title: Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway.
abstract: 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.
authors.affiliation: Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India.
authors.forename: P
authors.initials: P
authors.lastname: Elumalai
authors.forename: D N
authors.initials: DN
authors.lastname: Gunadharini
authors.forename: K
authors.initials: K
authors.lastname: Senthilkumar
authors.forename: S
authors.initials: S
authors.lastname: Banudevi
authors.forename: R
authors.initials: R
authors.lastname: Arunkumar
authors.forename: C S
authors.initials: CS
authors.lastname: Benson
authors.forename: G
authors.initials: G
authors.lastname: Sharmila
authors.forename: J
authors.initials: J
authors.lastname: Arunakaran
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D053222:Fas Ligand Protein / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D036701:Limonins / Q000494:pharmacology*; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009376:Neoplasms, Hormone-Dependent / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C494819:BAD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D036701:Limonins; D012333:RNA, Messenger; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; C042198:nimbolide; D045304:Cytochromes c; D020169:Caspases