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

Abstract

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.

Role of mitochondria and reactive oxygen species in dendritic cell differentiation and functions

“…We have used in this work rotenone, the inhibitor of the respiratory chain Complex I, to modulate mitochondrial basal ROS generation. This approach, which has been pursued in many laboratories, has produced conflicting results reporting that rotenone could elevate cellular ROS generation in some cases [16,30,31], but reduce ROS production in others [12,15,[32][33][34]. Data have recently been produced showing that ROS generated during the oxidation of NAD-dependent substrates are greatly enhanced by rotenone and exclusively released into the mitochondrial matrix [15,16], where they are dissipated by the matrix antioxidant defense.…”

Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production

“…The importance of mitochondrial respiratory chain complex I inhibitor-induced apoptosis was further recognized with the finding that tumor necrosis factor (TNF)-␣ could inhibit the mitochondrial respiratory chain at the mitochondrial complex I site (14). However, contradictory reports showed that rotenone could inhibit apoptosis in other systems (17)(18)(19).…”

“…Early reports showed that the complex I inhibitor rotenone and the complex b-c1 inhibitor antimycin could stimulate superoxide and hydrogen peroxide formation on submitochondrial particles (31,35). However, results of rotenone-induced mitochondrial ROS production measured at the cellular level appeared inconsistent with conflicting results reporting that rotenone could elevate cellular ROS production in some cases (11,36) while inhibiting cellular ROS production in others (17,37,38).…”

Effect of Supplementation Kayu Manis (Cinnamomum Burmannii) Extract in Neuronal Cell Death Protection in Wistar Rats Lir-Psychotic on Haloperidol Therapy

“…One of the important functions of apoptosis is the elimination of preneoplastic and neoplastic cells [12]. In most forms of cell suicide, the signaling cascade utilizes reactive oxygen species as essential intermediate messenger molecules [13]. This is the reason that antioxidants are capable of inhibiting apoptosis.…”