Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rαc‐grycero‐3‐phosphocholine in p53‐defective hepatocytes

The mechanism of induction of apoptosis by the novel anti‐cancer drug 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3) was investigated in p53‐defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 μM ET‐18‐OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET‐18‐OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET‐18‐OCH3 in the presence of the antioxidant α‐to‐copherol. Similar results were achieved when cells were switched acutely to choline‐deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET‐18‐OCH3 and CD decreased the phos‐phatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5′,6,6′‐tetramethylbenzimidazolcarbocyanine iodide (JC‐1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the in‐tracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET‐18‐OCH3 or CD. Taken together, these results suggest that the form of p53‐independent apoptosis induced by ET‐18‐OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.— Vrablic, A. S., Albright, C. D., Craciunescu, C. N., Salganik, R. I., Zeisel, S. H. Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1‐O‐octadecyl‐2‐methyl‐rac‐glycero‐3‐phosphocholine in p53‐defective hepatocytes. FASEB J. 15, 1739—1744 (2001)

[1]  J. Freyer,et al.  Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids. , 1997, American journal of physiology. Cell physiology.

[2]  D. Brenner,et al.  The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. , 1998, Biochimica et biophysica acta.

[3]  Luowei Li,et al.  Protein Kinase Cδ Targets Mitochondria, Alters Mitochondrial Membrane Potential, and Induces Apoptosis in Normal and Neoplastic Keratinocytes When Overexpressed by an Adenoviral Vector , 1999, Molecular and Cellular Biology.

[4]  E. Yeh,et al.  Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis , 1998, Oncogene.

[5]  C. Woodworth,et al.  Transformation of rat hepatocytes by transfection with simian virus 40 DNA to yield proliferating differentiated cells. , 1986, Cancer research.

[6]  C. Franceschi,et al.  A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). , 1993, Biochemical and biophysical research communications.

[7]  C. Woodworth,et al.  Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes , 1988, Molecular and cellular biology.

[8]  S. Zeisel,et al.  Choline deficiency induces apoptosis in SV40‐immortalized CWSV‐1 rat hepatocytes in culture 1 , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  J. Carson,et al.  Morphogenesis of the murine node and notochordal plate , 1994, Developmental dynamics : an official publication of the American Association of Anatomists.

[10]  D. Green,et al.  Apoptosis: checkpoint at the mitochondrial frontier. , 1999, Mutation research.

[11]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[12]  C. Chinopoulos,et al.  Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide‐Induced Oxidative Stress in Nerve Terminals , 1999, Journal of neurochemistry.

[13]  M. V. Heiden,et al.  Redox regulation of p53 during hypoxia , 2000, Oncogene.

[14]  M. Kates.  Techniques of Lipidology , 1972 .

[15]  S. C. Garner,et al.  Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats. , 1993, The Journal of biological chemistry.

[16]  B. Chaudhuri,et al.  Release of cytochrome c and decrease of cytochrome c oxidase in Bax‐expressing yeast cells, and prevention of these effects by coexpression of Bcl‐xL , 1997, FEBS letters.

[17]  Javier Naval,et al.  Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

[18]  S. Zeisel,et al.  Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes. , 1997, Carcinogenesis.

[19]  C. J. Barnes,et al.  Effects of iron supplementation and ET-18-OCH3 on MDA-MB 231 breast carcinomas in nude mice consuming a fish oil diet. , 1997, British Journal of Cancer.

[20]  Y Li,et al.  [Mitochondria and apoptosis]. , 2000, Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine].

[21]  J. Klaunig,et al.  Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[22]  H. Ishibashi,et al.  Rotenone, a mitochondrial NADH dehydrogenase inhibitor, induces cell surface expression of CD13 and CD38 and apoptosis in HL-60 cells. , 1996, Leukemia & lymphoma.

[23]  G. Rook,et al.  A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. , 1985, Journal of immunological methods.

[24]  C. Franceschi,et al.  JC‐1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis , 1997, FEBS letters.

[25]  S. Jackowski,et al.  Apoptosis Triggered by 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine Is Prevented by Increased Expression of CTP:Phosphocholine Cytidylyltransferase* , 1998, The Journal of Biological Chemistry.

[26]  D. Lane,et al.  T antigen is bound to a host protein in SY40-transformed cells , 1979, Nature.

[27]  D. Vance,et al.  A Genetic Defect in Phosphatidylcholine Biosynthesis Triggers Apoptosis in Chinese Hamster Ovary Cells* , 1996, The Journal of Biological Chemistry.

[28]  C D Woodworth,et al.  Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium , 1987, Molecular and cellular biology.

[29]  J. Casida,et al.  NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. Schägger,et al.  Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity. , 2000, Biochimica et biophysica acta.

[31]  G. Buettner,et al.  Increased generation of lipid-derived and ascorbate free radicals by L1210 cells exposed to the ether lipid edelfosine. , 1993, Cancer research.

[32]  Xiaodong Wang,et al.  Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c , 1996, Cell.

[33]  S. Zeisel,et al.  Methyl‐group donors cannot prevent apoptotic death of rat hepatocytes induced by choline‐deficiency , 1996, Journal of cellular biochemistry.

[34]  D. Hockenbery,et al.  Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. , 1998, Experimental cell research.

[35]  Seamus J. Martin,et al.  Cytochrome c activation of CPP32‐like proteolysis plays a critical role in a Xenopus cell‐free apoptosis system , 1997, The EMBO journal.

[36]  R. Schlapbach,et al.  Oxidants in mitochondria: from physiology to diseases. , 1995, Biochimica et biophysica acta.

[37]  S. Zeisel,et al.  Choline deficiency‐induced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[38]  P. Galle,et al.  Reactive Oxygen Intermediates Are Involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells* , 1997, The Journal of Biological Chemistry.

[39]  G. Kroemer,et al.  Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo , 1995, European journal of immunology.

[40]  E. Clementi,et al.  Endogenous and exogenous nitric oxide enhance the DNA strand scission induced by tert‐butylhydroperoxide in PC12 cells via peroxynitrite‐dependent and independent mechanisms, respectively , 2000, The European journal of neuroscience.

[41]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[42]  C. Rock,et al.  The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine. , 1998, Biochimica et biophysica acta.

[43]  F. Mollinedo,et al.  Selective induction of apoptosis in cancer cells by the ether lipid ET-18-OCH3 (Edelfosine): molecular structure requirements, cellular uptake, and protection by Bcl-2 and Bcl-X(L). , 1997, Cancer research.

[44]  G. Kroemer,et al.  Mitochondria in chemotherapy-induced apoptosis: a prospective novel target of cancer therapy (review). , 1998, International journal of oncology.

[45]  R. Harris,et al.  Metabolic fate of fatty acids in the carnitine cycle of brown adipose tissue mitochondria. , 1975, Biochimica et biophysica acta.