cytochrome-c-t and miltefosine

Miltefosine (MIL), an alkylphospholipid, is the first orally administrable anti-leishmanial drug. But due to its long half-life, miltefosine is highly vulnerable for resistance. Hence it is important to understand the mechanism of resistance and to elucidate its action on Leishmania. Here we investigate the miltefosine induced process of programmed cell death in wild type (miltefosine sensitive) and in laboratory generated resistant strains of Leishmania donovani. Results indicate that miltefosine induced apoptosis like death in a time and dose dependent manner in wild-type cells, but not in MIL-resistant cell line. The miltefosine resistant cells remained protected against miltefosine-induced loss of mitochondrial membrane potential, gradual ATP loss and cytochrome C release from mitochondria into the cytosol. Comparative transcriptomic study showed significantly increased expression of FeSODA and SIR2 genes, putatively involved in oxidative stress associated apoptotic cell death. We hypothesize that oxidative stress mediated apoptosis as an alternative mechanism of miltefosine resistance.

Topics: Adenosine Triphosphate; Animals; Antiprotozoal Agents; Apoptosis; Cell Line; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance; Gene Expression Profiling; Leishmania donovani; Leishmaniasis, Visceral; Macrophages; Membrane Potential, Mitochondrial; Mice; Oxidative Stress; Phosphorylcholine; Sirtuins; Superoxide Dismutase

The oxidative phosphorylation process is the main source of endogenous reactive oxygen species (ROS) such as superoxide in mitochondria. In mammals, manganese superoxide dismutase plays an important role in detoxification of superoxide before it interferes with mitochondrial function and causes programmed cell death. Here, we investigated the role of Leishmania donovani mitochondrial iron superoxide dismutase-A (LdFeSODA) in protecting the parasite from oxidative stress and in the control of programmed cell death events. We have shown that overexpression of LdFeSODA protects Leishmania donovani from miltefosine induced cytotoxicity and reduced mitochondrial-derived superoxide generation. Furthermore, parasites overexpressing LdFeSODA showed (i) lower level of phosphatidylserine exposure as measured by flow cytometry and fluorescent microscopy; and (ii) reduced level of TUNEL staining of parasites compared to the control parasites. Finally, prolonged incubation of the parasites with miltefosine induced the release of both cytochrome C and LdFeSODA into the cytosol as demonstrated by Western blotting and fluorescence microscopy indicating programmed cell death. The results indicate that LdFeSODA protects the mitochondria of Leishmania from oxidative stress thereby inhibiting programmed cell death.

Topics: Antiprotozoal Agents; Apoptosis; Cytochromes c; Cytosol; DNA Fragmentation; Leishmania donovani; Mitochondrial Proteins; Phosphorylcholine; Protozoan Proteins; Reactive Oxygen Species; Sulfhydryl Compounds; Superoxide Dismutase

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.

Topics: Animals; Antiprotozoal Agents; Apoptosis; Arsenites; Cytochromes c; DNA Damage; Dose-Response Relationship, Drug; Drug Resistance; In Situ Nick-End Labeling; Leishmania donovani; Membrane Potentials; Microscopy, Fluorescence; Microscopy, Interference; Mitochondria; Phosphorylcholine