Abstract:
This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes previously demonstrated to be safe and orally active against Visceral Leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and generation of reactive oxygen species triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in release of cytochrome c into the cytosol impairing ATP production. Oxidative stress caused depletion of reduced glutathione while pre-treatment with anti-oxidant N-acetyl-cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment suggesting that 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 microscopic studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations and displacement of organelles. Moreover, 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 towards development of K-09 as a potential drug candidate for VL.
