Induction of apoptosis-like cell death by pentamidine and doxorubicin through differential inhibition of topoisomerase II in arsenite-resistant L. donovani
Introduction
L. donovani infection is responsible for visceral leishmaniasis, the cause of considerable morbidity and mortality worldwide. The inadequate vector control, absence of vaccines and most importantly the generation of drug resistant parasites constantly challenges chemotherapy, the mainstay to fight the disease (Oullette et al., 2004). High cost, adverse reactions and acquired resistance to the operational drugs demands new anti-leishmanial drugs and identifying newer therapeutic targets. DNA topoisomerase (topo) II, due to its ability to control DNA topology; to remove torsional stress during vital cellular processes like replication, transcription and recombination; and its requirement to segregate intertwined daughter chromosomes, was successfully targeted by some of the most widely prescribed antibiotics and anticancer drugs (Burden and Osheroff, 1998, Wang, 2002). Apart from normal DNA metabolic activities, the presence of complex kinetoplast DNA (kDNA) network in kinetoplastids presents a unique topological problem that requires topo II activity and led it to be cloned and sequenced in these parasites including Leishmania (Das et al., 2006).
The topo II-DNA binary complex, in the enzyme reaction cycle, is a target of drugs that stabilize the protein–DNA association and causes double stranded DNA breaks which eventually trigger the process of programmed cell death or apoptosis in the targeted cells (Burden and Osheroff, 1998, Wang, 2002). Apoptosis, the genetically regulated physiological cell suicide process, has attracted considerable attention in unicellular parasitic organisms like Leishmania, not only in terms of its intriguing features but due to the purpose served (Duszenko et al., 2006). A variety of topoisomerase targeting drugs are reported to induce apoptosis in Leishmania (Sen et al., 2004a, Sen et al., 2004b, Singh et al., 2005, Duszenko et al., 2006). Despite of various common features of metazoan apoptosis observed in protozoans, phenomenon of drug induced apoptosis in the protozoan parasites is a matter of great interest for its evolutionary significance and therapeutic importance. Study of biochemical mechanism by which parasite death occurs, due to the drug treatment, ought to be important since it is one of the strategies employed against invading pathogens.
Model drug resistant phenotypes, such as arsenite-resistant L. donovani (Ld-As20) generated in our laboratory and used in the current study, have been widely used to understand and address drug resistance in parasites such as Leishmania (Singh et al., 2007). Our previous investigations had revealed an overexpressing and increased topo II activity in Ld-As20 (Singh et al., 2005). Ld-As20 displayed MDR-like phenotypes and showed cross-resistance to structurally unrelated drugs including pentamidine and doxorubicin (Kaur and Dey, 2000, Prasad et al., 2000). Pentamidine, an aromatic diamidine and drug of choice for visceral leishmaniasis has been shown to inhibit topo II in different parasitic organisms including Pneumocystis carinii, Giardia lambia and Trypanosoma spp. (Shapiro and Englund, 1990, Dykstra and Tidwell, 1991, Bell et al., 1993). Doxorubicin, an extensively used anti-cancer agent that exerts its cytotoxic effects primarily through topo II inhibition has been recently shown for its profound activity against Leishmania (Sett et al., 1992, Binaschi et al., 2001, Nitiss, 2002, Mukherjee et al., 2004).
In the work presented here we investigated the effect of pentamidine and doxorubicin on topo II activity from Ld-Wt and Ld-As20. The current study demonstrates the possibility of observed cross-resistance of Ld-As20 to pentamidine and doxorubicin to be, in part, implicated through differential inhibition of topo II. The treatment of promastigotes at concentrations of drugs inhibiting 50% of topo II activity results in induction of apoptosis-like cell death process.
Section snippets
Reagents
RPMI-1640 culture media was from Gibco BRL (Grand Island, NY, USA). Rabbit polyclonal cytochrome C and anti-COX-4 antibodies were purchased from BD PharMingen (San Diego, CA, USA). ApoAlert™ annexin V labeling kit, ApoAlert™ cell fractionation kit, ApoAlert™ caspase-3 colorimetric assay kit and ApoAlert™ DNA fragmentation assay kit were purchased from BD Biosciences Clontech (Palo Alto, CA, USA). Rabbit polyclonal anti-γ-tubulin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa
Inhibition of topoisomerase II activity by pentamidine and doxorubicin
Effect of pentamidine and doxorubicin on ATP-dependent topo II decatenation activity was investigated. Decatenation assay was performed in presence of increasing concentration of pentamidine (0.25–10 μM) and doxorubicin (0.75–6.0 μM) (Fig. 1A and B, respectively). As has been reported previously (Singh et al., 2005) the topo II activity was observed to be higher in Ld-As20 as compared to Ld-Wt (inset, Fig. 1A and B). Though a dose-dependent increase in inhibition of decatenation activity was
Discussion
The present study, conducted with sodium arsenite-resistant L. donovani strain (Ld-As20) exhibiting a MDR-phenotype, provides evidences that cross resistance and cell cytotoxicity inflicted by experimental anti-leishmanial agents such as, pentamidine and anti cancer agents such as, doxorubicin seems to be exerted by differential inhibition of topo II activity. As a consequence of topo II inhibition induction of cell death, displaying typical nuclear and cytoplasmic features of apoptosis were
Acknowledgements
We thank Dr. P. RamaRao, Director, NIPER, for his support in this work. We acknowledge the continuous technical support of Mr. Ranvir Singh during the process of executing experiments. G.S. is supported by Senior Research Fellowship from Council for Scientific and Industrial Research, New Delhi, Govt. of India. This work was supported by a grant (37(1103)\02\EMR-II) from Council for Scientific and Industrial Research, New Delhi, Govt. of India.
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