Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells Arti Parihar a , Mordhwaj S. Parihar b , Rafal Nazarewicz c , Pedram Ghafourifar d, ⁎ a Department of Biological Sciences, GDC College, Vikram University, Ujjain, MP, India b School of Studies in Biotechnology & Zoology, Vikram University, Ujjain, MP, India c Department of Medicine, Emory University, Atlanta, GA, USA d Tri-State Institute of Pharmaceutical Sciences, Huntington, WV, USA abstract article info Article history: Received 24 November 2009 Received in revised form 14 March 2010 Accepted 31 March 2010 Available online 8 April 2010 Keywords: Ceramide Cytochrome c Mitochondria Membrane potential Respiration Oxidative stress Mammary adenocarcinoma cells Background: Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides. Methods: The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry. Results: We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c. Conclusions: Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione. General significance: Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Ceramides are intracellular lipid mediator involved in various cellular responses including oxidative stress and apoptosis [1]. Ceramides are involved in cell death induced by variety of stimuli including tumor necrosis factor α [2], anthracyclines [3] and irradiation [4]. Elevated levels of ceramides induce apoptosis in various cells [5,6] including human mammary adenocarcinoma cells MCF-7 and MDA-MB-231 [7,8]. Mitochondria are the key organelles involved in apoptotic cell death [9–12]. In isolated mitochondria, ceramides affect the electron transport chain [13] and play a critical role in the release of cytochrome c (cyt c) through the mitochondrial permeability transition (MPT) pore [14,15]. Ceramides interact directly with mitochondria [16], exacerbate generation of reactive oxygen species (ROS) [17,18] and release cyt c [19] leading to perturbations and loss of mitochondrial functions [20,21]. GSH has also been implicated in protection against the induction of apoptosis and necrotic cell death in a variety of cell types. However, the exact mechanism underlying ceramides-induced cyt c release and role of mitochondrial GSH in this process is not fully understood. The present study shows that ceramides induce apoptosis in MCF-7 and MDA-MB- 231 mammary adenocarcinoma cells by exacerbating oxidative stress and interacting with oxidized cyt c (cyt oxy ). Our findings show the importance of the redox state of cyt c for the reaction of cyt c with ceramides and induction of apoptosis. The present findings also suggest that the level of mitochondrial GSH may be an important determinant of the sensitivity to cyt c release and cell death induced by ceramides. Biochimica et Biophysica Acta 1800 (2010) 646–654 Abbreviations: C-2 ceramide, N-Acetyl-D-sphingosine; C-6 ceramide, N-Caproyl- C18-sphingosine; DHC, Dihydroceramide; cyt c, cytochrome c; Δψ, mitochondrial transmembrane potential; cyt oxy , oxidized cytochrome c; cyt red , reduced cytochrome c; TBARS, thiobarbituric acid reactive substances; LPO, lipid peroxidation ⁎ Corresponding author. E-mail address: Pedram.Ghafourifar@tipswv.com (P. Ghafourifar). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.022 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen