title: Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway.
abstract: Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF-7 cells in a concentration- and time-dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF-10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl-serine externalization, DNA fragmentation, caspase-7 activation, and PARP cleavage. Apoptosis was associated with caspase-9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl-2 ratio. Pre-treatment with caspase-9 specific inhibitor (Z-LEHD-fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow-cytometric analysis revealed triggering of ROS in a time-dependent manner. Pre-treatment with ROS scavenger N-acetylcysteine (NAC) and glutathione markedly abrogated hesperetin-mediated apoptosis whereas carbonyl cyanide m-chlorophenylhydrazone (CCCP) pretreatment along with DHR123-based flow-cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre-treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin-mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin-mediated apoptosis suggesting that hesperetin-mediated apoptosis of MCF-7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA-MB-231 cells via intrinsic pathway via activation of caspase -9 and -3 and increase in Bax:Bcl-2 ratio.
authors.affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
authors.forename: Shreyasi
authors.initials: S
authors.lastname: Palit
authors.forename: Susanta
authors.initials: S
authors.lastname: Kar
authors.forename: Gunjan
authors.initials: G
authors.lastname: Sharma
authors.forename: Pijush K
authors.initials: PK
authors.lastname: Das
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006569:Hesperidin / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D006569:Hesperidin; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; C013015:hesperetin