title: Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene.
abstract: This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL.
authors: Electron Microscopy Unit, Sophisticated Analytical Equipment Facility, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Manoj
authors: M
authors: Kathuria
authors: Electron Microscopy Unit, Sophisticated Analytical Equipment Facility, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Arindam
authors: A
authors: Bhattacharjee
authors: Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India Academy of Scientific and Innovative Research, New Delhi, India.
authors: Koneni V
authors: KV
authors: Sashidhara
authors: Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, India.
authors: Suriya Pratap
authors: SP
authors: Singh
authors: Electron Microscopy Unit, Sophisticated Analytical Equipment Facility, CSIR-Central Drug Research Institute, Lucknow, India Academy of Scientific and Innovative Research, New Delhi, India k_mitra@cdri.res.in.
authors: Kalyan
authors: K
authors: Mitra
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D045785:Diterpenes, Clerodane / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D007893:Leishmania donovani / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D045785:Diterpenes, Clerodane; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D053148:Caspase 3; D053179:Caspase 7; D005978:Glutathione