Cell death in amastigote forms of Leishmania amazonensis induced by parthenolide

BMC Microbiology

Celso Vataru Nakamura¹  Antonio Alonso²  Tânia Ueda-Nakamura¹  Tatiana Shioji Tiuman¹ 

[1] Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, Paraná, Brazil;Instituto de Física, Universidade Federal de Goiás, Campus II, CEP 74001-970 Goiânia, Goiás, Brazil

关键词: Autophagy;    Cell death;    Parthenolide;    Amastigotes;    Leishmania amazonensis;   

Others  :  1140940
DOI  :  10.1186/1471-2180-14-152

 received in 2014-01-24, accepted in 2014-05-21,  发布年份 2014

【 摘 要 】

Background

Leishmania amazonensis infection results in diverse clinical manifestations: cutaneous, mucocutaneous or visceral leishmaniasis. The arsenal of drugs available for treating Leishmania infections is limited. Therefore, new, effective, and less toxic leishmaniasis treatments are still needed. We verified cell death in amastigote forms of Leishmania amazonensis induced by the sesquiterpene lactone parthenolide.

Results

The tested compound was able to concentration-dependently affect axenic and intracellular amastigotes, with IC₅₀ values of 1.3 μM and 2.9 μM, respectively after 72 h incubation. No genotoxic effects were observed in a micronucleus test in mice. Parthenolide induced morphological and ultrastructural changes in axenic amastigotes, including a loss of membrane integrity, swelling of the mitochondrion, cytoplasmic vacuoles, and intense exocytic activity in the region of the flagellar pocket. These results led us to investigate the occurrence of autophagic vacuoles with monodansylcadaverine and the integrity of the plasma membrane and mitochondrial membrane potential using flow cytometry. In all of the tests, parthenolide had positive results.

Conclusions

Our results indicate that the antileishmanial action of parthenolide is associated with autophagic vacuole appearance, a reduction of fluidity, a loss of membrane integrity, and mitochondrial dysfunction. Considering the limited repertoire of existing antileishmanial compounds, the products derived from medicinal plants has been one the greatest advances to help develop new chemotherapeutic approaches.

【 授权许可】

   
2014 Tiuman et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]WHO: Leishmaniasis: magnitude of the problem. World Health Org 2013. [http://www.who.int/leishmaniasis/burden/magnitude/burden_magnitude/en/ webcite]
• [2]Fernández-Guerrero ML, Robles P, Rivas P, Mójer F, Muñíz G, Górgolas M: Visceral leishmaniasis in immunocompromised patients with and without AIDS: a comparison of clinical features and prognosis. Acta Trop 2004, 90:11-16.
• [3]Carnaúba-Jr D, Konishi CT, Petri V, Martinez ICP, Shimizu L, Pereira-Chioccola VL: Atypical disseminated leishmaniasis similar to post-kala-azar dermal leishmaniasis in a Brazilian AIDS patient infected with Leishmania (Leishmania) infantum chagasi: a case report. Int J Infect Dis 2009, 13:504-507.
• [4]Barral A, Pedral-Sampaio D, Grimaldi Júnior G, Momen H, McMahon-Pratt D, Ribeiro De Jesus A, Almeida R, Badaro R, Barral-Netto M, Carvalho EM, Johnson Júnior WD: Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am J Trop Med Hyg 1991, 44:536-546.
• [5]Oliveira JPC, Fernandes F, Cruz AK, Trombela V, Monteiro E, Camargo AA, Barral A, Oliveira CI: Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping. Kinetoplastid Biol Dis 2007. doi:10.1186/1475-9292-6-5
• [6]Croft SL, Coombs GH: Leishmaniasis – current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 2003, 19:502-508.
• [7]Tiuman TS, Santos AO, Ueda-Nakamura T, Dias Filho BP, Nakamura CV: Recent advances in leishmaniasis treatment. Int J Infect Dis 2011, 15:e525-e532.
• [8]Croft SL, Sundar S, Fairlamb AH: Drug resistance in leishmaniasis. Clin Microbiol Rev 2006, 19:111-126.
• [9]Natera S, Machuca C, Padrón-Nieves M, Romero A, Díaz E, Ponte-Sucre A: Leishmania spp.: proficiency of drug-resistant parasites. Int J Antimicrob Agents 2007, 29:637-642.
• [10]Tiuman TS, Ueda-Nakamura T, Cortez DAG, Dias Filho BP, Morgado-Díaz JA, De Souza W, Nakamura CV: Antileishmanial activity of parthenolide, a sesquiterpene lactone isolated from Tanacetum parthenium. Antimicrob Agents Chemother 2005, 49:176-182.
• [11]Tiuman TS, Ueda-Nakamura T, Dias-Filho BP, Cortez DAG, Morgado-Díaz JA, Nakamura CV: Morphologic and ultrastructural alterations in Leishmania amazonensis induced by 4a,5β-epoxy-germacra-1(10),11(13)-dien-12,6a-olide. Acta Protozool 2007, 46:349-355.
• [12]Linsinger G, Wilhelm S, Wagner H, Häcker G: Uncouplers of oxidative phosphorylation can enhance a Fas death signal. Mol Cell Biol 1999, 19:3299-3311.
• [13]Jost PC, Griffith OH, Capaldi RA, Vanderkooi G: Evidence for boundary lipid in membranes. Proc Natl Acad Sci U S A 1973, 70:480-484.
• [14]Rotureau B: Are new world leishmaniases becoming anthroponoses? Med Hypotheses 2006, 67:1235-1241.
• [15]WHO: Urbanization: an increasing risk factor for leishmaniasis. WklyEpidemiol Rec 2002, 77:365-370.
• [16]Polonio T, Efferth T: Leishmaniasis: drug resistance and natural products (review). Int J Mol Med 2008, 22:277-286.
• [17]Sereno D, Lemesre JL: Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrob Agents Chemother 1997, 41:972-976.
• [18]Sen R, Chatterjee M: Plant derived therapeutics for the treatment of leishmaniasis. Phytomedicine 2011, 18:1056-1059.
• [19]Kayser O, Kiderlen AF, Croft SL: Natural products as antiparasitic drugs. Parasitol Res 2003, 90:S55-S62.
• [20]Sikkema J, De Bont JAM, Poolman B: Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 1995, 59:201-222.
• [21]Fumarola L, Spinelli R, Brandonisio O: In vitro assays for evaluation of drug activity against Leishmania spp. Res Microbiol 2004, 155:224-230.
• [22]Sereno D, Cordeiro Da Silva A, Mathieu-Daude F, Ouaissi A: Advances and perspectives in leishmania cell based drug-screening procedures. Parasitol Int 2007, 56:3-7.
• [23]Weniger B, Robledo S, Arango GJ, Deharo E, Aragón R, Muñoz V, Callapa J, Lobstein A, Anton R: Antiprotozoal activities of Colombian plants. J Ethnopharmacol 2001, 78:193-200.
• [24]Weniger B, Vonthron-Sénécheau C, Kaiser M, Brun R, Anton R: Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids. Phytomedicine 2006, 13:176-180.
• [25]Winter MJ, Ellis LCJ, Hutchinson TH: Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide. Mutat Res 2007, 629:89-99.
• [26]Costa MA, Ishida K, Kaplum V, Koslyk ED, de Mello JC, Ueda-Nakamura T, Dias Filho BP, Nakamura CV: Safety evaluation of proanthocyanidin polymer-rich fraction obtained from stem bark of Stryphnodendron adstringens (BARBATIMAO) for use as a pharmacological agent. Regul Toxicol Pharmacol 2010, 58:330-335.
• [27]Hayashi M, MacGregor JT, Gatehouse DG, Adler I, Blakey DH, Dertinger SD, Krishna G, Morita T, Russo A, Sutou S: In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. Environ Mol Mutagen 2000, 35:234-252.
• [28]Edinger AL, Thompson CB: Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 2004, 16:663-669.
• [29]Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X, Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT, Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu CT, Chung J, Clarke PG, et al.: Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008, 4:151-175.
• [30]Biederbick A, Kern HF, Elsässer HP: Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 1995, 66:3-14.
• [31]Mizushima N: Methods for monitoring autophagy. Int J Biochem Cell Biol 2004, 36:2491-2502.
• [32]Munafó DB, Colombo MI: A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 2001, 114:3619-3629.
• [33]Bera A, Singh S, Nagaraj R, Vaidya T: Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides. Mol Biochem Parasitol 2003, 127:23-35.
• [34]Cohen BE: Amphotericin B membrane action: role for two types of ion channels in eliciting cell survival and lethal effects. J Membr Biol 2010, 238:1-20.
• [35]Di Giorgio C, Faraut-Gambarelli F, Imbert A, Minodier P, Gasquet M, Dumon H: Flow cytometric assessment of amphotericin B susceptibility in Leishmania infantum isolates from patients with visceral leishmaniasis. J Antimicrob Chemother 1999, 44:71-76.
• [36]Dengler WA, Schulte J, Berger DP, Mertelsmann R, Fiebig HH: Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays. Anticancer Drugs 1995, 6:522-532.
• [37]Riccardi C, Nicoletti I: Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 2006, 1:1458-1461.
• [38]Scaduto RC Jr, Grotyohann LW: Measurement of mitochondrial membrane potential using fluorescent rhidanmine derivatives. Biophys J 1999, 76:469-477.
• [39]Menna-Barreto RFS, Goncalves RLS, Costa EM, Silva RSF, Pinto AV, Oliveira MF, Castro SL: The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction. Free Radic Biol Med 2009, 47:644-653.
• [40]Gottlieb E, Armour SM, Harris MH, Thompson CB: Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ 2003, 10:709-717.
• [41]Santa-Rita RM, Henriques-Pons A, Barbosa HS, Castro SL: Effect of the lysophospholipid analogues edelfosine, ilmofosine and miltefosine against Leishmania amazonensis. J Antimicrob Chemother 2004, 54:704-710.
• [42]Pozarowski P, Halicka DH, Darzykiewicz Z: NF-κB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures. Cytometry 2003, 54A:118-124.
• [43]Alonso A, Meirelles NC, Tabak M: Effect of hydration upon the fluidity of intercellular membranes of stratum corneum: an EPR study. Biochim Biophys Acta 1995, 1237:6-15.
• [44]Alonso A, Queiroz CS, Magalhães AC: Chilling stress leads to increased cell membrane rigidity in roots of coffee (Coffea arabica L.) seedlings. Biochim Biophys Acta 1997, 1323:75-84.
• [45]Nepomuceno MF, Alonso A, Pereira-da-Silva L, Tabak M: Inhibitory effect of dipyridamole and its derivatives on lipid peroxidation in mitochondria. Free Radic Biol Med 1997, 23:1046-1054.
• [46]Zilberstein D: The role of pH and temperature in the development of Leishmania parasites. Annu Rev Microbiol 1994, 48:449-470.
• [47]Ueda-Nakamura T, Attias M, Souza W: Megasome biogenesis in Leishmania amazonensis: a morphometric and cytochemical study. Parasitol Res 2001, 87:89-97.
• [48]Budil DE, Lee S, Saxena S, Freed JH: Nonlinear-least-squares analysis of slow-motional EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm. J Magn Reson 1996, A120:155-189.
• [49]Dos Anjos JLV, Neto DD, Alonso A: Effects of ethanol/L-menthol on the dynamics and partitioning of spin-labeled lipids in the stratum corneum. Eur J Pharm Biopharm 2007, 67:406-412.
• [50]Dos Anjos JLV, Alonso A: Terpenes increase the partitioning and molecular dynamics of an amphipathic spin label in stratum corneum membranes. Int J Pharm 2008, 350:103-112.