Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction

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Abstract

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.

Introduction

Leishmaniasis, one of the most dreaded parasitic diseases, continues to rely on chemotherapy in the absence of effective vaccines and efficient vector control measures. Conversely, the lack of effective and non-toxic drugs; variation in efficacy as result of intrinsic variation in drug sensitivity; and the emergence of drug resistance limits the arsenal of anti-leishmanial drugs (Croft and Coombs, 2003). Miltefosine, the first oral treatment of human visceral leishmaniasis, has shown remarkable activity and offers great promise for the treatment of parasitic infections including those caused by Leishmania sp. non-responsive to antimony (Sundar et al., 1998, Sundar et al., 1999). However, limited reports are available on the sensitivity of miltefosine towards drug-resistant Leishmania (Yardley et al., 2005).

Differential sensitivity of a drug results from the differences in net drug accumulation regulated by influx and/or efflux pathways. P-glycoproteins (Pgps) and multiple drug resistance (MDR) have been implicated in miltefosine resistance among different mammalian cell lines (Rybczynska et al., 2001a, Rybczynska et al., 2001b). Conversely, Pgp overexpressing cell lines displaying MDR have also been reported to be sensitive to miltefosine or other lysophospholipid analogues (Glasser et al., 1996, Verdonck and Van Hengten, 1997). In Leishmania, daunomycin-resistant L. tropica cells overexpressing a Pgp-like transporter have been found to be cross-resistant to miltefosine (Perez-Victoria et al., 2001b). However, an in vitro generated miltefosine-resistant strain of L. donovani was observed to have defective inward translocation of drug involving a P-type lipid translocase without any participation of Pgp-like proteins (Perez-Victoria et al., 2003a, Perez-Victoria et al., 2003b, Seifert et al., 2003). Thus, there is ambiguity in the role played by Pgp-like proteins in miltefosine resistance and the sensitivity of miltefosine among MDR/Pgp overexpressing Leishmania cell lines. An arsenite-resistant L. donovani (Ld-As20) strain generated in our laboratory has been reported to overexpress Pgp-like protein and display MDR-like phenotypes (Kaur and Dey, 2000). In an attempt to address these issues, we have used this strain.

Apoptosis involves series of morphological and biochemical changes in which mitochondria serves as a major regulator of apoptosis (Hengartner, 2000). Change in lipid content on oxidative damage leads to change in the permeabilization of mitochondrial membrane and release of proapoptotic proteins into the cytosol including cytochrome C (Hengartner, 2000). Relocation of cytochrome C serves as the irreversible commitment to death by activating cellular proteases. Different signaling pathways converge at this stage, leading to cleavage of multiple downstream substrates. Miltefosine has been found to induce apoptosis-like death in wild type L. donovani (Paris et al., 2004, Verma and Dey, 2004), although its mode of action on drug-resistant strains of Leishmania and pathways and the proteins implicated in apoptosis-like death process is not known. In the present study, we investigated the sensitivity of the arsenite-resistant L. donovani (Ld-As20) to miltefosine, and its cytotoxic effects, followed by death process.

Section snippets

Chemicals

Miltefosine was a kind gift from Zentaris (Frankfurt, Germany). RPMI1640 culture media was from Gibco BRL (Grand Island, NY, USA). Foetal calf serum was obtained from Biological Industries (Kibbutz Beit, Haemek, Israel). ApoAlert DNA fragmentation assay kit, ApoAlert Annexin-Vfluorescein isothiocynate (FITC) apoptosis kit, ApoAlert caspase-3 colorimetric assay kit and ApoAlert cell fractionation kit were purchased from BD Biosciences Clontech (Palo Alto, CA, USA). Mouse monoclonal anti-PARP

In vitro anti-leishmanial activity of miltefosine on Ld-As20

Cytotoxic potential of miltefosine on Ld-As20 was determined using MTT assay. Dose-dependent killing resulting almost 100% death of Ld-As20 promastigotes at around 30 μM miltefosine concentrations was observed (Fig. 1A). The cytotoxic potential of miltefosine was assessed for both Ld-Wt and Ld-As20 strains to rule out any variation in IC50 due to different drug aliquots, medium and batches of serum. There is significant difference between the IC50 of two strains. The IC50 of miltefosine for

Discussion

Miltefosine, the first oral drug for leishmaniasis, has been highly effective against visceral leishmaniasis and gave high cure rates in areas where antimony resistance is prevalent (Sundar et al., 1998, Sundar et al., 1999). However, limited data is available on the sensitivity of miltefosine towards drug-resistant Leishmania (Yardley et al., 2005). There are two major findings of this study. Miltefosine has a profound cytotoxic effect on arsenite-resistant L. donovani promastigotes and that

Acknowledgments

We thank C.L. Kaul, Ex-Director, NIPER and P. Ramarao, Director, NIPER for their keen interest in this study. We acknowledge K. G. Jayanarayan for his support in executing some experiments and helpful discussions. Mr. R. Singh is acknowledged for his assistance in the laboratory. N.K.V. was a Research Scientist of NIPER. G. S. is the recipient of Senior Research Fellowship from CSIR, India.

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