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 microM 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 alpha-tocopherol. 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 phosphatidylcholine (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 intracellular 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.
CITATION STYLE
VRABLIC, A. S., ALBRIGHT, C. D., CRACIUNESCU, C. N., SALGANIK, R. I., & ZEISEL, S. H. (2001). 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 FASEB Journal, 15(10), 1739–1744. https://doi.org/10.1096/fj.00-0300com
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