Experimental Parasitology 116 (2007) 1–13 www.elsevier.com/locate/yexpr 0014-4894/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.10.007 Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction Navin K. Verma, Gaganmeet Singh, Chinmoy S. Dey ¤ Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER) 1 , Punjab 160 062, India Received 13 March 2006; received in revised form 5 October 2006; accepted 13 October 2006 Available online 11 December 2006 Abstract The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leish- manial chemotherapy is emergence of drug resistance. Miltefosine, the Wrst orally administrable anti-leishmanial drug, has shown the poten- tial 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 eVect of mil- tefosine 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 pro- grammed 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 conse- quent 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.  2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Leishmania; Arsenite resistance; Miltefosine; Apoptosis; Dyskinetoplastidy 1. Introduction Leishmaniasis, one of the most dreaded parasitic dis- eases, continues to rely on chemotherapy in the absence of eVective vaccines and eYcient vector control measures. Conversely, the lack of eVective and non-toxic drugs; varia- tion in eYcacy as result of intrinsic variation in drug sensi- tivity; and the emergence of drug resistance limits the arsenal of anti-leishmanial drugs (Croft and Coombs, 2003). Miltefosine, the Wrst oral treatment of human vis- ceral leishmaniasis, has shown remarkable activity and oVers great promise for the treatment of parasitic infections including those caused by Leishmania sp. non-responsive to antimony (Sundar et al., 1998, 1999). However, limited reports are available on the sensitivity of miltefosine towards drug-resistant Leishmania (Yardley et al., 2005). DiVerential sensitivity of a drug results from the diVer- ences in net drug accumulation regulated by inXux and/or eZux pathways. P-glycoproteins (Pgps) and multiple drug resistance (MDR) have been implicated in miltefosine resis- tance among diVerent mammalian cell lines (Rybczynska et al., 2001a,b). 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 Leish- mania, 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 par- ticipation of Pgp-like proteins (Perez-Victoria et al., 2003a,b; Seifert et al., 2003). Thus, there is ambiguity in the * Corresponding author. E-mail address: csdey@niper.ac.in (C.S. Dey). 1 NIPER communication number: 387.