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Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi
Acta Tropica 75 (2000) 219 – 228 www.elsevier.com/locate/actatropica
Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi Ricardo M. Santa-Rita a, Helene Santos Barbosa b, Maria de Nazareth S.L. Meirelles b, Solange L. de Castro a,* a
Laborato´rio de Biologia Celular, DUBC, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, CP 926, 21045 -900 Rio de Janeiro, Brazil b Laborato´rio de Ultra-estrutura Celular, DUBC, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, CP 926, 21045 -900 Rio de Janeiro, Brazil Received 1 May 1999; received in revised form 1 November 1999; accepted 20 December 1999
Abstract Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH3, hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED50/24 h values were 13.4 92.8 mM and 11.7 9 0.6 mM for amastigotes and epimastigotes, respectively. ET-18-OCH3 (0.3 and 0.6 mM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40–57%. This drug (3.75–15 mM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED50 values of 14.3 9 4.2, 8.9 9 1.9 and 6.8 9 0.4 mM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma cruzi; Chemotherapy; Alkyl-lysophospholipids; Edelfosine; ET-18-OCH3
1. Introduction
* Corresponding author. Fax: +55-21-2604-434. E-mail address: [email protected] (S.L. de Castro)
Alkyl-lysophospholipids (ALPs) were first designed as potential immunomodulators and antimetabolites of phospholipid metabolism, and
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have more recently been shown to be cytotoxic for a wide variety of tumour and leukaemia cells (reviewed in Brachwitz and Vollgraf, 1995). The first studies with ET-18-OCH3, an alkylglycerophosphocholine prototype, showed growth inhibition of Ehrlich ascite tumour cells in mice (Munder et al., 1976) and Lewis lung carcinoma metastases formation (Berdel et al., 1980). Later, a thioether substituted phosphatidylcholine (PC) analogue, ilmofosine, was developed and proved also to be active against neoplastic cells (Berdel et al., 1983). Observing that the glycerol moiety was not essential for the biological effects, a series of similar ether phospholipid alkylphosphocholines was synthesized, with hexadecyIPC (mitelfosine) being the most studied derivative. This compound displays a pronounced antitumour effect in a variety of rodent and human tumour cell lines (Unger et al., 1989). In addition, many other ALPs are under clinical trials (reviewed in Berdel, 1990; Winkelmann et al., 1992; Planting et al., 1993; Vogler et al., 1996a). The antitumour activity of ALPs is thought to be mediated by inhibition of cell proliferation and invasion, differentiation, induction and activation of cytotoxic macrophages (Berdel, 1991). The toxicity of ALPs involve inhibition of PC synthesis (Modolell et al., 1979; Vogler et al., 1985, 1996b), inhibition of signal transduction enzymes such as phosphatidylinositol-phospholipase C (Seewald et al., 1990; Powis et al., 1992) and protein kinase C (Helfman et al., 1983; Daniel et al., 1987), and alterations of intracellular calcium levels (Lazenby et al., 1990; Bergmann et al., 1994). Furthermore, due to their highly lipophilic nature, ALPs affect the physical properties of neoplastic cells by permeabilizing and increasing plasma membrane fluidity (Noseda et al., 1988). The development of ALPs as anticancer agents for humans ensures data on the pharmacology, toxicology and tolerance of these compounds, factors which are of importance for reducing the cost of drug development against tropical parasitic diseases. It is worth mentioning that hexadecyIPC showed promising results in clinical studies for the treatment of visceral leishmaniasis (Sundar et al., 1998). As part of a programme to investigate the antitrypanosomal activities of ALPs, we have in-
vestigated, in the present work, the effect of these compounds on Trypanosoma cruzi, the etiologic agent of Chagas’ disease. This disease constitutes one of the most important public health problems in many South American countries, affecting about 16–18 million persons (Moncayo, 1993). There is an urgent need of more efficient and safe drugs for the treatment of this disease, as benznidazole and nifurtimox have restricted applicability in chronic patients, besides presenting severe side effects (de Castro, 1993; Croft et al., 1997).
2. Materials and methods
2.1. Parasites and cell cultures Bloodstream trypomastigotes of T. cruzi (Y strain) were obtained from infected albino Swiss mice. Amastigotes were collected from the supernatant of infected J-774G-8 macrophage cultures (de Castro et al., 1987). The epimastigotes (Y strain and clone Dm28C) were maintained in LIT medium supplemented with 10% foetal calf serum (FCS) (Camargo, 1964) and harvested during the exponential phase of growth. Primary heart muscle cell cultures (HMC) were obtained from 18-day-old mouse embryos, after incubation with trypsin and collagenase as previously described (Meirelles et al., 1986).
2.2. Chemicals 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was purchased from Bachem (UK), hexadecylphosphocholine (hexadecylPC) was purchased from Sigma Chemical Co. (USA) and ilmofosine (1-hexadecylthio-2methoxymethyl - rac - glycero - 3 - phosphocholine) was a gift of Boehringer-Mannheim (Mannheim, Germany). FCS was obtained from Sigma Chemical Co. All other chemicals were of analytical grade.
2.3. Treatment of the three forms of T. cruzi with the ALPs Stock solutions of the compounds were prepared at 100 mM in phosphate-buffered saline (PBS).
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Trypomastigotes were resuspended in Dulbecco’s modified Eagle medium plus 10% FCS (DMES) to a final parasite concentration of 107 cells/ml in the absence or presence of 5% mice blood. The parasite suspension (100 ml) was added in a volume ratio of 1:1 to the compounds, and the mixture incubated for 24 h at 4 or 37°C. Untreated and crystal violet-treated parasites were used as controls. Amastigote and epimastigote forms (Y strain) were resuspended in DMES and LIT, respectively, and plated in 24-well plates at 5 × 106 cells/ml, in the absence or presence of different ALP concentrations at 28°C. For trypomastigote cultures, cell counts were performed after 24 h; for amastigote and epimastigote, they were performed from 4 h to 4 days. In all experiments, the ED50 was calculated.
2.6. Effect of ET-18 -OCH3 on the interaction between T. cruzi and heart muscle cells
2.4. Ultrastructural analysis
3. Results
Epimastigotes were treated with different concentrations of ALPs (2.5 – 10 mM), at 28°C in LIT. After 24 h, control and treated parasites were fixed (60 min/4°C) in a solution containing 2.5% glutaraldehyde, 2.5 mM CaCl2 and 0.1 M cacodylate buffer, pH 7.2 (Tcaco). After washing in cacodylate buffer, the cells were treated for 1 h with the Tcaco solution containing 1% OsO4, 0.8% potassium ferricyanide and 2.5 mM CaCl2. They were then dehydrated in acetone and embedded in Epon resin. Thin sections, stained with uranyl acetate and lead citrate, were examined in an EM 10C Zeiss transmission electron microscope.
The effect of the three compounds (ET-18OCH3, hexadecyIPC and ilmofosine) on trypomastigote viability was assayed after 24 h of incubation at 4 and 37°C, in the presence or absence of blood (Fig. 1). The major lytic activity was observed at the higher temperature and in the absence of blood. The ED50 values are presented in Table 1, and we can observed that at both 4 and 37°C, the addition of 5% blood decreased the trypanocidal activity two to 3.5 times. Comparison of the lytic activity of ALPs with that of crystal violet (ED50/24 h= 459.5951.9 mM), in standard conditions (4°C and in presence of blood) (de Castro et al., 1994), showed that they are about 2.5 to four times more active than the reference drug in inducing lysis of the parasite. Assays with the proliferative forms were performed at 28°C, using DMES for amastigotes and LIT medium for epimastigotes. ET-18-OCH3 showed a similar effect on the kinetics of the proliferation of both forms, with the ED50/24 h being 13.492.8 and 11.79 0.6 mM for amastigotes and epimastigotes, respectively (Table 2). The drugs were, respectively, 4–8.8 and 11.1–17.9 times more active against epimastigotes and amastigotes then nifurtimox (De Conti et al., 1996).
2.5. Effect of ET-18 -OCH3 on the metacyclogenesis process Epimastigote forms (clone Dm28C) were harvested from the LIT medium, incubated for 2 h in artificial triatomine urine (TAU) (Contreras et al., 1988) and then incubated in TAU supplemented with 10 mM L-proline, 50 mM sodium glutamate and 2 mM sodium aspartate (TAU3AAG) in the presence or absence of ET-18-OCH3. After 96 h, epimastigotes and trypomastigotes in the supernatant were counted in a Neubauer chamber.
HMC were incubated for 24 h with trypomastigotes (10:1, parasite:HMC ratio). The noninternalized parasites were removed, and fresh DMES, with or without ET-18-OCH3 (6.3–50 mM), added to the cultures and changed every 2 days. At specified intervals, the cultures were fixed (Bouin’s solution), stained (Giemsa), and counted (Zeiss photomicroscope). In another protocol, prior to the infection, the trypomastigotes were incubated (1 h at 37°C) with or without ALPs (1.9–60 mM). After washing (twice with DMES), the parasites were counted and added to HMC cultures (10:1, parasite:HMC ratio). After 24 h, the cultures were processed as already described.
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Fig. 1. Trypanocidal effect of ALPs on bloodstream trypomastigotes of T. cruzi (Y strain) in the absence (a,c) and presence (b,d) of 5% blood (4 and 37°C): ( ) ET-18-OCH3, (") ilmofosine and () hexadeylPC. Table 1 Values of ED50/24 h (mM) of the ALPs on T. cruzi bloodstream trypomastigotes in different experimental conditions 4°C/DMES
37°C/DMES
–
+5% whole mice blood
–
ET-18-OCH3 Ilmofosine
59.496.2a 44.29 7.0
141.99 3.1 177.69 11.6
29.0 92.8 29.5 9 3.6
HexadecyIPC
62.79 3.1
196.69 15.0
55.4 97.6
a
Mean9 S.D. of at least three experiments.
+5% whole mice blood 66.99 2.0 61.8 912.0 130.0 92.1
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Table 2 Values of ED50 (mM) of the ALPs on the proliferation of T. cruzi 4h Epimastigotes ET-18-OCH3 Ilmofosine HexadecyIPC
41.792.3a – –
Nifurtimoxb Amastigotes ET-18-OCH3 HexadecyIPC Ilmofosine
b
11.790.6 26.6 9 3.7 17.491.2
48 h
72 h
96 h
5.4 90.9 9.3 9 0.3 9.2 90.0
4.4 9 0.2 6.4 9 0.8 8.0 9 0.0
4.0 90.8 3.4 91.1 7.7 90.0
3.8 9 1.4 4.5 90.1 7.9 91.9
3.4 9 0.4 4.2 90.1 7.4 9 2.5
2.3 90.6 2.5 90.6 4.8 90.4
103.79 4.1 59.69 10.4 – –
Nifurtimoxb a
24 h
13.492.8 11.69 0.1 18.69 6.8 207.39 35.4
Mean9 S.D. of at least three experiments. De Conti et al. (1996).
The ALP activity against T. cruzi was much more pronounced when the incubations were performed in the absence of serum. In appropriate culture medium, the ED50 after 4 h for amastigote and epimastigotes was 59.6 and 41.7 mM, while, in PBS after 20 min, these values decreased to 7.5 and 25.7 mM, respectively. While the range of ED50 for trypomastigotes after 24 h was about 30 mM, after 30 min at 30 mM in PBS, total lysis of the parasites occurred. The ultrastructural analysis of epimastigotes treated with the ALPs showed initial alterations in cell swelling and extensive blebing of the flagellar membrane. These blebs were a characteristic effect of the three compounds (Fig. 2b,c and Fig. 3a – c). A marked swelling of the kinetoplast – mitochondrion system with loss of the inner membrane organization and disappearance of the electron-dense bodies of the matrix was also observed (Fig. 2b). These structural changes were drug-concentration dependent, as shown by the extensive disruption of the cellular matrix, disintegration of organelles and appearance of ghost parasites (data not shown). The kinetoplast (Fig. 3c), nucleus and subpellicular microtubules (Fig. 3a,b,d) were the most unaltered structures. In four independent experiments, the metacyclogenesis was inhibited by 40 – 57%, at 0.3 and 0.6 mM ET-18-OCH3 (Fig. 4). These concentra-
tions did not interfere with the total number of parasites (epimastigotes plus trypomastigotes). To analyse the effect of ET-18-OCH3 on both the kinetics of the infection and on the intracellular differentiation of amastigotes, the drug (3.75– 15 mM) was added to HMC and maintained throughout the experiment, leading to a dose-de-
Fig. 2. Ultrastructural alterations in epimastigote forms treated for 24 h with ET-18-OCH3. (a) Control cell with normal morphology (N, nucleus; k, kinetoplast; F, flagellum; FP, flagellar pocket; G, Golgi apparatus; M, mitochondrion) (10 000 × ); (b,c) ET-18-OCH3 (5 and 10 mM) resulted in swelling of the mitochondrion with loss of the inner membrane organization resembling a fibrous network (arrow), and also blebs of the plasma membrane most commonly in the flagellum (arrowhead) (12 600 × and 16 500 ×).
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Fig. 3. Ultrastructural alterations on epimastigote forms treated for 24 h with the ALPs: (a,b) ilmofosine (2.5 and 5 mM) showing similar effect on the flagellar membrane (arrowhead) (18 000 × and 19 000 × ); (c,d) hexadecylPC (5 and 10 mM) (9600 × and 14 500 ×). The micrographs show different effects of the damage at the flagellum (arrowhead). Some parasites showed extensive vacuolization around the flagellar pocket (arrows).
and 3 days of treatment, respectively. At 30 mM, this ALP caused damage to the host cell. After 1 and 2 days of treatment, the percentage of cells containing parasites in differentiation was similar to the control (Fig. 5b), indicating no significant effect on the differentiation of amastigotes, although a marked decrease in the infection index. In another approach, the parasites were treated with ET-18-OCH3 (2–60 mM), for 1 h, washed and added to the cultures. After 18 h of interaction, inhibition of parasite interiorization in HMC was observed, in the range 10–60% (data not shown). It is important to note that no alteration on motility of the trypomastigotes or other damage, as determined by light microscopy, was observed during the 1 h treatment with the ALP.
Fig. 4. Inhibition of the T. cruzi (clone Dm28c) metacyclogenesis by ET-18-OCH3. After 3 days of treatment with 0.3 and 0.6 mM ALP, differential countings of epimastigotes and trypomastigotes were performed. The total number of parasites was maintained inaltered during the experiments.
pendent inhibition of the infection (Fig. 5a). The mean values of ED50 for three experiments were 14.3 94.2, 8.991.9 and 6.89 0.4 mM, for 1, 2
Fig. 5. Effect of ET-18-OCH3 on the infection of HMC with T. cruzi. (a) Dose- and time-dependent effects of the drug in the range 3.2 – 15 mM; (b) similar percentage levels of intracellular differentiation of amastigotes to trypomastigotes was observed between control and treated cells (3.2 and 7.5 mM).
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4. Discussion The effect of ALPs on trypomastigotes was assayed in four different conditions, by combining temperature (4 and 37°C) with addition or not of mouse blood to DMES medium. Experiments were performed at 4°C in the presence of blood due to the potential use of these compounds for the prophylaxis of banked blood. The decrease of the lytic activity of different compounds against T. cruzi due to the presence of blood components has been already described (Lopes et al., 1978; Rovai et al., 1990). In all four conditions, the ALPs presented a higher lytic activity than crystal violet, the standard compound. Mitelfosine was less active that the other two drugs. This could be explained by structural differences between these compounds; while ET-18-OCH3 and ilmofosine are alkyl-glycerophosphocholines, hexadecyIPC presents a simpler backbone constituted only of a phosphocholine polar moiety and a fatty alkyl chain. When tissue culture-derived trypomastigotes were used, the drugs showed lytic activity lower than that of crystal violet (Croft et al., 1996). The lower lytic effect of ALPs against trypomastigotes at 4°C when compared with 37°C (in the range 1.1– 3.0 times) could be due to alterations with membrane activities. In leukaemia cells, it has been already determined that higher temperatures are determinant factors for susceptibility, leading to higher ALPs uptake and enrichment in the membrane (Kelley et al., 1993). Addition of whole blood caused a decrease of ALPs lytic activity, as shown by an increase in the ED50 between 2.1 and 4.0 times for the three compounds. This inhibition could be associated with the low performance of these ALPs in invivo experiments (Croft et al., 1996). The influence of serum components was more clearly observed when the parasites were incubated with the ALP in PBS buffer. Plasma protein binding is not surprising for hydrophobic compounds such as the ALPs. A ‘protecting effect’ of serum against the cytotoxicity induced by ALPs against T. cruzi was also reported for tumour cells (Petersen et al., 1992; Lohmeyer and Workman, 1993; Heesbeen et al., 1995).
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For all three forms of T. cruzi, all compounds showed similar lytic activity, with ED50/24 h in the range 11–30 mM. The only exception was hexadecyIPC acting on trypomastigotes (standard conditions), whose ED50/24 h was 55.4 mM. In comparison, the ED50/24 h for susceptible leukaemic cells (HL-60) was about 10 times lower (Diomede et al., 1993; Mollinedo et al., 1993), probably reflecting different mechanisms of action between T. cruzi and tumoral cells. The mode of action of ALP against several pathogenic micro-organisms is under investigation. The activity of a series of ALPs against cilliates and funghi was observed to present a good correlation with the effect on tumour cells (Tsushima et al., 1982). There are several reports on the effect of ALPs on trypanosomatids, African trypanosomes (Croft et al., 1996; Konstantinov et al., 1997) being more resistant to the drugs than T. cruzi or Leishmania (Kuhlencord et al., 1992; Bourass et al., 1996; Croft et al., 1996). Analysis of the ultrastructural alterations induced by the three drugs on the parasites showed a characteristic effect, which was the damage of the flagellar membrane, with extensive blebing. These alterations localized only in the flagellar membrane could be associated with interference caused by the ALPs on the phospholipid composition (Urbina, personal communication), as it is already known that the body and flagellar membranes of T. cruzi have different composition (reviewed in De Souza, 1984), Alterations in the mitochondrion, with loss of the inner membrane organization and absence of the cristae, was also frequently observed. The effect of the drugs was progressive and, with higher concentrations, an uncharacteristic effect on the cytoplasm, with extensive vacuolization, was also detected. Inhibitory effect of ALPs on lipid synthesis in tumour cells is well documented, involving PC in most cases, associated with inhibition of the translocation of PC cytidyltransferase (Modolell et al., 1979; Wieder et al., 1995; Vogler et al., 1996b). Preliminary results from our group show that the treatment of epimastigotes with ET-18OCH3 after incubation with 32P lead to a decrease of PC levels of about 60%, while alterations in phospho-inositides were also observed (data not shown).
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Due to their structure, ALPs have the potential to physically interact with the plasma membrane so as to bring about physical disruption. In tumour cells, ALP accumulation into cell membranes and perturbation of fluidity (Van Blitterswijk et al., 1987) and lipid metabolism have been previously described (Tidwell et al., 1981). Comparing the alterations caused by ALPs on T. cruzi and tumour cells, it seems that the membrane in both cases is a prime target (Berdel et al., 1983; Noseda et al., 1989; Vagnetti et al., 1990). Treatment of HL-60 leukaemic cells with ET-18-OCH3 at similar conditions, as used in the present work (2.5 mM/24 h), led to the formation of blebs and holes in the plasma membrane, cytoplasm vacuolization and nuclei piknosis (Noseda et al., 1989). On the other hand, in relation to ilmofosine, morphological alterations in cells isolated from leukaemia patients occurred at concentrations about 10 times higher than that observed with T. cruzi (Berdel et al., 1983). Alterations involving destruction of mitochondrial cristae, vacuolization and rarefaction of the cytoplasm were also observed after treatment of lymphoma cells with ET-18-OCH3 (Vagnetti et al., 1990). The observed inhibitory effect of ET-18-OCH3 on the differentiation of epimastigotes to trypomastigotes could involve the adenylate cyclase/ cyclic AMP system. Interference with this signalling system was previously associated with inhibition of differentiation of T. cruzi, including the metacyclogenesis process (de Castro et al., 1987; Rangel-Aldao et al., 1987; Gonzales-Perdomo et al., 1988). The treatment with ET-18-OCH3 of infected cultures, in the range 3 – 15 mM, caused a dose-dependent inhibition of HMC infection, while at 30 mM, this drug caused damage to the host cells, with extensive vacuolization of the cytoplasm and a decrease in the number of adhered cells. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by ET-18-OCH3. Drug doses inhibiting proliferation of amastigotes internalized in heart cells were in the same range as those that inhibited the proliferation of this form in axenic medium. On the other hand, amastigotes inside heart cells were less susceptible to ET-18-OCH3 than in
macrophages, the ED50/3 days being 6.8 and 1.4 mM, respectively (Croft et al., 1996). The pretreatment of the parasite with the drug leading to inhibition of its interiorization in HMC was an interesting finding, reinforcing the active role played by T. cruzi in this process. The effect of ET-18-OCH3 could be on the calcium homeostasis of the parasite, since, in preliminary experiments, we observed an enhancement of this ion in trypomastigotes on incubation with the drug (data not shown). The fundamental role of calcium levels of the T. cruzi in its interaction with host cells has already been reported (Moreno et al., 1994). The present results show the direct lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. As described in tumour cells, the membrane seems to be a primordial target of the drugs. Our next step will be the investigation of the effect of ALPs on T. cruzi signalling transduction involving both adenylate cyclase and phospholipase C.
Acknowledgements This investigation received financial support from INCO-DC (IC18-CT96-0084), CNPq and FIOCRUZ. The authors are grateful S.L. Croft and J.A. Urbina for helpful discussions, and to Dr M.J. Soares and Dr H. Moomen for the critical reading of this manuscript.
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Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi Ricardo M. Santa-Rita a, Helene Santos Barbosa b, Maria de Nazareth S.L. Meirelles b, Solange L. de Castro a,* a
Laborato´rio de Biologia Celular, DUBC, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, CP 926, 21045 -900 Rio de Janeiro, Brazil b Laborato´rio de Ultra-estrutura Celular, DUBC, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, CP 926, 21045 -900 Rio de Janeiro, Brazil Received 1 May 1999; received in revised form 1 November 1999; accepted 20 December 1999
Abstract Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH3, hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED50/24 h values were 13.4 92.8 mM and 11.7 9 0.6 mM for amastigotes and epimastigotes, respectively. ET-18-OCH3 (0.3 and 0.6 mM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40–57%. This drug (3.75–15 mM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED50 values of 14.3 9 4.2, 8.9 9 1.9 and 6.8 9 0.4 mM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma cruzi; Chemotherapy; Alkyl-lysophospholipids; Edelfosine; ET-18-OCH3
1. Introduction
* Corresponding author. Fax: +55-21-2604-434. E-mail address: [email protected] (S.L. de Castro)
Alkyl-lysophospholipids (ALPs) were first designed as potential immunomodulators and antimetabolites of phospholipid metabolism, and
0001-706X/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 0 ) 0 0 0 5 2 - 8
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have more recently been shown to be cytotoxic for a wide variety of tumour and leukaemia cells (reviewed in Brachwitz and Vollgraf, 1995). The first studies with ET-18-OCH3, an alkylglycerophosphocholine prototype, showed growth inhibition of Ehrlich ascite tumour cells in mice (Munder et al., 1976) and Lewis lung carcinoma metastases formation (Berdel et al., 1980). Later, a thioether substituted phosphatidylcholine (PC) analogue, ilmofosine, was developed and proved also to be active against neoplastic cells (Berdel et al., 1983). Observing that the glycerol moiety was not essential for the biological effects, a series of similar ether phospholipid alkylphosphocholines was synthesized, with hexadecyIPC (mitelfosine) being the most studied derivative. This compound displays a pronounced antitumour effect in a variety of rodent and human tumour cell lines (Unger et al., 1989). In addition, many other ALPs are under clinical trials (reviewed in Berdel, 1990; Winkelmann et al., 1992; Planting et al., 1993; Vogler et al., 1996a). The antitumour activity of ALPs is thought to be mediated by inhibition of cell proliferation and invasion, differentiation, induction and activation of cytotoxic macrophages (Berdel, 1991). The toxicity of ALPs involve inhibition of PC synthesis (Modolell et al., 1979; Vogler et al., 1985, 1996b), inhibition of signal transduction enzymes such as phosphatidylinositol-phospholipase C (Seewald et al., 1990; Powis et al., 1992) and protein kinase C (Helfman et al., 1983; Daniel et al., 1987), and alterations of intracellular calcium levels (Lazenby et al., 1990; Bergmann et al., 1994). Furthermore, due to their highly lipophilic nature, ALPs affect the physical properties of neoplastic cells by permeabilizing and increasing plasma membrane fluidity (Noseda et al., 1988). The development of ALPs as anticancer agents for humans ensures data on the pharmacology, toxicology and tolerance of these compounds, factors which are of importance for reducing the cost of drug development against tropical parasitic diseases. It is worth mentioning that hexadecyIPC showed promising results in clinical studies for the treatment of visceral leishmaniasis (Sundar et al., 1998). As part of a programme to investigate the antitrypanosomal activities of ALPs, we have in-
vestigated, in the present work, the effect of these compounds on Trypanosoma cruzi, the etiologic agent of Chagas’ disease. This disease constitutes one of the most important public health problems in many South American countries, affecting about 16–18 million persons (Moncayo, 1993). There is an urgent need of more efficient and safe drugs for the treatment of this disease, as benznidazole and nifurtimox have restricted applicability in chronic patients, besides presenting severe side effects (de Castro, 1993; Croft et al., 1997).
2. Materials and methods
2.1. Parasites and cell cultures Bloodstream trypomastigotes of T. cruzi (Y strain) were obtained from infected albino Swiss mice. Amastigotes were collected from the supernatant of infected J-774G-8 macrophage cultures (de Castro et al., 1987). The epimastigotes (Y strain and clone Dm28C) were maintained in LIT medium supplemented with 10% foetal calf serum (FCS) (Camargo, 1964) and harvested during the exponential phase of growth. Primary heart muscle cell cultures (HMC) were obtained from 18-day-old mouse embryos, after incubation with trypsin and collagenase as previously described (Meirelles et al., 1986).
2.2. Chemicals 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was purchased from Bachem (UK), hexadecylphosphocholine (hexadecylPC) was purchased from Sigma Chemical Co. (USA) and ilmofosine (1-hexadecylthio-2methoxymethyl - rac - glycero - 3 - phosphocholine) was a gift of Boehringer-Mannheim (Mannheim, Germany). FCS was obtained from Sigma Chemical Co. All other chemicals were of analytical grade.
2.3. Treatment of the three forms of T. cruzi with the ALPs Stock solutions of the compounds were prepared at 100 mM in phosphate-buffered saline (PBS).
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Trypomastigotes were resuspended in Dulbecco’s modified Eagle medium plus 10% FCS (DMES) to a final parasite concentration of 107 cells/ml in the absence or presence of 5% mice blood. The parasite suspension (100 ml) was added in a volume ratio of 1:1 to the compounds, and the mixture incubated for 24 h at 4 or 37°C. Untreated and crystal violet-treated parasites were used as controls. Amastigote and epimastigote forms (Y strain) were resuspended in DMES and LIT, respectively, and plated in 24-well plates at 5 × 106 cells/ml, in the absence or presence of different ALP concentrations at 28°C. For trypomastigote cultures, cell counts were performed after 24 h; for amastigote and epimastigote, they were performed from 4 h to 4 days. In all experiments, the ED50 was calculated.
2.6. Effect of ET-18 -OCH3 on the interaction between T. cruzi and heart muscle cells
2.4. Ultrastructural analysis
3. Results
Epimastigotes were treated with different concentrations of ALPs (2.5 – 10 mM), at 28°C in LIT. After 24 h, control and treated parasites were fixed (60 min/4°C) in a solution containing 2.5% glutaraldehyde, 2.5 mM CaCl2 and 0.1 M cacodylate buffer, pH 7.2 (Tcaco). After washing in cacodylate buffer, the cells were treated for 1 h with the Tcaco solution containing 1% OsO4, 0.8% potassium ferricyanide and 2.5 mM CaCl2. They were then dehydrated in acetone and embedded in Epon resin. Thin sections, stained with uranyl acetate and lead citrate, were examined in an EM 10C Zeiss transmission electron microscope.
The effect of the three compounds (ET-18OCH3, hexadecyIPC and ilmofosine) on trypomastigote viability was assayed after 24 h of incubation at 4 and 37°C, in the presence or absence of blood (Fig. 1). The major lytic activity was observed at the higher temperature and in the absence of blood. The ED50 values are presented in Table 1, and we can observed that at both 4 and 37°C, the addition of 5% blood decreased the trypanocidal activity two to 3.5 times. Comparison of the lytic activity of ALPs with that of crystal violet (ED50/24 h= 459.5951.9 mM), in standard conditions (4°C and in presence of blood) (de Castro et al., 1994), showed that they are about 2.5 to four times more active than the reference drug in inducing lysis of the parasite. Assays with the proliferative forms were performed at 28°C, using DMES for amastigotes and LIT medium for epimastigotes. ET-18-OCH3 showed a similar effect on the kinetics of the proliferation of both forms, with the ED50/24 h being 13.492.8 and 11.79 0.6 mM for amastigotes and epimastigotes, respectively (Table 2). The drugs were, respectively, 4–8.8 and 11.1–17.9 times more active against epimastigotes and amastigotes then nifurtimox (De Conti et al., 1996).
2.5. Effect of ET-18 -OCH3 on the metacyclogenesis process Epimastigote forms (clone Dm28C) were harvested from the LIT medium, incubated for 2 h in artificial triatomine urine (TAU) (Contreras et al., 1988) and then incubated in TAU supplemented with 10 mM L-proline, 50 mM sodium glutamate and 2 mM sodium aspartate (TAU3AAG) in the presence or absence of ET-18-OCH3. After 96 h, epimastigotes and trypomastigotes in the supernatant were counted in a Neubauer chamber.
HMC were incubated for 24 h with trypomastigotes (10:1, parasite:HMC ratio). The noninternalized parasites were removed, and fresh DMES, with or without ET-18-OCH3 (6.3–50 mM), added to the cultures and changed every 2 days. At specified intervals, the cultures were fixed (Bouin’s solution), stained (Giemsa), and counted (Zeiss photomicroscope). In another protocol, prior to the infection, the trypomastigotes were incubated (1 h at 37°C) with or without ALPs (1.9–60 mM). After washing (twice with DMES), the parasites were counted and added to HMC cultures (10:1, parasite:HMC ratio). After 24 h, the cultures were processed as already described.
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Fig. 1. Trypanocidal effect of ALPs on bloodstream trypomastigotes of T. cruzi (Y strain) in the absence (a,c) and presence (b,d) of 5% blood (4 and 37°C): ( ) ET-18-OCH3, (") ilmofosine and () hexadeylPC. Table 1 Values of ED50/24 h (mM) of the ALPs on T. cruzi bloodstream trypomastigotes in different experimental conditions 4°C/DMES
37°C/DMES
–
+5% whole mice blood
–
ET-18-OCH3 Ilmofosine
59.496.2a 44.29 7.0
141.99 3.1 177.69 11.6
29.0 92.8 29.5 9 3.6
HexadecyIPC
62.79 3.1
196.69 15.0
55.4 97.6
a
Mean9 S.D. of at least three experiments.
+5% whole mice blood 66.99 2.0 61.8 912.0 130.0 92.1
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Table 2 Values of ED50 (mM) of the ALPs on the proliferation of T. cruzi 4h Epimastigotes ET-18-OCH3 Ilmofosine HexadecyIPC
41.792.3a – –
Nifurtimoxb Amastigotes ET-18-OCH3 HexadecyIPC Ilmofosine
b
11.790.6 26.6 9 3.7 17.491.2
48 h
72 h
96 h
5.4 90.9 9.3 9 0.3 9.2 90.0
4.4 9 0.2 6.4 9 0.8 8.0 9 0.0
4.0 90.8 3.4 91.1 7.7 90.0
3.8 9 1.4 4.5 90.1 7.9 91.9
3.4 9 0.4 4.2 90.1 7.4 9 2.5
2.3 90.6 2.5 90.6 4.8 90.4
103.79 4.1 59.69 10.4 – –
Nifurtimoxb a
24 h
13.492.8 11.69 0.1 18.69 6.8 207.39 35.4
Mean9 S.D. of at least three experiments. De Conti et al. (1996).
The ALP activity against T. cruzi was much more pronounced when the incubations were performed in the absence of serum. In appropriate culture medium, the ED50 after 4 h for amastigote and epimastigotes was 59.6 and 41.7 mM, while, in PBS after 20 min, these values decreased to 7.5 and 25.7 mM, respectively. While the range of ED50 for trypomastigotes after 24 h was about 30 mM, after 30 min at 30 mM in PBS, total lysis of the parasites occurred. The ultrastructural analysis of epimastigotes treated with the ALPs showed initial alterations in cell swelling and extensive blebing of the flagellar membrane. These blebs were a characteristic effect of the three compounds (Fig. 2b,c and Fig. 3a – c). A marked swelling of the kinetoplast – mitochondrion system with loss of the inner membrane organization and disappearance of the electron-dense bodies of the matrix was also observed (Fig. 2b). These structural changes were drug-concentration dependent, as shown by the extensive disruption of the cellular matrix, disintegration of organelles and appearance of ghost parasites (data not shown). The kinetoplast (Fig. 3c), nucleus and subpellicular microtubules (Fig. 3a,b,d) were the most unaltered structures. In four independent experiments, the metacyclogenesis was inhibited by 40 – 57%, at 0.3 and 0.6 mM ET-18-OCH3 (Fig. 4). These concentra-
tions did not interfere with the total number of parasites (epimastigotes plus trypomastigotes). To analyse the effect of ET-18-OCH3 on both the kinetics of the infection and on the intracellular differentiation of amastigotes, the drug (3.75– 15 mM) was added to HMC and maintained throughout the experiment, leading to a dose-de-
Fig. 2. Ultrastructural alterations in epimastigote forms treated for 24 h with ET-18-OCH3. (a) Control cell with normal morphology (N, nucleus; k, kinetoplast; F, flagellum; FP, flagellar pocket; G, Golgi apparatus; M, mitochondrion) (10 000 × ); (b,c) ET-18-OCH3 (5 and 10 mM) resulted in swelling of the mitochondrion with loss of the inner membrane organization resembling a fibrous network (arrow), and also blebs of the plasma membrane most commonly in the flagellum (arrowhead) (12 600 × and 16 500 ×).
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Fig. 3. Ultrastructural alterations on epimastigote forms treated for 24 h with the ALPs: (a,b) ilmofosine (2.5 and 5 mM) showing similar effect on the flagellar membrane (arrowhead) (18 000 × and 19 000 × ); (c,d) hexadecylPC (5 and 10 mM) (9600 × and 14 500 ×). The micrographs show different effects of the damage at the flagellum (arrowhead). Some parasites showed extensive vacuolization around the flagellar pocket (arrows).
and 3 days of treatment, respectively. At 30 mM, this ALP caused damage to the host cell. After 1 and 2 days of treatment, the percentage of cells containing parasites in differentiation was similar to the control (Fig. 5b), indicating no significant effect on the differentiation of amastigotes, although a marked decrease in the infection index. In another approach, the parasites were treated with ET-18-OCH3 (2–60 mM), for 1 h, washed and added to the cultures. After 18 h of interaction, inhibition of parasite interiorization in HMC was observed, in the range 10–60% (data not shown). It is important to note that no alteration on motility of the trypomastigotes or other damage, as determined by light microscopy, was observed during the 1 h treatment with the ALP.
Fig. 4. Inhibition of the T. cruzi (clone Dm28c) metacyclogenesis by ET-18-OCH3. After 3 days of treatment with 0.3 and 0.6 mM ALP, differential countings of epimastigotes and trypomastigotes were performed. The total number of parasites was maintained inaltered during the experiments.
pendent inhibition of the infection (Fig. 5a). The mean values of ED50 for three experiments were 14.3 94.2, 8.991.9 and 6.89 0.4 mM, for 1, 2
Fig. 5. Effect of ET-18-OCH3 on the infection of HMC with T. cruzi. (a) Dose- and time-dependent effects of the drug in the range 3.2 – 15 mM; (b) similar percentage levels of intracellular differentiation of amastigotes to trypomastigotes was observed between control and treated cells (3.2 and 7.5 mM).
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4. Discussion The effect of ALPs on trypomastigotes was assayed in four different conditions, by combining temperature (4 and 37°C) with addition or not of mouse blood to DMES medium. Experiments were performed at 4°C in the presence of blood due to the potential use of these compounds for the prophylaxis of banked blood. The decrease of the lytic activity of different compounds against T. cruzi due to the presence of blood components has been already described (Lopes et al., 1978; Rovai et al., 1990). In all four conditions, the ALPs presented a higher lytic activity than crystal violet, the standard compound. Mitelfosine was less active that the other two drugs. This could be explained by structural differences between these compounds; while ET-18-OCH3 and ilmofosine are alkyl-glycerophosphocholines, hexadecyIPC presents a simpler backbone constituted only of a phosphocholine polar moiety and a fatty alkyl chain. When tissue culture-derived trypomastigotes were used, the drugs showed lytic activity lower than that of crystal violet (Croft et al., 1996). The lower lytic effect of ALPs against trypomastigotes at 4°C when compared with 37°C (in the range 1.1– 3.0 times) could be due to alterations with membrane activities. In leukaemia cells, it has been already determined that higher temperatures are determinant factors for susceptibility, leading to higher ALPs uptake and enrichment in the membrane (Kelley et al., 1993). Addition of whole blood caused a decrease of ALPs lytic activity, as shown by an increase in the ED50 between 2.1 and 4.0 times for the three compounds. This inhibition could be associated with the low performance of these ALPs in invivo experiments (Croft et al., 1996). The influence of serum components was more clearly observed when the parasites were incubated with the ALP in PBS buffer. Plasma protein binding is not surprising for hydrophobic compounds such as the ALPs. A ‘protecting effect’ of serum against the cytotoxicity induced by ALPs against T. cruzi was also reported for tumour cells (Petersen et al., 1992; Lohmeyer and Workman, 1993; Heesbeen et al., 1995).
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For all three forms of T. cruzi, all compounds showed similar lytic activity, with ED50/24 h in the range 11–30 mM. The only exception was hexadecyIPC acting on trypomastigotes (standard conditions), whose ED50/24 h was 55.4 mM. In comparison, the ED50/24 h for susceptible leukaemic cells (HL-60) was about 10 times lower (Diomede et al., 1993; Mollinedo et al., 1993), probably reflecting different mechanisms of action between T. cruzi and tumoral cells. The mode of action of ALP against several pathogenic micro-organisms is under investigation. The activity of a series of ALPs against cilliates and funghi was observed to present a good correlation with the effect on tumour cells (Tsushima et al., 1982). There are several reports on the effect of ALPs on trypanosomatids, African trypanosomes (Croft et al., 1996; Konstantinov et al., 1997) being more resistant to the drugs than T. cruzi or Leishmania (Kuhlencord et al., 1992; Bourass et al., 1996; Croft et al., 1996). Analysis of the ultrastructural alterations induced by the three drugs on the parasites showed a characteristic effect, which was the damage of the flagellar membrane, with extensive blebing. These alterations localized only in the flagellar membrane could be associated with interference caused by the ALPs on the phospholipid composition (Urbina, personal communication), as it is already known that the body and flagellar membranes of T. cruzi have different composition (reviewed in De Souza, 1984), Alterations in the mitochondrion, with loss of the inner membrane organization and absence of the cristae, was also frequently observed. The effect of the drugs was progressive and, with higher concentrations, an uncharacteristic effect on the cytoplasm, with extensive vacuolization, was also detected. Inhibitory effect of ALPs on lipid synthesis in tumour cells is well documented, involving PC in most cases, associated with inhibition of the translocation of PC cytidyltransferase (Modolell et al., 1979; Wieder et al., 1995; Vogler et al., 1996b). Preliminary results from our group show that the treatment of epimastigotes with ET-18OCH3 after incubation with 32P lead to a decrease of PC levels of about 60%, while alterations in phospho-inositides were also observed (data not shown).
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Due to their structure, ALPs have the potential to physically interact with the plasma membrane so as to bring about physical disruption. In tumour cells, ALP accumulation into cell membranes and perturbation of fluidity (Van Blitterswijk et al., 1987) and lipid metabolism have been previously described (Tidwell et al., 1981). Comparing the alterations caused by ALPs on T. cruzi and tumour cells, it seems that the membrane in both cases is a prime target (Berdel et al., 1983; Noseda et al., 1989; Vagnetti et al., 1990). Treatment of HL-60 leukaemic cells with ET-18-OCH3 at similar conditions, as used in the present work (2.5 mM/24 h), led to the formation of blebs and holes in the plasma membrane, cytoplasm vacuolization and nuclei piknosis (Noseda et al., 1989). On the other hand, in relation to ilmofosine, morphological alterations in cells isolated from leukaemia patients occurred at concentrations about 10 times higher than that observed with T. cruzi (Berdel et al., 1983). Alterations involving destruction of mitochondrial cristae, vacuolization and rarefaction of the cytoplasm were also observed after treatment of lymphoma cells with ET-18-OCH3 (Vagnetti et al., 1990). The observed inhibitory effect of ET-18-OCH3 on the differentiation of epimastigotes to trypomastigotes could involve the adenylate cyclase/ cyclic AMP system. Interference with this signalling system was previously associated with inhibition of differentiation of T. cruzi, including the metacyclogenesis process (de Castro et al., 1987; Rangel-Aldao et al., 1987; Gonzales-Perdomo et al., 1988). The treatment with ET-18-OCH3 of infected cultures, in the range 3 – 15 mM, caused a dose-dependent inhibition of HMC infection, while at 30 mM, this drug caused damage to the host cells, with extensive vacuolization of the cytoplasm and a decrease in the number of adhered cells. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by ET-18-OCH3. Drug doses inhibiting proliferation of amastigotes internalized in heart cells were in the same range as those that inhibited the proliferation of this form in axenic medium. On the other hand, amastigotes inside heart cells were less susceptible to ET-18-OCH3 than in
macrophages, the ED50/3 days being 6.8 and 1.4 mM, respectively (Croft et al., 1996). The pretreatment of the parasite with the drug leading to inhibition of its interiorization in HMC was an interesting finding, reinforcing the active role played by T. cruzi in this process. The effect of ET-18-OCH3 could be on the calcium homeostasis of the parasite, since, in preliminary experiments, we observed an enhancement of this ion in trypomastigotes on incubation with the drug (data not shown). The fundamental role of calcium levels of the T. cruzi in its interaction with host cells has already been reported (Moreno et al., 1994). The present results show the direct lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. As described in tumour cells, the membrane seems to be a primordial target of the drugs. Our next step will be the investigation of the effect of ALPs on T. cruzi signalling transduction involving both adenylate cyclase and phospholipase C.
Acknowledgements This investigation received financial support from INCO-DC (IC18-CT96-0084), CNPq and FIOCRUZ. The authors are grateful S.L. Croft and J.A. Urbina for helpful discussions, and to Dr M.J. Soares and Dr H. Moomen for the critical reading of this manuscript.
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