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-o ctadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigate d in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 1 2 muM ET-18-OCH3 for 36 h induced apoptosis as determined using classical m orphological features and agarose gel electrophoresis of genomic DNA. Incre ased levels of reactive oxygen species (ROS) were detected spectrophotometr ically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-1 8-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 achiev ed when cells were switched acutely to choline- deficient (CD) medium in th e presence of the antioxidant. The possible role of mitochondria in the gen eration of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosp hatidylcholine (PC) content of mitochondrial and associated membranes, whic h correlated with depolarization of the mitochondrial membrane as analyzed using 5,5', 6,6'- tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sens itive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intra cellular 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-independe nt apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondr ial membrane PC, a loss of mitochondrial membrane potential, and the releas e of ROS, resulting in completion of apoptosis.