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Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole
Experimental Parasitology 124 (2010) 167–171
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole Rodrigo López-Muñoz a, Mario Faúndez d, Sebastián Klein d, Sebastián Escanilla d, Gloria Torres a, Dasfne Lee-Liu a, Jorge Ferreira a, Ulrike Kemmerling b, Myriam Orellana a, Antonio Morello a, Arturo Ferreira c, Juan D. Maya a,* a
Program of Molecular and Clinical Pharmacology, ICBM, School of Medicine, University of Chile, Chile Program of Anatomy and Developmental Biology, ICBM, School of Medicine, University of Chile/School of Health Sciences, Talca University, Chile Program of Immunology, ICBM, School of Medicine, University of Chile, Chile d Facultad de Química, Pontificia Universidad Católica de Chile, Chile b c
a r t i c l e
i n f o
Article history: Received 9 March 2009 Received in revised form 1 September 2009 Accepted 2 September 2009 Available online 6 September 2009 Keywords: Trypanosoma cruzi Aspirin Nifurtimox Benznidazole Synergism
a b s t r a c t Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor b and PGE2 activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE2 synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction In Latin America, Chagas’ disease, caused by the protozoan Trypanosoma cruzi, represents a health threat for an estimated 10–20 million people and is the second highest burden of disease among Tropical Diseases in the Americas (Reithinger et al., 2009). Current Chagas’ disease treatment is limited to nifurtimox and benznidazole. However, these drugs have limited efficacy and frequent and significant side effects. Hundreds of natural and synthetic compounds have been tested against T. cruzi. However, very few are devoid of host’s cytotoxic activity and are more effective than nifurtimox and benznidazole, especially against the intracellular amastigotes (Maya et al., 2007). During primoinfection, T. cruzi trypomastigotes invade macrophages, and transform in the intracellular amastigote forms. In this setting, macrophages mount an antiparasitic response through an inflammatory activity including increasing nitric oxide production and oxidative stress through peroxynitrite formation (Borges et al.,
* Corresponding author. Address: University of Chile, Faculty of Medicine, Molecular and Clinical Pharmacology Program, P.O. Box 70000, Santiago 7, Chile. Fax: +562 7355580. E-mail address: [email protected] (J.D. Maya). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.09.005
1998; Peluffo et al., 2004). Simultaneously, T. cruzi induces an antiinflammatory response through activation of cellular mediators such as tumor growth factor b (TGF-b) (Li et al., 2006) and prostaglandins, specifically, PGE2 (Abdalla et al., 2008). TGF-b and PGE2 decrease iNOS activity and, consequently, increase polyamine production, by increasing L-arginine availability (Freire-de-Lima et al., 2006; Peluffo et al., 2004). Polyamines are essential for T. cruzi amastigote proliferation and trypanothione (T(SH)2) synthesis. T(SH)2 is the most important oxidative aggression scavenger in trypanosomatids (Maya et al., 2007). Thus, the parasite evades the innate immune response attenuating the antiparasitic response of macrophages by decreasing nitric oxide production, improving polyamine availability and providing a favorable intracellular redox environment. Freire-de-Lima et al. (2000) showed that prostaglandin synthesis inhibition in T. cruzi infected macrophages, which have ingested apoptotic bodies, produced a significant decrease in infection. Further evidence indicates that prostaglandin synthesis inhibition could be important in acute infection therapy (Freire-de-Lima et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005; Pinge-Filho et al., 1999). In this work, we show that a COX inhibitor like aspirin (ASA) increases the trypanocidal activity of nifurtimox and benznidazole in
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an in vitro model of T. cruzi infection. Thus, the experimental basis for a potentially new therapeutic strategy for the control of this disease is established.
treatment and centrifuged at 500 g during 5 min. Supernatants were discarded, pellets resuspended in 1 ml of fresh RPMI 1640 and trypomastigotes were counted using direct microscopy (Freire-de-Lima et al., 2000; Nunes et al., 1998).
2. Materials and methods 2.6. Statistical analysis 2.1. Cell culture and in vitro infection with T. cruzi RAW (1 105) 264.7 cells/cm2 were cultured in 0.22% sodium bicarbonate, 5% fetal bovine serum supplemented RMPI 1640 medium in humidified air with 5% CO2, at 37 °C. RAW 264.7 cells were infected with Dm28c trypomastigotes, at a 1:3 (cell:parasite) ratio. T. cruzi trypomastigotes were initially obtained from primary cultures of peritoneal macrophage from chagasic mice.
IC50 and growth rate calculations were analyzed by non-linear regression. When necessary, data were also analyzed through two-way ANOVA. The drug combination treatments were analyzed by isobologram constructions through sum-of-squares plus Fisher test. Values of p equal or less than 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism 5.0. All experiments were done in triplicate and results correspond to means ± SD from at least three independent experiments.
2.2. Cell viability assay 3. Results The effect of drug treatments on RAW 264.7 cells was evaluated through the MTT assay as viability test (Mosmann, 1983). Briefly, 10 ll of 5 mg/ml tetrazolium dye (MTT; 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) plus 0.22 mg/ml Phenazine metosulfate (electron carrier), were added to each well containing RAW 264.7 cell culture in 100 ll RPMI 1640 without phenol red. Drugs, dissolved in DMSO, were added to culture media at the concentrations shown in figures and tables. DMSO final concentration was less than 0.25% v/v. After incubation for 4 h at 37 °C, the generated water insoluble formazan dye was dissolved by addition of 100 ll of 10% w/v SDS in 0.01 M HCl. The plates were further incubated overnight at 37 °C, and optical density (OD) of the wells was determined using a microplate reader (Labsystems Multiskan MS, Finlandia) at 570 nm. Under these conditions, the OD is directly proportional to the viable cell number in each well. All experiments were performed at least three times and data are shown as the means and their standard deviations from triplicate cultures.
3.1. Aspirin decreases trypomastigote release to culture supernatants from T. cruzi-infected RAW 264.7 cells At fourth day of RAW cells infection, trypomastigotes began to be released to the supernatants. Aspirin showed a concentrationdependent effect, with an IC50 of 313.1 ± 36.0 lM (Table 1). This concentration is below the steady-state concentration (Css, 1.1– 2.2 mM after oral administration of 3–4 g/day) found in humans treated with aspirin at high concentrations, as is the highest aspirin concentration used in other experiments (1 mM) (Anderson et al., 2002; Keystone et al., 1982). This result is in agreement with previous reports, showing that aspirin and other COX inhibitors decreased T. cruzi infection (Abdalla et al., 2008; Freire-de-Lima et al., 2000; Kubata et al., 2002; Michelin et al., 2005; Paiva et al., 2007). 3.2. Aspirin improves the activity of nifurtimox and benznidazole upon infected RAW 264.7 cells in a synergistic way
2.3. Prostaglandin E2 measurement The PGE2 levels were measured in the supernatant of an infected RAW cells culture. The ‘‘Prostagladin E2 EIA Kit – Monoclonal” (CaymanÒ Chemical Co., USA) kit was used, according to the manufacturer’s instructions. RAW 264.7 cells were infected with T. cruzi Dm28 clone trypomastigotes. After 72 h post-infection, 50 ll supernatant were tested using this immunoassay. 2.4. Nitric oxide measurement NO synthesis was evaluated indirectly, through nitrite content in supernatants from T. cruzi-infected RAW 264.7 cultures. Griess’ reactive was used to measure colorimetrically the nitrite content at 570 nm. In each experiment, nitrite concentration was calculated from a NaNO2 standard curve (Tsikas, 2007). 2.5. Effect of drugs on infected RAW 264.7 cells The effect of aspirin, nifurtimox and benznidazole on T. cruzi-infected RAW 264.7 cells was assessed by the number of trypomastigotes released to the culture supernatants. Twenty-four hours after infection, aspirin, nifurtimox, benznidazole treatments and their combinations were started at concentrations described in results. Every 24 h culture media was removed and fresh medium was added together with the drugs at the same concentrations. To show the influence upon NOS and COX, aminoguanidine (5 mM) and PGE2 (1 mM), respectively, were added in some experiments. Cell culture medium was harvested at the third day post
As expected, nifurtimox and benznidazole had a concentrationdependent effect on trypomastigote release with an IC50 of 0.083 lM and 0.336 lM, respectively (Table 1). The addition of a low concentration of aspirin (IC12.5 of aspirin) reduces the IC50 of nifurtimox from 0.083 to 0.023 lM (reduction by 72.3%) while the IC50 of benznidazole was reduced by 61.7%. In addition, when aspirin was combined with nifurtimox or benznidazole (at its IC12.5 concentrations), the IC50 value for aspirin was decreased by 57.2% and 76.7%, respectively. The observed IC50 value is significantly lower than that expected for the combinations if the interaction was additive (p < 0.002) (Table 1). The graphical representation of this interaction is shown in the isobolograms of Fig. 1. These isobolograms allow the comparison of theoretical additive and observed experimental effects. Combinatory indexes (Chou, 2006) equal, lower or larger than 1, correspond to additive, synergistic or antagonic behavior, respectively. Combinatory indexes for aspirin and nifurtimox or benznidazole combinations are described in Table 1. Neither aspirin, nifurtimox, benznidazole nor their combinations, at the highest trypanocidal concentrations, showed cytotoxicity against uninfected RAW 264.7 cells (data not shown). 3.3. Aspirin effect on infected RAW cells is not dependent of PGE2 synthesis inhibition Trypanosoma cruzi-infected RAW 264.7 cells presented a significantly higher prostaglandin (PGE2) production compared to uninfected RAW cells. ASA reverted prostaglandin levels to baseline in a dose–response way (Fig. 2A). Note that the maximal effect of ASA
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Table 1 Combinatory index of the aspirin effect together with nifurtimox and benznidazole upon RAW 264.7 cells infected with T. cruzi. The effect of drugs (IC50 values) was determined by trypomastigote release from T. cruzi-infected RAW 264.7 cells. IC50 (lM)
IC50 of the combination (lM)
Combinatory index
Theoreticala
Observed
p-Value
Aspirin Nifurtimox
313.1 ± 36.0 0.083 ± 0.005
250 0.066
134.0 ± 13.4 0.023 ± 0.008
<0.001 0.002
0.71
Aspirin Benznidazole
313.1 ± 36.0 0.34 ± 0.002
250 0.27
73.1 ± 11.4 0.13 ± 0.02
<0.0001 <0.0001
0.61
a Theoretical IC50 corresponds to the value predicted from isobolograms depicted in Fig. 1. Nifurtimox, aspirin and benznidazole were added at their estimated IC12.5 concentrations. p values correspond to the comparisons between theoretical and observed IC50 values through sum-of-squares plus F test. Combinatory index was calculated according to Chou (2006). Results are expressed as means ± SD from three independent experiments.
Fig. 1. Isobolographic analysis of the effect of aspirin, nifurtimox, benznidazole and their combinations on trypomastigote release. RAW 264.37 cells were infected with T. cruzi Dm28c trypomastigotes and incubated with (A) aspirin alone, aspirin plus nifurtimox at their IC12.5 and, nifurtimox plus aspirin at their IC12.5; (B) aspirin alone, aspirin plus benznidazole at their IC12.5 and, benznidazole plus aspirin at their IC12.5. After 3 days of treatment, trypomastigotes released to the supernatant were counted by direct microscopy. Continuous line corresponds to the predicted positions of the experimental points for additive effects. Dots in the discontinuous line correspond to the experimental findings depicted in Table 1.
over the PGE2 synthesis is achieved at 0.25 mM, a dose–response fit of this data showed an IC50 of 7.7 ± 2.54 lM. PGE2 was added to reverse the effect of ASA 0.25 mM. The lack of effect of PGE2 over ASA treatment might indicate that PGE2 is not involved in the ASA effect on infected cells (Fig. 2B).
Fig. 2. Relationship between prostaglandin synthesis inhibition and anti-T. cruzi aspirin effect on infected RAW 264.7 cells. (A) Effect of aspirin upon prostaglandin production in RAW 264.7 cells. Macrophages were infected with T. cruzi trypomastigotes and PGE2 was measured in the supernatant, after 72 h treatment with aspirin (ASA, at 0.065, 0.125, 0.25, 0.5 mM). Results are expressed as means ± SD from three independent experiments, ***p < 0.001 from Tukey post-test. (B) Effect of PGE2 upon trypomastigote release in aspirin treated RAW 264.7 cells infected with T. cruzi. Macrophages were infected with T. cruzi trypomastigotes and, after 72 h treatment with 0.25 mM ASA alone or combined with 1 mM PGE2, trypomastigote release to supernatants were measured. Results are expressed as means ± SD from three independent experiments. ***p < 0.001 from Tukey post-test. ns, no significant differences.
3.4. Aspirin restores NO levels in T. cruzi-infected RAW 264.7 cells The effect of aspirin upon NO production was evaluated by nitrite detection in supernatants from T. cruzi-infected RAW 264.7 cells by the Griess reaction. While T. cruzi infection decreases NO
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Fig. 3. Effect of aspirin upon nitrite production and trypomastigote release in T. cruzi-infected RAW 264.7 cells. (A) Nitrite levels (white bars) and trypomastigote release (black bars) in aspirin treated Raw 264.7 cells infected with T. cruzi trypomastigotes. After 72 h nitrite levels in supernatant were determined by the Griess reaction. §p < 0.001 compared with uninfected control (RAW). àp < 0001 compared with infected control (ASA 0 mM). (B) Effect of aminoguanidine (AMG) upon trypomastigote release in aspirin-treated T. cruzi-infected RAW 264.7 cells. Macrophages were infected with T. cruzi trypomastigotes, and after 72 h treatment with 1 mM aspirin (ASA), 5 mM AMG or ASA plus AMG trypomastigote release to supernatants was measured. Results are expressed as means ± SD from three independent experiments. *p < 0.05; ***p < 0.001 from Tukey post-test.
production in RAW 264.7 cells, aspirin restores it (Fig. 3A). The increase in NO production, induced by aspirin has a dose–response behavior, similar to trypomastigote release inhibition (Fig. 3A). At 1 mM aspirin, the highest effective concentration evaluated, NO production was similar to non-infected cells (p < 0.001) (Fig. 3A). To corroborate that the effect of aspirin on macrophage activity depends upon restoring NO levels, we assessed the trypomastigote release to culture medium when RAW 264.7 cells were incubated with aminoguanidine (AMG), an iNOS inhibitor (Fig. 3C). Thus, 5 mM AMG reverts aspirin effect by 50%.
4. Discussion Different studies have shown the effect of COX inhibitors, such as aspirin, on several T. cruzi infection models (Freire-de-Lima et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005; Pinge-Filho et al., 1999). However, COX inhibitors concentrationdependent responses and mechanisms affecting T. cruzi proliferation inside host cells have not been studied. Herein, we studied the improvement of the macrophage antiparasitic activity induced by aspirin, the synergy of this effect with the activity of the antichagasic drugs nifurtimox and benznidazole and, possible mechanisms involved in those effects.
Attention can be drawn to the low reported IC50 values of nifurtimox (0.0830 lM) and benznidazole (0.336 lM). However, this might be due to the low parasite density used in the experiments. In previous experiments carried out on VERO cells (Faundez et al., 2005), parasite density was an order of magnitude higher and the highest concentration used for nifurtimox and benznidazole was 1 lM. Nevertheless, this situation is not an obstacle to perform synergism studies with other drugs. Herein, the relationship of aspirin with classic antichagasic therapy is further substantiated. Thus, the IC50 value of aspirin in infected macrophages is several folds higher than those determined for nifurtimox and benznidazole (Table 1). However, from the isobolographic analysis, we can conclude that aspirin acts synergistically with these classical drugs. In fact, the IC50 of both nifurtimox and benznidazole, decreased in average by 67% when combined with aspirin (Table 1). However, the observed synergy is not related to a direct effect of both drugs upon the same target or pathway, i.e. parasite viability. Instead, the combined effects of aspirin on the macrophage and the toxic effects of antichagasic drugs on the parasite are complementary mechanisms that explain the isobolographic result. This concept is in agreement with synergistic mechanisms reported for other drug combinations (Jia et al., 2009). Fig. 2A shows that PGE2 production increases significantly in T. cruzi infected macrophages as compared with healthy macrophages. Thus, PGE2 synthesis inhibition using aspirin could improve macrophage response against T. cruzi infection. In fact, ASA reduces PGE2 levels in a dose–response way, with an IC50 = 7.7 lM. This value is 40-fold lower than the ASA IC50 observed on trypomastigote release experiments (IC50 = 313 lM). Therefore, complete PGE2 synthesis inhibition is achieved at concentration levels that have no significant effect on trypomastigote release. Moreover, there is no evidence supporting that PGE2 itself could increase T. cruzi infection. We attempted to determine whether PGE2 is involved in the effect of ASA upon T. cruzi infected cells. We did not find reversion of ASA effect, even at PGE2 concentrations as high as 1 mM (Fig. 2B), indicating that PGE2 does not have a main role on ASA effect. Other COX inhibitors, such as indomethacin and meloxicam were tested, both inhibiting trypomastigote release (IC50 = 97.6 ± 11.1 and 42.7 ± 0.36, respectively). These data suggest a pivotal function of COX in intracellular amastigote proliferation. However, there might be other prostaglandins playing key roles in parasite proliferation. Cardoni and Antunez (2004) showed that T. cruzi infected mice have increased levels of other COX metabolites, such as PGI2 and TXA2, yet their role on parasite infection is still unknown (Cardoni and Antunez, 2004). Trypanosoma cruzi induces TGF-b and PGE2 production by macrophages, especially in the presence of apoptotic bodies (Freire-deLima et al., 2000). Thus, the parasite induces an anti-inflammatory response, attenuating macrophage activation and evading its antiparasitic activity. In macrophages exposed to apoptotic bodies, TGF-b increases PGE2 synthesis and decreases NO (Freire-de-Lima et al., 2006). Inhibition of COX activity may increase NO levels, thus restoring the antiparasitic activity of macrophages. Our results are in agreement with this proposal (Fig. 3A and B). It is important to note that all these experiments were performed without exposure to apoptotic bodies. Consequently, T. cruzi itself can suppress macrophage antiparasitic activity in an apoptosis mimicry fashion, through, perhaps, T. cruzi calreticulin–C1q interactions (Ferreira et al., 2004). Thus, macrophage antiparasitic activity restoration can be pharmacologically modulated. Finally, in T. cruzi infected macrophages, COX is related to the increase of ornithine decarboxylase (ODC) activity (Freire-de-Lima et al., 2000), which might increase the polyamine content in macrophages. Since T. cruzi uses these polyamines to synthesize trypanothione, the inhibition of COX by ASA, indirectly contribute to
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decrease trypanothione synthesis in T. cruzi. This mechanism, together with the restoring of NO levels in macrophages through COX inhibition, could be involved in the activity of ASA on the macrophage response to T. cruzi infection. In conclusion, the observed synergic effect of aspirin with nifurtimox and benznidazole is explained by the capability of aspirin to improve the antiparasitic activity of macrophages. Acknowledgments We thank Dr. Norbel Galanti (Cellular and Molecular Biology Program, ICBM, Faculty of Medicine, University of Chile) for critical review of this paper. FONDECYT 1090078, PBCT ANILLO ACT 29 and REDES-07, Chilean grants supported this research. References Abdalla, G.K., Faria, G.E., Silva, K.T., Castro, E.C., Reis, M.A., Michelin, M.A., 2008. Trypanosoma cruzi: the role of PGE2 in immune response during the acute phase of experimental infection. Experimental Parasitology 118, 514–521. Anderson, P.O., Knoben, J.E., Troutman, W.G., 2002. Handbook of Clinical Drug Data. McGraw-Hill Medical Pub. Division, New York. Borges, M.M., Kloetzel, J.K., Andrade Jr., H.F., Tadokoro, C.E., Pinge-Filho, P., Abrahamsohn, I., 1998. Prostaglandin and nitric oxide regulate TNF-alpha production during Trypanosoma cruzi infection. Immunology Letters 63, 1–8. Cardoni, R.L., Antunez, M.I., 2004. Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections. Mediators of Inflammation 13, 235–240. Chou, T.C., 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 58, 621–681. Faundez, M., Pino, L., Letelier, P., Ortiz, C., Lopez, R., Seguel, C., Ferreira, J., Pavani, M., Morello, A., Maya, J.D., 2005. Buthionine sulfoximine increases the toxicity of nifurtimox and benznidazole to Trypanosoma cruzi. Antimicrobial Agents and Chemotherapy 49, 126–130. Ferreira, V., Valck, C., Sanchez, G., Gingras, A., Tzima, S., Molina, M.C., Sim, R., Schwaeble, W., Ferreira, A., 2004. The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi. Journal of Immunology 172, 3042–3050. Freire-de-Lima, C.G., Nascimento, D.O., Soares, M.B., Bozza, P.T., Castro-Faria-Neto, H.C., de Mello, F.G., DosReis, G.A., Lopes, M.F., 2000. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 403, 199–203. Freire-de-Lima, C.G., Xiao, Y.Q., Gardai, S.J., Bratton, D.L., Schiemann, W.P., Henson, P.M., 2006. Apoptotic cells, through transforming growth factor-beta, coordinately induce anti-inflammatory and suppress pro-inflammatory
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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole Rodrigo López-Muñoz a, Mario Faúndez d, Sebastián Klein d, Sebastián Escanilla d, Gloria Torres a, Dasfne Lee-Liu a, Jorge Ferreira a, Ulrike Kemmerling b, Myriam Orellana a, Antonio Morello a, Arturo Ferreira c, Juan D. Maya a,* a
Program of Molecular and Clinical Pharmacology, ICBM, School of Medicine, University of Chile, Chile Program of Anatomy and Developmental Biology, ICBM, School of Medicine, University of Chile/School of Health Sciences, Talca University, Chile Program of Immunology, ICBM, School of Medicine, University of Chile, Chile d Facultad de Química, Pontificia Universidad Católica de Chile, Chile b c
a r t i c l e
i n f o
Article history: Received 9 March 2009 Received in revised form 1 September 2009 Accepted 2 September 2009 Available online 6 September 2009 Keywords: Trypanosoma cruzi Aspirin Nifurtimox Benznidazole Synergism
a b s t r a c t Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor b and PGE2 activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE2 synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction In Latin America, Chagas’ disease, caused by the protozoan Trypanosoma cruzi, represents a health threat for an estimated 10–20 million people and is the second highest burden of disease among Tropical Diseases in the Americas (Reithinger et al., 2009). Current Chagas’ disease treatment is limited to nifurtimox and benznidazole. However, these drugs have limited efficacy and frequent and significant side effects. Hundreds of natural and synthetic compounds have been tested against T. cruzi. However, very few are devoid of host’s cytotoxic activity and are more effective than nifurtimox and benznidazole, especially against the intracellular amastigotes (Maya et al., 2007). During primoinfection, T. cruzi trypomastigotes invade macrophages, and transform in the intracellular amastigote forms. In this setting, macrophages mount an antiparasitic response through an inflammatory activity including increasing nitric oxide production and oxidative stress through peroxynitrite formation (Borges et al.,
* Corresponding author. Address: University of Chile, Faculty of Medicine, Molecular and Clinical Pharmacology Program, P.O. Box 70000, Santiago 7, Chile. Fax: +562 7355580. E-mail address: [email protected] (J.D. Maya). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.09.005
1998; Peluffo et al., 2004). Simultaneously, T. cruzi induces an antiinflammatory response through activation of cellular mediators such as tumor growth factor b (TGF-b) (Li et al., 2006) and prostaglandins, specifically, PGE2 (Abdalla et al., 2008). TGF-b and PGE2 decrease iNOS activity and, consequently, increase polyamine production, by increasing L-arginine availability (Freire-de-Lima et al., 2006; Peluffo et al., 2004). Polyamines are essential for T. cruzi amastigote proliferation and trypanothione (T(SH)2) synthesis. T(SH)2 is the most important oxidative aggression scavenger in trypanosomatids (Maya et al., 2007). Thus, the parasite evades the innate immune response attenuating the antiparasitic response of macrophages by decreasing nitric oxide production, improving polyamine availability and providing a favorable intracellular redox environment. Freire-de-Lima et al. (2000) showed that prostaglandin synthesis inhibition in T. cruzi infected macrophages, which have ingested apoptotic bodies, produced a significant decrease in infection. Further evidence indicates that prostaglandin synthesis inhibition could be important in acute infection therapy (Freire-de-Lima et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005; Pinge-Filho et al., 1999). In this work, we show that a COX inhibitor like aspirin (ASA) increases the trypanocidal activity of nifurtimox and benznidazole in
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an in vitro model of T. cruzi infection. Thus, the experimental basis for a potentially new therapeutic strategy for the control of this disease is established.
treatment and centrifuged at 500 g during 5 min. Supernatants were discarded, pellets resuspended in 1 ml of fresh RPMI 1640 and trypomastigotes were counted using direct microscopy (Freire-de-Lima et al., 2000; Nunes et al., 1998).
2. Materials and methods 2.6. Statistical analysis 2.1. Cell culture and in vitro infection with T. cruzi RAW (1 105) 264.7 cells/cm2 were cultured in 0.22% sodium bicarbonate, 5% fetal bovine serum supplemented RMPI 1640 medium in humidified air with 5% CO2, at 37 °C. RAW 264.7 cells were infected with Dm28c trypomastigotes, at a 1:3 (cell:parasite) ratio. T. cruzi trypomastigotes were initially obtained from primary cultures of peritoneal macrophage from chagasic mice.
IC50 and growth rate calculations were analyzed by non-linear regression. When necessary, data were also analyzed through two-way ANOVA. The drug combination treatments were analyzed by isobologram constructions through sum-of-squares plus Fisher test. Values of p equal or less than 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism 5.0. All experiments were done in triplicate and results correspond to means ± SD from at least three independent experiments.
2.2. Cell viability assay 3. Results The effect of drug treatments on RAW 264.7 cells was evaluated through the MTT assay as viability test (Mosmann, 1983). Briefly, 10 ll of 5 mg/ml tetrazolium dye (MTT; 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) plus 0.22 mg/ml Phenazine metosulfate (electron carrier), were added to each well containing RAW 264.7 cell culture in 100 ll RPMI 1640 without phenol red. Drugs, dissolved in DMSO, were added to culture media at the concentrations shown in figures and tables. DMSO final concentration was less than 0.25% v/v. After incubation for 4 h at 37 °C, the generated water insoluble formazan dye was dissolved by addition of 100 ll of 10% w/v SDS in 0.01 M HCl. The plates were further incubated overnight at 37 °C, and optical density (OD) of the wells was determined using a microplate reader (Labsystems Multiskan MS, Finlandia) at 570 nm. Under these conditions, the OD is directly proportional to the viable cell number in each well. All experiments were performed at least three times and data are shown as the means and their standard deviations from triplicate cultures.
3.1. Aspirin decreases trypomastigote release to culture supernatants from T. cruzi-infected RAW 264.7 cells At fourth day of RAW cells infection, trypomastigotes began to be released to the supernatants. Aspirin showed a concentrationdependent effect, with an IC50 of 313.1 ± 36.0 lM (Table 1). This concentration is below the steady-state concentration (Css, 1.1– 2.2 mM after oral administration of 3–4 g/day) found in humans treated with aspirin at high concentrations, as is the highest aspirin concentration used in other experiments (1 mM) (Anderson et al., 2002; Keystone et al., 1982). This result is in agreement with previous reports, showing that aspirin and other COX inhibitors decreased T. cruzi infection (Abdalla et al., 2008; Freire-de-Lima et al., 2000; Kubata et al., 2002; Michelin et al., 2005; Paiva et al., 2007). 3.2. Aspirin improves the activity of nifurtimox and benznidazole upon infected RAW 264.7 cells in a synergistic way
2.3. Prostaglandin E2 measurement The PGE2 levels were measured in the supernatant of an infected RAW cells culture. The ‘‘Prostagladin E2 EIA Kit – Monoclonal” (CaymanÒ Chemical Co., USA) kit was used, according to the manufacturer’s instructions. RAW 264.7 cells were infected with T. cruzi Dm28 clone trypomastigotes. After 72 h post-infection, 50 ll supernatant were tested using this immunoassay. 2.4. Nitric oxide measurement NO synthesis was evaluated indirectly, through nitrite content in supernatants from T. cruzi-infected RAW 264.7 cultures. Griess’ reactive was used to measure colorimetrically the nitrite content at 570 nm. In each experiment, nitrite concentration was calculated from a NaNO2 standard curve (Tsikas, 2007). 2.5. Effect of drugs on infected RAW 264.7 cells The effect of aspirin, nifurtimox and benznidazole on T. cruzi-infected RAW 264.7 cells was assessed by the number of trypomastigotes released to the culture supernatants. Twenty-four hours after infection, aspirin, nifurtimox, benznidazole treatments and their combinations were started at concentrations described in results. Every 24 h culture media was removed and fresh medium was added together with the drugs at the same concentrations. To show the influence upon NOS and COX, aminoguanidine (5 mM) and PGE2 (1 mM), respectively, were added in some experiments. Cell culture medium was harvested at the third day post
As expected, nifurtimox and benznidazole had a concentrationdependent effect on trypomastigote release with an IC50 of 0.083 lM and 0.336 lM, respectively (Table 1). The addition of a low concentration of aspirin (IC12.5 of aspirin) reduces the IC50 of nifurtimox from 0.083 to 0.023 lM (reduction by 72.3%) while the IC50 of benznidazole was reduced by 61.7%. In addition, when aspirin was combined with nifurtimox or benznidazole (at its IC12.5 concentrations), the IC50 value for aspirin was decreased by 57.2% and 76.7%, respectively. The observed IC50 value is significantly lower than that expected for the combinations if the interaction was additive (p < 0.002) (Table 1). The graphical representation of this interaction is shown in the isobolograms of Fig. 1. These isobolograms allow the comparison of theoretical additive and observed experimental effects. Combinatory indexes (Chou, 2006) equal, lower or larger than 1, correspond to additive, synergistic or antagonic behavior, respectively. Combinatory indexes for aspirin and nifurtimox or benznidazole combinations are described in Table 1. Neither aspirin, nifurtimox, benznidazole nor their combinations, at the highest trypanocidal concentrations, showed cytotoxicity against uninfected RAW 264.7 cells (data not shown). 3.3. Aspirin effect on infected RAW cells is not dependent of PGE2 synthesis inhibition Trypanosoma cruzi-infected RAW 264.7 cells presented a significantly higher prostaglandin (PGE2) production compared to uninfected RAW cells. ASA reverted prostaglandin levels to baseline in a dose–response way (Fig. 2A). Note that the maximal effect of ASA
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Table 1 Combinatory index of the aspirin effect together with nifurtimox and benznidazole upon RAW 264.7 cells infected with T. cruzi. The effect of drugs (IC50 values) was determined by trypomastigote release from T. cruzi-infected RAW 264.7 cells. IC50 (lM)
IC50 of the combination (lM)
Combinatory index
Theoreticala
Observed
p-Value
Aspirin Nifurtimox
313.1 ± 36.0 0.083 ± 0.005
250 0.066
134.0 ± 13.4 0.023 ± 0.008
<0.001 0.002
0.71
Aspirin Benznidazole
313.1 ± 36.0 0.34 ± 0.002
250 0.27
73.1 ± 11.4 0.13 ± 0.02
<0.0001 <0.0001
0.61
a Theoretical IC50 corresponds to the value predicted from isobolograms depicted in Fig. 1. Nifurtimox, aspirin and benznidazole were added at their estimated IC12.5 concentrations. p values correspond to the comparisons between theoretical and observed IC50 values through sum-of-squares plus F test. Combinatory index was calculated according to Chou (2006). Results are expressed as means ± SD from three independent experiments.
Fig. 1. Isobolographic analysis of the effect of aspirin, nifurtimox, benznidazole and their combinations on trypomastigote release. RAW 264.37 cells were infected with T. cruzi Dm28c trypomastigotes and incubated with (A) aspirin alone, aspirin plus nifurtimox at their IC12.5 and, nifurtimox plus aspirin at their IC12.5; (B) aspirin alone, aspirin plus benznidazole at their IC12.5 and, benznidazole plus aspirin at their IC12.5. After 3 days of treatment, trypomastigotes released to the supernatant were counted by direct microscopy. Continuous line corresponds to the predicted positions of the experimental points for additive effects. Dots in the discontinuous line correspond to the experimental findings depicted in Table 1.
over the PGE2 synthesis is achieved at 0.25 mM, a dose–response fit of this data showed an IC50 of 7.7 ± 2.54 lM. PGE2 was added to reverse the effect of ASA 0.25 mM. The lack of effect of PGE2 over ASA treatment might indicate that PGE2 is not involved in the ASA effect on infected cells (Fig. 2B).
Fig. 2. Relationship between prostaglandin synthesis inhibition and anti-T. cruzi aspirin effect on infected RAW 264.7 cells. (A) Effect of aspirin upon prostaglandin production in RAW 264.7 cells. Macrophages were infected with T. cruzi trypomastigotes and PGE2 was measured in the supernatant, after 72 h treatment with aspirin (ASA, at 0.065, 0.125, 0.25, 0.5 mM). Results are expressed as means ± SD from three independent experiments, ***p < 0.001 from Tukey post-test. (B) Effect of PGE2 upon trypomastigote release in aspirin treated RAW 264.7 cells infected with T. cruzi. Macrophages were infected with T. cruzi trypomastigotes and, after 72 h treatment with 0.25 mM ASA alone or combined with 1 mM PGE2, trypomastigote release to supernatants were measured. Results are expressed as means ± SD from three independent experiments. ***p < 0.001 from Tukey post-test. ns, no significant differences.
3.4. Aspirin restores NO levels in T. cruzi-infected RAW 264.7 cells The effect of aspirin upon NO production was evaluated by nitrite detection in supernatants from T. cruzi-infected RAW 264.7 cells by the Griess reaction. While T. cruzi infection decreases NO
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Fig. 3. Effect of aspirin upon nitrite production and trypomastigote release in T. cruzi-infected RAW 264.7 cells. (A) Nitrite levels (white bars) and trypomastigote release (black bars) in aspirin treated Raw 264.7 cells infected with T. cruzi trypomastigotes. After 72 h nitrite levels in supernatant were determined by the Griess reaction. §p < 0.001 compared with uninfected control (RAW). àp < 0001 compared with infected control (ASA 0 mM). (B) Effect of aminoguanidine (AMG) upon trypomastigote release in aspirin-treated T. cruzi-infected RAW 264.7 cells. Macrophages were infected with T. cruzi trypomastigotes, and after 72 h treatment with 1 mM aspirin (ASA), 5 mM AMG or ASA plus AMG trypomastigote release to supernatants was measured. Results are expressed as means ± SD from three independent experiments. *p < 0.05; ***p < 0.001 from Tukey post-test.
production in RAW 264.7 cells, aspirin restores it (Fig. 3A). The increase in NO production, induced by aspirin has a dose–response behavior, similar to trypomastigote release inhibition (Fig. 3A). At 1 mM aspirin, the highest effective concentration evaluated, NO production was similar to non-infected cells (p < 0.001) (Fig. 3A). To corroborate that the effect of aspirin on macrophage activity depends upon restoring NO levels, we assessed the trypomastigote release to culture medium when RAW 264.7 cells were incubated with aminoguanidine (AMG), an iNOS inhibitor (Fig. 3C). Thus, 5 mM AMG reverts aspirin effect by 50%.
4. Discussion Different studies have shown the effect of COX inhibitors, such as aspirin, on several T. cruzi infection models (Freire-de-Lima et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005; Pinge-Filho et al., 1999). However, COX inhibitors concentrationdependent responses and mechanisms affecting T. cruzi proliferation inside host cells have not been studied. Herein, we studied the improvement of the macrophage antiparasitic activity induced by aspirin, the synergy of this effect with the activity of the antichagasic drugs nifurtimox and benznidazole and, possible mechanisms involved in those effects.
Attention can be drawn to the low reported IC50 values of nifurtimox (0.0830 lM) and benznidazole (0.336 lM). However, this might be due to the low parasite density used in the experiments. In previous experiments carried out on VERO cells (Faundez et al., 2005), parasite density was an order of magnitude higher and the highest concentration used for nifurtimox and benznidazole was 1 lM. Nevertheless, this situation is not an obstacle to perform synergism studies with other drugs. Herein, the relationship of aspirin with classic antichagasic therapy is further substantiated. Thus, the IC50 value of aspirin in infected macrophages is several folds higher than those determined for nifurtimox and benznidazole (Table 1). However, from the isobolographic analysis, we can conclude that aspirin acts synergistically with these classical drugs. In fact, the IC50 of both nifurtimox and benznidazole, decreased in average by 67% when combined with aspirin (Table 1). However, the observed synergy is not related to a direct effect of both drugs upon the same target or pathway, i.e. parasite viability. Instead, the combined effects of aspirin on the macrophage and the toxic effects of antichagasic drugs on the parasite are complementary mechanisms that explain the isobolographic result. This concept is in agreement with synergistic mechanisms reported for other drug combinations (Jia et al., 2009). Fig. 2A shows that PGE2 production increases significantly in T. cruzi infected macrophages as compared with healthy macrophages. Thus, PGE2 synthesis inhibition using aspirin could improve macrophage response against T. cruzi infection. In fact, ASA reduces PGE2 levels in a dose–response way, with an IC50 = 7.7 lM. This value is 40-fold lower than the ASA IC50 observed on trypomastigote release experiments (IC50 = 313 lM). Therefore, complete PGE2 synthesis inhibition is achieved at concentration levels that have no significant effect on trypomastigote release. Moreover, there is no evidence supporting that PGE2 itself could increase T. cruzi infection. We attempted to determine whether PGE2 is involved in the effect of ASA upon T. cruzi infected cells. We did not find reversion of ASA effect, even at PGE2 concentrations as high as 1 mM (Fig. 2B), indicating that PGE2 does not have a main role on ASA effect. Other COX inhibitors, such as indomethacin and meloxicam were tested, both inhibiting trypomastigote release (IC50 = 97.6 ± 11.1 and 42.7 ± 0.36, respectively). These data suggest a pivotal function of COX in intracellular amastigote proliferation. However, there might be other prostaglandins playing key roles in parasite proliferation. Cardoni and Antunez (2004) showed that T. cruzi infected mice have increased levels of other COX metabolites, such as PGI2 and TXA2, yet their role on parasite infection is still unknown (Cardoni and Antunez, 2004). Trypanosoma cruzi induces TGF-b and PGE2 production by macrophages, especially in the presence of apoptotic bodies (Freire-deLima et al., 2000). Thus, the parasite induces an anti-inflammatory response, attenuating macrophage activation and evading its antiparasitic activity. In macrophages exposed to apoptotic bodies, TGF-b increases PGE2 synthesis and decreases NO (Freire-de-Lima et al., 2006). Inhibition of COX activity may increase NO levels, thus restoring the antiparasitic activity of macrophages. Our results are in agreement with this proposal (Fig. 3A and B). It is important to note that all these experiments were performed without exposure to apoptotic bodies. Consequently, T. cruzi itself can suppress macrophage antiparasitic activity in an apoptosis mimicry fashion, through, perhaps, T. cruzi calreticulin–C1q interactions (Ferreira et al., 2004). Thus, macrophage antiparasitic activity restoration can be pharmacologically modulated. Finally, in T. cruzi infected macrophages, COX is related to the increase of ornithine decarboxylase (ODC) activity (Freire-de-Lima et al., 2000), which might increase the polyamine content in macrophages. Since T. cruzi uses these polyamines to synthesize trypanothione, the inhibition of COX by ASA, indirectly contribute to
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decrease trypanothione synthesis in T. cruzi. This mechanism, together with the restoring of NO levels in macrophages through COX inhibition, could be involved in the activity of ASA on the macrophage response to T. cruzi infection. In conclusion, the observed synergic effect of aspirin with nifurtimox and benznidazole is explained by the capability of aspirin to improve the antiparasitic activity of macrophages. Acknowledgments We thank Dr. Norbel Galanti (Cellular and Molecular Biology Program, ICBM, Faculty of Medicine, University of Chile) for critical review of this paper. FONDECYT 1090078, PBCT ANILLO ACT 29 and REDES-07, Chilean grants supported this research. References Abdalla, G.K., Faria, G.E., Silva, K.T., Castro, E.C., Reis, M.A., Michelin, M.A., 2008. Trypanosoma cruzi: the role of PGE2 in immune response during the acute phase of experimental infection. Experimental Parasitology 118, 514–521. Anderson, P.O., Knoben, J.E., Troutman, W.G., 2002. Handbook of Clinical Drug Data. McGraw-Hill Medical Pub. Division, New York. Borges, M.M., Kloetzel, J.K., Andrade Jr., H.F., Tadokoro, C.E., Pinge-Filho, P., Abrahamsohn, I., 1998. Prostaglandin and nitric oxide regulate TNF-alpha production during Trypanosoma cruzi infection. Immunology Letters 63, 1–8. Cardoni, R.L., Antunez, M.I., 2004. Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections. Mediators of Inflammation 13, 235–240. Chou, T.C., 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 58, 621–681. Faundez, M., Pino, L., Letelier, P., Ortiz, C., Lopez, R., Seguel, C., Ferreira, J., Pavani, M., Morello, A., Maya, J.D., 2005. Buthionine sulfoximine increases the toxicity of nifurtimox and benznidazole to Trypanosoma cruzi. Antimicrobial Agents and Chemotherapy 49, 126–130. Ferreira, V., Valck, C., Sanchez, G., Gingras, A., Tzima, S., Molina, M.C., Sim, R., Schwaeble, W., Ferreira, A., 2004. The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi. Journal of Immunology 172, 3042–3050. Freire-de-Lima, C.G., Nascimento, D.O., Soares, M.B., Bozza, P.T., Castro-Faria-Neto, H.C., de Mello, F.G., DosReis, G.A., Lopes, M.F., 2000. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 403, 199–203. Freire-de-Lima, C.G., Xiao, Y.Q., Gardai, S.J., Bratton, D.L., Schiemann, W.P., Henson, P.M., 2006. Apoptotic cells, through transforming growth factor-beta, coordinately induce anti-inflammatory and suppress pro-inflammatory
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