Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex.
Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of DeltaPsim and generates ROS inside cells. Loss of DeltaPsim leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I-DNA complex is necessary to amplify apoptotic process.
Sen
N
N
Division of Infectious Diseases, Indian Institute of Chemical Biology, 4, Raja SC Mullick Road, Kolkata 700 032, India.
Banerjee
B
B
Das
B B
BB
Ganguly
A
A
Sen
T
T
Pramanik
S
S
Mukhopadhyay
S
S
Majumder
H K
HK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects; D053837:DNA Cleavage / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D016054:DNA, Protozoan / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D007893:Leishmania donovani / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000737:chemistry / Q000494:pharmacology; D054358:Withanolides
D016054:DNA, Protozoan; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D054358:Withanolides; D045304:Cytochromes c; D011493:Protein Kinase C; D020169:Caspases; D004264:DNA Topoisomerases, Type I; D005978:Glutathione; D019311:Staurosporine; C009684:withaferin A; D004875:Ergosterol