Chemico-Biological Interactions 167 (2007) 19–30 Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation V. Senthil a , S. Ramadevi a , V. Venkatakrishnan a , P. Giridharan b , B.S. Lakshmi a,∗ , R.A. Vishwakarma b , A. Balakrishnan b a Centre For Biotechnology, Anna University, Chennai 600025, Tamil Nadu, India b Nicholas Piramal Research Centre, Nirlon Complex, Goregoan (E), Mumbai 400063, India Received 4 November 2006; received in revised form 25 December 2006; accepted 2 January 2007 Available online 12 January 2007 Abstract The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12 h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24 h, suggesting the induction of apoptotic cell death in HL-60 cells. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: HL-60 cells; Withania somnifera; Apoptosis; Bcl-2; Mitochondria; Cytochrome c; Caspase 3; DNA fragmentation; Flow cytometry 1. Introduction Medicinal plants have an advantage in drug dis- covery based on their use by humans for more than thousands of years. The bioactive compounds obtained from these plants are normally non-toxic or less toxic to humans [1]. Withania somnifera is a well-known Indian medicinal plant commonly known as Ashwagandha belonging to the family Solanaceae. It is widely used in many ayurvedic preparations all over the world. Several ∗ Corresponding author. Tel.: +91 44 22350772; fax: +91 44 22350299. E-mail address: lakshmibs@annauniv.edu (B.S. Lakshmi). reports have demonstrated the anti-tumor activity [2,3], anti-arthritic [4], anti-pyretic and anti-inflammatory [5] activity of this plant. The root extract has shown to have immunomodulatory activity [6], antistress effect [7] and induce iNOS expression [8]. The methanolic root extract of W. somnifera is a rich source of steroidal lactones called withanolides [9]. Similarly the methanolic leaf extracts also include a variety of withanolides [10] and very few studies have investigated the molecular mech- anisms of the effect of leaf extracts. These withanolides from leaves are reported to have an antiproliferative activity [11,12] and possess an inhibitory effect on the cyclooxygenase-2 (COX-2) enzyme [13]. Apoptosis or programmed cell death is a geneti- cally regulated process that plays an essential role in 0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2007.01.004