPAs have been intensivelyinvestigated as potential inhibitors of enzymes involvedin the synthesis, breakdown or modification of membranelipids (Wieder et al., 1999; Wright et al., 2004). The mostthoroughly investigated are the effects of LPAs on the denovo synthesis of phosphatidylcholine (PC): it has beenshown that these compounds inhibit the synthesis of thisessential phospholipid in many cell types, specifically atthe level of CTP: phosphocholine citidyltransferase (CCT; EC2.7.7.15; Wright et al., 2004) (Haase et al., 1991; Wiederet al., 1995a, 1995b), which catalyses the limiting step ofthe main de novo PC biosynthesis pathway in mammaliancells (the Kennedy or CDP-choline pathway; Wright et al.,2004). Several studies have demonstrated consistently acorrelation between inhibition of PC biosynthesis and block-ade of cell proliferation (Wieder et al., 1995a, 1999). Thecholine head group of LPAs is essential for inhibition of CCTand for the antiproliferative effects as hexadecylphospho-ethanolamine and hexadecylphosphoserine were not activeas inhibitors of PC biosynthesis or inducers of cell death(Geilen et al., 1994; Wieder et al., 1998). Recent studieshave supported a causal relationship between the blockadeof PC biosynthesis induced by LPAs and their antiprolifera-tive action by showing that:

1. Restoration of PC biosynthesis by lyso-PC overrides thegrowth arrest induced by edelfosine (Boggs et al., 1995).

2. Leukaemic cell lines which express a mutant form of CCTare resistant to LPAs (Vogler et al., 1996).

Although PC is clearly an essential component of cellularmembranes and the source of many intracellular signallingmolecules, the precise molecular link between a blockadeof PC biosynthesis and arrest of cell proliferation is stillunder intense debate (Cui and Houweling, 2002; Wrightet al., 2004). However, several independent studies haveshown that a reduction of PC synthesis alone can triggerprogrammed cell death (apoptosis) in many cell lines (Cuiand Houweling, 2002; Wieder et al., 1998, 1999; Wright etal., 2004); this will be discussed in detail in the next section.

Along these lines, one possible mechanism is that adecrease of PC synthesis will depress the production of sph-ingomyelin (SM), as these biosynthetic pathways are stronglycoupled (Hampton and Morand, 1989), which in turn will leadto the elevation of the intracellular levels of ceramide, aknown inducer of apoptosis (Hannun and Obeid, 1995; Wrightet al., 2004). Wieder et al. (1998) showed that in HaCaTcells this is indeed the case, as incubation with sublyticlevels of miltefosine inhibited the incorporation of radio-labelled choline in PC and at a later time point in to SM;this was confirmed by metabolic labelling of SM with radi-olabelled serine, which also showed a significant increasein the cellular level of free ceramide. Another possibil-

ity, deduced from studies in yeast (Wright et al., 2004;Zaremberg and McMaster, 2002), is that the intracellularlevels of phosphatidic acid, produced from PC hydrolysis byphospholipase D, are the key regulators of cell growth in thatorganism.

pholipid composition of Trypanosoma cruzi epimastigotes. Themolar percentage composition of control (untreated) epimastig-otes is compared with those incubated with various concentra-tions of edelfosine (EDEL), miltefosine (MILT) or ilmofosine (ILM)for 120 h. Adapted from Lira et al. (2001).

S12 J.A. Urbina

Figure 3 Biosynthetic pathways for phosphatidylcholine biosynthesis in lower eukaryotes. The main biosynthetic route is thePE N-methylation (Brenmer-Greenberg) route. The sites of action of known phospholipid biosynthesis inhibitors are indicated byl difenc perm

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he antiparasitic activity of these compounds (Lira et al.,001).

The observation that the IC50s of edelfosine and milte-osine required to inhibit PC biosynthesis in T. cruzi epi-astigotes (Lira et al., 2001) were 10—20-fold lower than

hose required to induce the same effects in mammalianells (Haase et al., 1991; Wieder et al., 1995a, 1995b) couldrovide an explanation for the selective in vivo antipara-itic activities of these compounds, as has been proposedor the selective antifungal activity of some PC biosynthesisnhibitors such as iprobenfos and edifenphos (Robson et al.,990).

LPAs also induced a modification of the free sterol com-osition of T. cruzi epimastigotes, through an inhibition ofterol �22 desaturase, most probably a secondary effectesulting from the altered phospholipid composition (Lira etl., 2001). This finding led to an investigation of the possiblentiproliferative synergism of LPAs and specific ergosteroliosynthesis inhibitors, such as ketoconazole (Urbina andocampo, 2003), as the latter compounds also induce an

nversion of the PC/PE ratio in T. cruzi epimastigotes, proba-ly secondary to the altered sterol composition (Contreras etl., 1997). The results indicated that there is indeed a strongynergism of the antiproliferative effects of the two types ofrugs when used in combination against epimastigotes (Lira

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t al., 2001); this finding has been recently extended to thelinically relevant intracellular amastigote form of the par-site (Santa-Rita et al., 2005).

. Mechanisms of action: apoptosis

here is now a general consensus that LPAs, at concentra-ions below their non-specific cytotoxic levels, induce pro-rammed cell death (apoptosis) in susceptible mammalianells and in most cases this process appears to be linkedo the inhibition of the de novo biosynthesis of PC at theevel of CCT (Engelmann et al., 1996; Wieder et al., 1999).mong the experimental evidence that supports this conclu-ion is the observation that miltefosine induced an increasef diacylglycerol and triacylglycerol biosynthesis in KB cells,orrelated with a reduction of PC levels, prior to DNA frag-entation — this suggests a causal relationship betweenCT inhibition by the drug and cell death (Engelmann etl., 1996). It has been proposed the proximal cause of

iltefosine-induced apoptosis is an increase in the cellu-

ar levels of ceramide resulting from reduced biosynthesisf SM due to low levels of its precursor, PC (Wieder etl., 1998, 1999). This conclusion was based on the resultsf detailed studies on phospholipid biosynthesis in treated

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cells (see previous section) and was further supported by thefact that miltefosine-induced cell death was prevented byfumonisin-B1, an inhibitor of ceramide biosynthesis (Wiederet al., 1998).

The results of several other studies in recent years sup-port the notion of a causal relationship between inhibitionof PC biosynthesis and programmed cell death:

1. Inhibition of PC biosynthesis results in an arrest of thecell cycle in many cell types (Cui and Houweling, 2002;Wieder et al., 1998; Wright et al., 2004).

2. Cells carrying temperature-sensitive mutations of CCTwere driven into apoptosis and loss of cell viability whenshifted to the non-permissive growth temperature (Cuiet al., 1996; Howe et al., 2002).

3. Apoptosis can be induced by short-chain ceramides, suchas C2-ceramide, which block PC biosynthesis at the levelof CCT or cholinephosphotransferase (CPT) and theseeffects can be reversed by PC, but not by PE, phos-phatidylserine, phosphatidylinositol or phosphatidic acid(Cui and Houweling, 2002; Ramos et al., 2000).

4. Programmed cell death is also induced by farnesol, ger-anylgeranol and chelerythrine, known inhibitors of CPTand PC biosynthesis, and PC is able to rescue cells treatedwith these compounds (Cui and Houweling, 2002).

In contraposition to these findings, van der Sanden etal. (2004) claim that miltefosine-induced apoptosis in CHO-K1 cells differs in several ways from apoptosis observedin CCT-deficient MT58 cells and conclude that inhibition ofPC synthesis is not the primary mechanism of LPA-inducedcell death. However, the concentrations of miltefosine usedto treat CHO-K1 cells were very high (75 �M in serum-containing medium, 10 �M in serum-free media) and led torapid phosphatidyl-serine scrambling in the plasma mem-brane (within 5 min) and cell death (within 12 h), not asso-ciated with a significant reduction of PC content or the denovo synthesis of proteins (van der Sanden et al., 2004).These facts strongly suggest that the miltefosine-inducedcell death in these studies was due to non-specific cytotoxiceffects, as discussed above (see Introduction).

The existence of apoptosis in single-celled eukaryotes hasbeen the subject of controversy, but recent studies havepresented evidence of classical programmed cell death insuch organisms, including trypanosomatid parasites such asL. donovani and T. cruzi (Ameisen et al., 1996; Das et al.,2001; Debrabant et al., 2003; Lee et al., 2002; Paris et al.,2004; Piacenza et al., 2001; Verma and Dey, 2004). Paris etal. (2004) showed that miltefosine induced a cell death pro-cess in L. donovani promastigotes characterized by typicalapoptotic phenomena such as cell shrinkage, DNA fragmen-tation and phosphatidylserine exposure, with preservationof plasma membrane integrity (Paris et al., 2004). Thesephenomena were absent in a miltefosine-resistant strain(HePC-R40), showing that they resulted from specific druginteraction with the cells, and some of them were preventedin wild-type cells by specific caspase inhibitors (Paris et al.,

2004). Very similar findings were obtained by Verma and Dey(2004) in both promastigotes and amastigotes of the sameorganism. Taken together, these observations suggest thatthe particularly potent inhibition of PC biosynthesis inducedby LPAs in trypanosomatid parasites (see previous section)

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ould lead to programmed cell death in these organismsnd explain the selective antiparasitic effects of these com-ounds in vivo.

. Mechanisms of action: inhibition of cellignalling pathways

ue to the many potential interactions of LPAs, particularlyith components of membrane structures, the effects of

hese compounds on cell signalling pathways have been theubject of many studies (reviewed in Arthur and Bittman,998; Wieder et al., 1999; Wright et al., 2004). Recent stud-es have shown that disruption of membrane lipid rafts,pecialized structures composed of sphingolipids, phospho-ipids with saturated fatty acids and specific proteins, amonghem components of many signal transduction pathwaysGuan, 2004; Simons and Ikonen, 1997), severely reducesdelfosine-induced inhibition of PC and cytotoxicity (vaner Luit et al., 2002, 2003). This indicates that associationf LPAs with rafts is required for their internalization andubsequent effects on PC biosynthesis (van der Luit et al.,002, 2003). Other studies have suggested that edelfosinean directly induce apoptosis through intracellular activa-ion of the Fas/CD95 death receptor (Gajate and Mollinedo,001; Mollinedo et al., 2004).

The fact that edelfosine and other LPAs associate withipid rafts provides an explanation for the capacity of theseompounds to perturb a wide variety of signal transductionathways (Arthur and Bittman, 1998; Wieder et al., 1999;right et al., 2004). Among the most studied effects are

he inhibitory effects of LPAs on cellular signalling pathwayshat involve phospholipase-C and protein kinase C, which aremportant regulators of cell proliferation (Nishizuka, 1992).owever, the relevance of protein kinase C inhibition forhe antiproliferative effects of LPAs has been questionedecause of the observation that, in contrast to PC biosyn-hesis, there is no consistent correlation between proteininase C inhibition and growth arrest (Wieder et al., 1999).n T. cruzi epimastigotes a correlation between inhibitionf phospholipase C and growth arrest was observed with73122 but not with edelfosine (Malaquias and Oliveira,999).

In summary, although many cell-signalling pathways aren fact targeted by LPAs, it has been difficult to estab-ish strict correlations between most of these biochemicalffects and the antiproliferative effects of the drugs.

. Conclusions

he chemical nature of LPAs leads naturally to their inter-ction with a wide variety of subcellular structures andiochemical pathways, and these effects have been amplyocumented. However, those biochemical interactions thattrictly correlate with growth inhibition and programmedell death are much more restricted, and in most mam-alian cells these effects seem to be associated with a

lockade of PC biosynthesis at the level of CCT (Kennedyathway), probably through an increase of cellular ceramideevels due to depressed SM synthesis.

Although in trypanosomatid parasites much less informa-ion is available, inhibition of PC biosynthesis by LPAs has

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lso been demonstrated but at the level of PEMT (Green-erg’s pathway), as well as LPA-induced classical apoptotichenomena. However, the IC50s for the inhibition of PCiosynthesis in parasites are 10—20-fold lower that thoseound in mammalian cells, probably due to different bio-hemical pathways used in the two types of cells, and thisould provide an explanation for the selective antiparasiticctivities of LPAs in vivo.

onflicts of interest statementhe author has no conflicts of interest concerning the workeported in this paper.

cknowledgements

ork carried out at the author’s laboratory received supportrom the European Commission (INCO-DC, contract IC18-T96-0084), the Howard Hughes Medical Institute (grant5000620) and the Instituto Venezolano de Investigacionesientificas (IVIC).

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Mechanisms of action of lysophospholipid analogues

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aremberg, V., McMaster, C.R., 2002. Differential partitioningof lipids metabolized by separate yeast glycerol-3-phosphateacyltransferases reveals that phospholipase D generation ofphosphatidic acid mediates sensitivity to choline-containinglysolipids and drugs. J. Biol. Chem. 277, 39035—39044.