Antimonial-induced Increase in Intracellular Ca2+ through Non-selective Cation Channels in the Host and the Parasite Is Responsible for Apoptosis of Intracellular Leishmania donovani Amastigotes

Abstract

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca 2؉ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca 2؉ in both the cell types and reduced amastigote death, thus establishing a central role of Ca 2؉ in intracellular parasite clearance. This influx of Ca 2؉ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites.

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

“…6C). These alterations in redox potential are consistent with recent reports (34,35) that cells enter an apoptotic-like state in response to Sb III . The generation of excessive reactive oxygen species is seen as an important factor in apoptosis.…”

“…1C). Third, similar decreases in specific activities of intracellular 35 S-labeled GSH and T[SH] 2 are observed with or without Sb III throughout the incubations. Thus turnover through degradation and re-synthesis of these pools are unaffected by Sb III .…”

“…Sb III also inhibits trypanothione reductase in vitro, but the significance of this observation until now has remained obscure (9). More recent studies (34,35) have reported that Sb III -treated amastigotes undergo death by an apoptotic-like mechanism involving DNA fragmentation and externalization of phosphatidylserine by a caspase-independent mechanism. Three key observations arising from this study are relevant to the mode of action of antimonials against drug-sensitive L. donovani (Fig.…”

Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani

“…The pellet was discarded and the suspension was loaded on percoll gradient to isolate amastigotes from macrophages. 51 The purity was verified under phase contrast microscope.…”

Drug Resistance in Leishmaniasis

“…However, the specific targets in these pathways have not been identified. More recent studies have reported apoptosis in Sb(III)-treated amastigotes involving DNA fragmentation and externalization of phosphatidylserine on the outer surface of the plasma membrane (130,139). However, these effects do not involve the classical caspasemediated pathway (130) and do not meet the more recent stringent definition of apoptosis (85).…”