1. ¹Division of Infectious Diseases, Indian Institute of Chemical Biology, 4, Raja SC Mullick Road, Kolkata 700 032, India
2. ²Department of Biochemistry, Dr BC Roy Postgraduate Institute of Basic Medical Sciences, Calcutta University, 244, B, AJC Bose Road, Kolkata 700 020, India
3. ³Department of Chemistry, Indian Institute of Chemical Biology, 4, Raja SC Mullick Road, Kolkata 700 032, India

Correspondence: HK Majumder, Division of Infectious Diseases, Molecular Parasitology Laboratory, Indian Institute of Chemical Biology. 4, Raja SC Mullick Road, Kolkata 700 032, India. Tel: +91 33 2412 3207; Fax: +91 33 2473 5197; E-mail: hkmajumder@iicb.res.in

Received 16 January 2006; Revised 15 May 2006; Accepted 25 May 2006; Published online 14 July 2006.

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 _m and generates ROS inside cells. Loss of _m 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.