Oxidants such as hydrogen peroxide (H2O2) are implicated in mediating a wide array of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. Oxidants contribute to disease processes by causing damage to biomolecules and altering cellular metabolism. Key among the targets for oxidative damage are structural proteins and enzymes. In order to understand how oxidative stress can cause disease, it is important to discover which proteins become affected by oxidative stress, to what degree they are modified, and the functional consequences of the modifications. One place where protein oxidation could play a significant physiological role would be in tumor cells induced to die by cancer chemotherapy drugs. It has been hypothesized that intracellular oxidants (reactive oxygen species; ROS) are generated in cells treated with cancer chemotherapy drugs and that these are an essential component of the drug-induced apoptotic process. If so, then these oxidants likely act by modifying intracellular molecules that are required for cell viability. We are particularly interested in finding specific proteins that might be modified by apoptosis-associated oxidants as these are likely to catalyze the reactions that propagate or inhibit the apoptotic cascade. Our studies involve use of human B lymphoma and breast cancer cells as tumor models and we employ cancer chemotherapy drugs which have sometimes been cited as working through a pro-oxidant mechanism such as doxorubicin (adriamycin), VP-16 (etoposide), and cisplatin. Protein oxidation is assessed by measuring (1) protein carbonyl groups by Western blot immunoassay, (2) protein methionine sulfoxide residues by amino acid analysis, (3) protein sulfhydryl oxidation by Western blot immunoassay or fluorescence activated cell sorting (FACS). We also measure formation of F2-isoprostanes as a marker of lipid peroxidation, and intracellular ROS production using the oxidant-sensitive dyes DCFDA and DHR 123. Our results show that chemotherapy drugs induce extensive apoptosis in the absence of any detectable protein or lipid oxidation, measured in both the cytosolic and mitochondrial compartments of the cell. H2O2, which kills the cells by non-apoptotic pathways, causes increases in both protein and lipid oxidation. Three different antioxidant compounds (N-acetyl cysteine, Tempol, and MnTBAP) fail to inhibit drug-induced apoptosis while inhibiting H2O2-induced cell death. We conclude that drug-induced apoptosis occurs using a mechanism that does not involve oxidants and does not require protein oxidation. In vivo studies using a mouse pre-clinical model for treatment of breast cancer showed further that inclusion of an antioxidant in the treatment protocol does not interfere with the ability of doxorubicin to inhibit tumor growth. These finding may have important ramifications for improving chemotherapy protocols because they suggest that oxidant-mediated side-effects from cancer chemotherapy might safely be diminished by addition of dietary antioxidants to the treatment protocols.
