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.
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.
Vrablic
A S
AS
Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7400, USA.
Albright
C D
CD
Craciunescu
C N
CN
Salganik
R I
RI
Zeisel
S H
SH
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002461:Cell Line, Transformed; D004587:Electrophoresis, Agar Gel; D022781:Hepatocytes / Q000502:physiology* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000737:chemistry / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D008930:Mitochondria, Liver / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D009580:Nitroblue Tetrazolium; D010713:Phosphatidylcholines / Q000032:analysis; D010742:Phospholipid Ethers / Q000494:pharmacology*; D051381:Rats; D011916:Rats, Inbred F344; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000502:physiology*; D014810:Vitamin E / Q000494:pharmacology
D000952:Antigens, Polyomavirus Transforming; D000975:Antioxidants; D010713:Phosphatidylcholines; D010742:Phospholipid Ethers; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D014810:Vitamin E; C026659:edelfosine; D009580:Nitroblue Tetrazolium