title: Calpain and reactive oxygen species targets Bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis.
abstract: Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.
authors.affiliation: Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Sobhan
authors.forename: Mahendra
authors.initials: M
authors.lastname: Seervi
authors.forename: Lokesh
authors.initials: L
authors.lastname: Deb
authors.forename: Saneesh
authors.initials: S
authors.lastname: Varghese
authors.forename: Anjana
authors.initials: A
authors.lastname: Soman
authors.forename: Jeena
authors.initials: J
authors.lastname: Joseph
authors.forename: Krupa Ann
authors.initials: KA
authors.lastname: Mathew
authors.forename: Godi
authors.initials: G
authors.lastname: Raghu
authors.forename: George
authors.initials: G
authors.lastname: Thomas
authors.forename: Sreekumar
authors.initials: S
authors.lastname: E
authors.forename: Manjula
authors.initials: M
authors.lastname: S
authors.forename: Santosh Kumar T
authors.initials: SK
authors.lastname: R
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology* / Q000633:toxicity; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D002843:Chromatin; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D012717:Sesquiterpenes; D051028:bcl-2-Associated X Protein; C403304:zerumbone; D045304:Cytochromes c; D002154:Calpain; D020169:Caspases; D002118:Calcium