Abstract
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 ΔΨm and generates ROS inside cells. Loss of ΔΨm 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.
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Abbreviations
- ROS:
-
reactive oxygen species
- PI:
-
propidium iodide
- ΔΨm:
-
mitochondrial transmembrane potential
- NAC:
-
N-acetyl-cysteine
- DMSO:
-
dimethyl sulfoxide
- CPT:
-
camptothecin
- DEVD-AFC:
-
Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin
- TUNEL:
-
terminal deoxynucleotidyltransferase enzyme-mediated dUTP end labeling
- VAD-fmk:
-
Val-Ala-Asp-fluoromethyl ketone
- FITC:
-
fluorescein isothiocyanate
- DTT:
-
dithiothreitol
- H2DCFDA:
-
5-(and -6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate acetyl ester
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Acknowledgements
We thank Professor S Roy, the director of our institute, for his interest in this work. NS is supported by Senior Research Fellowship from the Council for Scientific and Industrial Research, Government of India. This work was supported by grants from Network Project SMM-003 of Council of Scientific and Industrial Research (CSIR), Government of India to HKM.
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Sen, N., Banerjee, B., Das, B. et al. Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I–DNA complex. Cell Death Differ 14, 358–367 (2007). https://doi.org/10.1038/sj.cdd.4402002
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DOI: https://doi.org/10.1038/sj.cdd.4402002
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