JAC Advance Access published online on June 21, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm219
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Tamoxifen is effective against Leishmania and induces a rapid alkalinization of parasitophorous vacuoles harbouring Leishmania (Leishmania) amazonensis amastigotes
1 Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo 05508-900 SP, Brazil 2 Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04023-062 SP, Brazil
* Corresponding author. Tel: +55-11-30917334; Fax: +55-11-30917417; E-mail: srbulian{at}icb.usp.br
Received 8 February 2007; returned 6 April 2007; revised 7 May 2007; accepted 24 May 2007
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Objectives: This study was performed to investigate the activity of tamoxifen, an antioestrogen widely used in the treatment of breast cancer, against Leishmania.
Methods: Drug activity was assessed in vitro against axenically grown promastigotes and amastigotes through cell counting or by measuring the cleavage of MTT, and against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of intracellular parasites. Intravacuolar pH changes induced inside parasitophorous vacuoles of Leishmania (Leishmania) amazonensis-infected macrophages were evaluated using the fluorescent probes SNAFL-calcein and Acridine Orange.
Results: Tamoxifen killed L. (L.) amazonensis promastigotes and amastigotes with 50% inhibitory concentrations (IC50) of 16.4 ± 0.2 and 11.1 ± 0.2 µM, respectively. The drug was also effective against Leishmania (Viannia) braziliensis, Leishmania (Leishmania) major, Leishmania (Leishmania) chagasi and Leishmania (Leishmania) donovani with IC50 values ranging from 9.0 to 20.2 µM. Tamoxifen induced a rapid and long-lasting alkalinization of the vacuolar environment. We also provide evidence that tamoxifen is more effective against promastigotes and amastigotes at pH 7.5 when compared with cultures at pH 4.5.
Conclusions: Tamoxifen effectively kills several Leishmania species and its activity against the parasite is increased by a modulation of the host cell intravacuolar pH induced by the drug.
Key Words: chemotherapy , pH , oestradiol , Acridine Orange , SNAFL-calcein
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Introduction |
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Human leishmaniasis is an endemic disease in 88 countries distributed in tropical and subtropical regions of the world. Clinical manifestations of this parasitic illness vary from self-healing cutaneous ulcers to very aggressive and disfiguring cases of diffuse or mucocutaneous diseases or to the life-threatening visceral form. More than 90% of cutaneous leishmaniasis cases occur in Iran, Afghanistan, Syria, Saudi Arabia, Brazil and Peru.1 In Brazil, Leishmania (Viannia) braziliensis is the most common agent for cutaneous and mucocutaneous leishmaniasis, whereas Leishmania (Leishmania) amazonensis is the most important causative agent of the diffuse form of the disease. Chemotherapy of leishmaniasis relies mainly on the administration of pentavalent antimonials that are toxic and poorly tolerated, require daily injections and are becoming ineffective due to the proliferation of resistant parasites.2,3 Therefore, alternative drugs are in pressing need.
Leishmania life cycle consists of two distinct forms: promastigotes, flagellated extracellular parasites of the digestive tract of sand flies, and amastigotes, non-flagellated, non-motile stages that live inside parasitophorous vacuoles (PVs) in macrophages of mammalian hosts. The PV encloses a strongly hydrolytic, acid environment—pH around 5.0—and the amastigote does not attenuate this hostile milieu.4–6 Amastigotes have been shown to carry out vital processes (i.e. cellular respiration, nutrient uptake and DNA and RNA synthesis) under these conditions.7
Tamoxifen is a triphenylethylene widely used in the treatment and in the prevention of breast cancer due to its activity as an oestrogen receptor modulator.8 However, it has become evident that many biological effects of tamoxifen are independent of the oestrogen machinery, such as modulation of calmodulin, caspases and kinases,9 interference in ceramide metabolism,10,11 inhibition of the acidification of intracellular organelles12–14 and partitioning into lipids where it exerts membrane fluidizing and antioxidant activities.15
While oestrogen receptor modulated responses have not been described in Leishmania, some of the effects observed with tumour cell lines suggested a potential activity of tamoxifen against these parasites. Particularly interesting to us were the findings of Altan et al.13 showing that adriamycin resistance in breast cancer cells was reversed by tamoxifen as a result of the redistribution of cellular adriamycin from acidic organelles to the nucleus. These authors established that tamoxifen was able to inhibit the acidification of organelles in several cell types in an oestrogen-independent way. Since Leishmania amastigotes are well adapted to survive in acidic PV, it seemed plausible to test the activity of tamoxifen against these parasites. In this paper, we describe the activity of tamoxifen against promastigotes and amastigotes of Leishmania in vitro. We also show that tamoxifen is able to modify the intravacuolar pH of L. (L.) amazonensis-infected macrophages inducing a state where the drug activity against the parasite is increased.
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Cells
Promastigotes of L. (L.) amazonensis (MHOM/BR/1973/M2269), L. (V.) braziliensis (MHOM/BR/1975/M2903), Leishmania (Leishmania) chagasi (MHOM/BR/1974/M2682), Leishmania (Leishmania) donovani (LD-15/MHOM/SD/00) and Leishmania (Leishmania) major (MHOM/IL/1981/Friedlin) were grown in Medium 199 (Invitrogen) supplemented with 10% or 20% heat-inactivated fetal calf serum (FCS; Invitrogen) and incubated at 25°C. Liquid cultures of L. (V.) braziliensis and L. (L.) chagasi were also supplemented with 2% sterile male human urine. L. (L.) amazonensis amastigotes were obtained from experimentally infected BALB/c mice as described previously.16 Amastigotes purified from lesions were kept at 33°C in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS in an atmosphere of 5% CO2. Parasites retained the typical amastigote morphology and were viable in these conditions for at least 48 h.
Resident peritoneal macrophages were obtained by peritoneal lavage of 25- to 30-day-old female BALB/c mice with 3–5 mL of RPMI 1640 medium repeated three times. The collected cell suspension was centrifuged at 300 g for 10 min at 4°C, the pellet was resuspended in RPMI 1640 medium supplemented with 10% FCS and gentamicin 50 mg/L and cultivated at 37°C in an atmosphere of 5% CO2.
The continuous line of human foreskin fibroblasts (HFF) was grown in RPMI 1640 supplemented with 10% FCS and cultivated at 37°C in an atmosphere of 5% CO2.
Tamoxifen, 17ß-oestradiol and amphotericin B were purchased from Sigma-Aldrich, St Louis, MO, USA. Stock solutions of tamoxifen and 17ß-oestradiol were prepared in ethanol and amphotericin B stock was prepared in DMSO (5 mM final concentration). Tamoxifen and amphotericin B were stored at –20°C and 17ß-oestradiol at room temperature.
In vitro antiproliferative activity assays
Cell viability was evaluated in vitro by cultivating promastigotes (5 x 106) or amastigotes (1 x 107) in 199 medium or RPMI 1640, respectively. Parasites were incubated in the presence of increasing concentrations of tamoxifen (2.5–30 µM), amphotericin B (as a control drug, 0.05–0.30 µM) or 17ß-oestradiol in 24-well culture dishes (Corning Life Sciences, Corning, NY, USA) for 24 h. Quantification of viable cells was assessed either by cell counting or by measuring the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) by metabolically active cells as described previously.17 In brief, cells were incubated in 3-(N-morpholino) propanesulfonic acid (MOPS)-buffered saline [30 mM MOPS (pH 7.2), 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose] containing MTT 5 mg/mL for 2 h (at 25°C for promastigotes and 33°C for amastigotes). MTT cleavage was assessed by using a multiwell scanning spectrophotometer (Labsystems; Multiskan EX) with a reference wavelength of 690 nm and a test wavelength of 595 nm. Assays were performed in triplicate and results are expressed as the mean percentage reduction of parasite numbers compared with untreated control wells calculated for at least three independent experiments. The 50% and 90% inhibitory concentrations (IC50 and IC90) were determined from sigmoidal regression of the concentration–response curves using Scientific Graphing and Analysis Software ORIGIN 7.5.
Alternatively, cell viability and growth was analysed by incubating promastigotes (5 x 106) in the presence of increasing concentrations of tamoxifen for 24 h; parasites were then washed in PBS [10 mM Na2PO4, 1.76 mM KH2PO4 (pH 7.4), 137 mM NaCl and 2.7 mM KCl] and grown in 199 medium for additional 24 h in the absence of drug. At the end of this period, cell density was determined by counting aliquots of the cultures in a haemocytometer.
In vitro cytotoxicity was evaluated by cultivating 5 x 105 HFF in 24-well plates for 24 h in the presence of increasing concentrations of tamoxifen (5–80 µM). Cell viability was assessed by the MTT assay as described above and results are expressed as percentage reduction in cell viability compared with untreated control cultures. The 50% cytotoxic concentration (CC50) was determined as described above for IC50 values.
Drug activity against intracellular amastigotes
Killing of intracellular L. (L.) amazonensis amastigotes was assayed by analyses of the number of infected cells in macrophage monolayers. Peritoneal macrophages were plated on round glass coverslips inside the wells of 24-well culture dishes, at a density of 4 x 105 cells per coverslip, in RPMI 1640 supplemented with 10% FCS and gentamicin 50 mg/L. After 2 h of incubation at 37°C in an atmosphere of 5% CO2, L. (L.) amazonensis stationary-phase promastigotes (in a ratio of 7 parasites:1 macrophage) or lesion-derived amastigotes (1:1) were added to the wells and the cultures were incubated at 33°C in a 5% CO2 atmosphere. After 3 h, parasites were removed by extensive washing with RPMI medium without FCS and infected cultures were treated with increasing concentrations of tamoxifen (5–20 µM) or amphotericin B (0.01–0.30 µM) for 48 h or with ammonium chloride (NH4Cl, 5 or 10 mM) for 24 h. The monolayers were washed, fixed and stained with the Instant Prov kit (Newprov, Pinhais, Brazil). The percentage of infected macrophages was assessed by light microscopy observation by counting 100 cells in triplicate coverslips. IC50 and IC90 were calculated from data obtained in three independent experiments.
Intravacuolar pH alterations in response to tamoxifen
Intracellular and intravacuolar pH were evaluated using two different fluorescent probes: SNAFL-calcein and Acridine Orange (AO) (Sigma-Aldrich).
Peritoneal BALB/c macrophages (8 x 105) were cultivated on 0.15 mm thick coverslips in Delta T dishes (Bioptechs, Butler, PA, USA) in RPMI 1640 medium at 37°C in a 5% CO2 atmosphere. After 3 h, amastigotes of L. (L.) amazonensis purified from mice lesions were used for in vitro macrophage infections. Infected cultures were kept at 33°C in a 5% CO2 atmosphere and samples were treated with 10 µM tamoxifen, ethanol (0.25%) or left untreated. Cells were loaded with the pH-sensitive ratiometric probe SNAFL-calcein (Molecular Probes).18 Experiments were performed as previously described.19 Lasersharp software version 3.2 TC was used for image acquisition and basic processing.
Peritoneal BALB/c macrophages, plated on round glass coverslips as described above, were infected with L. (L.) amazonensis amastigotes. Infected and uninfected cells were incubated in RPMI 1640 medium with or without drugs for the times indicated in the figure legends. Tamoxifen treatment was performed at 10 µM for 30 min. For time–lapse experiments, tamoxifen-treated cultures (30 min) were washed and incubated with RPMI without drug for 30 min, 4 or 24 h. NH4Cl was used at 5 or 10 mM concentrations for 30 min, 4 or 24 h. Cells were then incubated with 10 µM AO in PBS for 10 min at room temperature. Culture samples were immediately analysed using a Nikon-Microphot SX Fluorescence Microscope. ACT-1 Control Software was used for image acquisition.
pH effect on parasite viability and tamoxifen activity
L. (L.) amazonensis promastigotes (5 x 106) or freshly purified lesion-derived amastigotes (1 x 107) were incubated in RPMI medium pH 4.5 or 7.5 supplemented with 5% FCS for 4 or 24 h in the absence or in the presence of increasing concentrations of tamoxifen. Quantification of cell growth was evaluated by counting aliquots of the cultures in a haemocytometer.
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Activity of tamoxifen against Leishmania in vitro
Tamoxifen was tested for its effect on the viability of L. (L.) amazonensis promastigotes in vitro. Cell viability, evaluated through MTT cleavage, decreased as a function of drug concentration. After 24 h of incubation, the calculated IC50 against L. (L.) amazonensis was 16.4 ± 0.2 µM (Table 1). As a control, activity of amphotericin B against L. amazonensis promastigotes was calculated as being 0.15 ± 0.06 µM, in agreement with previously published data.20 These results were confirmed by cell counting. The effect of tamoxifen was irreversible since parasites incubated with the drug and left in normal culture media for 24 h did not recover (data not shown).
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The activity of tamoxifen against other Leishmania species was analysed by testing promastigotes of L. (V.) braziliensis and L. (L.) major, aetiological agents of cutaneous leishmaniasis and L. (L.) chagasi and L. (L.) donovani, species that cause visceral leishmaniasis. A dose-related response was detected when promastigotes of all the species tested were incubated with increasing concentrations of tamoxifen. The sensitivity of different Leishmania species was uniform with IC50 values ranging from 9.0 to 20.2 µM as depicted in Table 1.
Viability of L. (L.) amazonensis amastigotes, obtained from lesions and axenically cultivated for 24 h at 33°C, was also inhibited by tamoxifen at an IC50 of 11.1 ± 0.2 µM (Table 1).
Cytotoxicity assays were performed treating HFF cultures with increasing concentrations of tamoxifen, allowing the determination of a CC50 of 22.8 ± 2.6 µM. Treatment of HFF or peritoneal macrophage cultures with 10 µM tamoxifen for 24 h resulted in 100% and 85% survival, respectively.
Effect of tamoxifen on intracellular amastigote survival
The activity of the drug against the parasite in the intracellular environment was tested by treating peritoneal BALB/c macrophage cultures infected with L. (L.) amazonensis. The IC50 of tamoxifen against intracellular amastigotes was 5.46 ± 0.12 µM and the IC90 was calculated as 6.73 µM (Figure 1 and data not shown). Parasitism was unaltered in control-infected cells incubated in the presence of ethanol (the drug diluent). Testing of amphotericin B in the same assays allowed the calculation of an IC50 of 0.050 ± 0.003 µM for intracellular amastigotes.
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Oestradiol does not alter the viability of L. (L.) amazonensis promastigotes or the activity of tamoxifen
A search for oestrogen receptor homologues in the L. (L.) major genome database did not produce positive hits, indicating that tamoxifen's activity on these parasites is unlikely to be linked to the oestrogen receptor. To verify whether this was indeed the case, L. (L.) amazonensis promastigotes were incubated with increasing concentrations of 17ß-oestradiol and with equimolar concentrations of oestradiol plus tamoxifen. 17ß-Oestradiol-treated parasites did not exhibit changes in growth or survival while parasites incubated with the combination of equimolar concentrations of 17ß-oestradiol and tamoxifen were as sensitive to the drug as parasites incubated with tamoxifen alone (Figure 2). The same was observed for lesion-derived L. (L.) amazonensis amastigotes which exhibited the same pattern of growth in the absence or in the presence of 17ß-oestradiol while incubation with 17ß-oestradiol did not modify the activity of tamoxifen (Figure 2), confirming that the leishmanicidal activity of tamoxifen is independent of the oestrogen receptor machinery.
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Intravacuolar pH alterations in response to tamoxifen
In vitro infections were analysed using non-invasive ratiometric confocal microscopy with dual-label SNAFL-calcein-AM probe before and after incubation with 10 µM tamoxifen. SNAFL-calcein-AM compartmentalizes into acid vacuoles and the fluorescence is emitted in distinct wavelengths according to H+ concentrations—the more acidic the intravacuolar pH, the greener the organelle appears, whereas a more alkaline environment appear in tones towards red. As shown in Figure 3, images were obtained by merging the 550 and 640 nm emission channels. In infected and untreated macrophages, SNAFL-calcein-AM was detected mainly as yellow and green granules concentrated in the PV. Upon addition of tamoxifen and as incubation time elapsed, detection of SNAFL-calcein-AM in PVs was more intense in the 640 nm emission channel, resulting in red fluorescence (Figure 3). This suggested a rapid alkalinization of the PVs, detectable as soon as 10 min after drug addition. The presence of parasites inside PVs was confirmed by the phase contrast images also shown in Figure 4. Infected macrophages treated with 10 µM tamoxifen for 24 h and incubated with SNAFL-calcein-AM exhibited large vacuoles devoid of amastigotes and more alkaline than PVs in untreated cells (data not shown).
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Measurements of intravacuolar pH were also carried out in infected macrophages treated with ethanol in the concentration necessary to deliver the highest tamoxifen concentration used in the tests. Changes in pH were not observed in these cells (data not shown) indicating that the effect obtained with tamoxifen was not due to the drug diluent.
These findings were therefore consistent with the hypothesis of reduced acidification of intracellular compartments induced by tamoxifen, previously observed in other systems.
The effect of tamoxifen on intravacuolar acidification was also tested labelling peritoneal BALB/c macrophages infected with L. (L.) amazonensis amastigotes with AO, a weakly basic fluorescent probe. AO is taken up by living cells and accumulates in acidic compartments. Fluorescence of AO is green at low concentrations, whereas at high concentrations the fluorescence changes to orange.21 Nuclei in infected or non-infected cells (Figure 4a and i) stained green. In the cytoplasm of infected cells, AO accumulated inside vacuoles containing amastigotes resulting in red fluorescence (Figure 4a). Upon addition of 10 µM tamoxifen for 30 min, a decrease of the red AO fluorescence was observed in PVs of infected cells (Figure 4b). To verify whether this effect was lasting, infected macrophages treated with tamoxifen for 30 min were washed and kept at 33°C for 30 min, 4 or 24 h in culture media without drug (Figure 4c–e). Cells were then stained with AO for 10 min and examined. The pH in PVs of infected macrophages remained alkaline, even 24 h after drug removal, indicating that tamoxifen's effect on these cells was probably irreversible.
Tamoxifen's alkalinizing effect was not exclusive to infected cells, since non-infected macrophages also showed a remarkable reduction of orange-stained microvesicles (Figure 4i and j).
Alkalinization of the PV is not sufficient to control the infection
To verify whether parasites were killed by tamoxifen's direct action or by an indirect effect of the drug on the host cell, two different approaches were taken. The alkalinizing agent NH4Cl was used to treat infected macrophages for 30 min, 4 or 24 h. As expected, 5 or 10 mM NH4Cl induced a pH shift observed in infected cells as AO stained PVs green (Figure 4f–h and data not shown). However, the treatment of Leishmania-infected macrophages with 5 or 10 mM NH4Cl for 30 min or 24 h did not induce any reduction in the infection rates (Figure 4f–h and Table 2), indicating that the alkaline PV is not sufficient to arrest the infection.
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The second approach used to test whether tamoxifen's direct effect was required was to pre-treat uninfected macrophage cultures with tamoxifen for 24 h. Cells were then washed, infected and the cultures were grown for additional 24 h in the absence of tamoxifen. Pre-treated macrophages were as permissive to the infection as naive cells and there was no reduction in the percentage of infection compared with control untreated cells (Table 2), indicating that tamoxifen is directly active against the intracellular parasite.
Tamoxifen's activity is increased at neutral pH
To verify whether a neutral–alkaline pH in the PV would have any impact on drug activity, promastigotes and freshly lesion-purified amastigotes were submitted to tamoxifen treatment for 4 and 24 h at pH 4.5 or 7.5. A striking increase in drug activity was observed against promastigotes grown at pH 7.5 as compared with cultures kept at pH 4.5 (Figure 5a and b). A less pronounced but nonetheless clear increase in effect was also observed against amastigotes kept at neutral pH conditions (Figure 5c and d). The increased parasite killing at neutral pHs was not due to the pH itself since, after 4 and 24 h, both control promastigotes and amastigotes, grown in the absence of drugs, maintained unaltered their metabolic rate and viability in either pH as judged by similar optical densities in the MTT test (data not shown).
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This confirms that tamoxifen, with a pKa of 6.9, is more effective in its neutral form, attained at neutral and alkaline pHs. Consequently, activity against intracellular amastigotes is probably intensified by the increase in PV pH verified after addition of tamoxifen.
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We have shown that tamoxifen is effective against Leishmania. Susceptibility of microorganisms to tamoxifen in vitro has only been described for Candida albicans22 and Coccidioides immitis.23 There are, as yet, no reports available regarding the activity of tamoxifen against protozoan parasites.
The effect of tamoxifen against Leishmania is remarkable, given the absence of response to oestrogen in these organisms. On the other hand, several studies have reported biological properties of tamoxifen that cannot be explained by oestrogen receptor binding, such as the inhibition of acidification of organelles reported in tumour cells.14 Using resident peritoneal macrophages, we demonstrated that 10 µM tamoxifen reduced parasitism by more than 90% in 24 h. We have also shown that PVs of L. (L.) amazonensis-infected macrophages undergo a rapid pH shift upon addition of tamoxifen.
The rapid change in PV pH, however, is not directly responsible for killing amastigotes, as shown by the evaluation of parasite survival in pH 4.5 or 7.5. It does, however, have a major impact on tamoxifen's effectiveness against the parasite. That is most likely explained by the properties of tamoxifen: the neutral molecule partitions into the membrane and flips more easily into the interior than would the charged molecule. It is tempting to speculate that, once inside the acid PV, tamoxifen gets protonated, the pH shifts and this is followed by an increase in the influx of Cl–, as shown in microsomes isolated from MCF-7/ADR cells.14 That would bring about the regeneration of neutral tamoxifen that may now combine with the amastigote membrane.
Besides the ability to accumulate in the lipid phase, tamoxifen has also been shown to be a potent inhibitor of ATP-dependent membrane acid transport.24,25 Vacuoles containing Leishmania are rapidly acidified and reach pH 5.0 in <30 min after phagocytosis.26 The acidic luminal pH is sustained by a vacuolar H+-ATPase of macrophage origin in the PV membrane. Amastigotes also express a P-type H+-ATPase in their plasma membrane and may take part in the PV acidification.27 This P-type H+-ATPase is vital for the parasite to maintain a neutral internal pH.28 H+ ATPases both at the PV membrane and at the amastigote surface could be the targets of tamoxifen's action resulting initially in vacuole alkalinization followed by disruption of the ionic metabolic steady-state of the parasite. Omeprazole, a potent ATPase inhibitor at acidic pHs, has been suggested as a potential antileishmanial drug. It has been shown to kill intracellular L. donovani in vitro through inhibition of the parasite K+, H+ ATPase.29
Interestingly, not only intracellular amastigotes were killed by tamoxifen but also extracellular promastigotes. In fact, we showed that tamoxifen inhibits the survival of promastigotes of a wide range of Leishmania species, aetiological agents of distinct clinical forms of leishmaniasis, in a low micromolar concentration range. Plasma membrane P-type H+ ATPases have also been shown to regulate intracellular pH in L. (L.) amazonensis promastigotes.30 Further studies are needed to verify whether tamoxifen's mechanism of action against Leishmania depends on ATPase inhibition.
Tamoxifen is highly lipophilic and has been shown to be strongly incorporated in biomembranes, reported to disrupt the structure of model and artificial membranes and to induce the permeabilization of mitochondria.31 It is also possible, therefore, that the effect on Leishmania is derived from drug partition into the parasite membranes. It has been observed that the partition of tamoxifen into membranes is modulated by several factors, including membrane composition: for example, partition of tamoxifen into membranes decreases as cholesterol concentration is raised.32 Leishmania do not synthesize cholesterol. Ergosterol makes up the main sterol in the plasma membrane in these parasites.33 Amastigotes have been shown to be able to incorporate cholesterol from their hosts but they are also able to synthesize ergosterol (D. C. Arruda and S. R. B. Uliana, unpublished results). Distinct parasite and host cell membrane compositions could result in preferential partitioning into the parasite's membrane. Further studies are necessary to verify whether tamoxifen is indeed incorporated into Leishmania membranes and if the presence of ergosterol favours the partition of tamoxifen.
Breast cancer accounts for 31% of all incident cancers in women in North America. It is estimated that more than 214 000 new cases of breast cancer were diagnosed in the USA in 200534 and tamoxifen represents the cornerstone of breast cancer treatment in women with tumours bearing hormone receptors. Since these patients are treated with tamoxifen for at least 5 years, much clinical experience has been gained on the safety and undesirable effects of the drug. Tamoxifen is well tolerated and side effects, of which the most serious is an increased risk for endometrial cancer, appear only after prolonged use. The doses usually administered for the treatment of breast cancer, of 20 mg/kg body weight, achieve serum concentrations of 0.2–0.8 µM.35,36 Due to the high hydrophobicity of tamoxifen, in human tissues concentrations are 10- to 60-fold higher than in serum, with particularly high levels detected in the liver and lung.37 Tamoxifen also accumulates in the skin and bone tissue.37 This distribution pattern is relevant considering parasite localization in cutaneous and visceral leishmaniasis. Tissue concentrations attainable with currently used regimens are at least equivalent to the effective concentrations that kill Leishmania intracellular amastigotes in our study. Furthermore, it is to be expected that antileishmanial therapy with tamoxifen would not require such prolonged use. In conclusion, our results suggest that tamoxifen has a great potential to be tested as a therapeutic agent against leishmaniasis.
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This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). D. C. M. is the recipient of a post-graduate fellowship from FAPESP.
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None to declare.
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We are grateful to Dr Alejandro M. Katzin for providing tamoxifen for the initial experiments, and to Alexandre Santos de Moura, Dr A. Tania Bijovsky and Dr Maria Julia M. Alves for helping with the microscopy and for the critical reading of the manuscript.
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References |
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