Toxicology Letters 215 (2012) 131–142 Contents lists available at SciVerse ScienceDirect Toxicology Letters jou rn al h om epage: www.elsevier.com/locate/toxlet Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway P. Elumalai, D.N. Gunadharini, K. Senthilkumar, S. Banudevi, R. Arunkumar, C.S. Benson, G. Sharmila, J. Arunakaran ∗ Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India h i g h l i g h t s ◮ Nimbolide inhibits the breast cancer (MCF-7 and MDA MB-231) cell pro- liferation. ◮ It increased the levels of pro- apoptotic proteins. ◮ Anti-apoptotic proteins were decreased in nimbolide treated cells. ◮ It activates caspase-8, caspase-9, caspase-3 and cleavage of PARP. ◮ Nimbolide induced apoptosis medi- ated by both extrinsic and intrinsic pathway. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 16 July 2012 Received in revised form 8 October 2012 Accepted 11 October 2012 Available online xxx Keywords: Nimbolide Breast cancer cell lines Apoptosis Intrinsic and extrinsic a b s t r a c t We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy. © 2012 Elsevier Ireland Ltd. All rights reserved. ∗ Corresponding author. Tel.: +91 44 2454 7043; fax: +91 44 2454 0709. E-mail address: j arunakaran@hotmail.com (J. Arunakaran). 1. Introduction Breast cancer is the most common malignancy in women. The incidence of breast cancer in India is on the rise and is rapidly becoming the number one cancer in females pushing the cervical 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2012.10.008