Detection and imaging of the free radical DNA in cells—Site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy

Oxidative stress-related damage to the DNA macromolecule produces lesions that are implicated in various diseases. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Measurement of DNA damage has been a matter of debate as most of the available methods measure the end product of a sequence of events and provide limited information on the initial free radical formation. We report a measurement of free radical damage in DNA induced by a Cu(II)-H2O2 oxidizing system using immuno-spin trapping supplemented with electron paramagnetic resonance. In this investigation, the short-lived radical generated is trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) immediately upon formation. The DMPO adduct formed is initially electron paramagnetic resonance active, but is subsequently oxidized to the stable nitrone adduct, which can be detected and visualized by immuno-spin trapping and has the potential to be further characterized by other analytical techniques. The radical was found to be located on the 20-deoxyadenosine (dAdo) moiety of DNA. The nitrone adduct was repaired on a time scale consistent with DNA repair. In vivo experiments for the purpose of detecting DMPO–DNA nitrone adducts should be conducted over a range of time in order to avoid missing adducts due to the repair processes

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells

Background: Topoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide (•NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide. Methods: Here, we have evaluated the roles of •NO/•NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of •NO on the ATPase activity of topo II. Results: Treatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. •NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells. Conclusion: PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells. General significance: As tumors express nitric oxide synthase and generate •NO, inhibition of topo II functions by •NO/•NO-derived species could render tumors resistant to certain topo II-poisons in the clinic

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion(MPP<SUP>+</SUP>): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP<SUP>+</SUP>) has been studied using spin-trapping techniques. Incubation of MPP<SUP>+</SUP> with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP<SUP>+</SUP> was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP<SUP>+</SUP> in comparison to paraquat, a lung toxin containing two pyridinium moieties

Molecular Mechanisms of Cytotoxicity of NCX4040, the Non-Steroidal Anti-Inflammatory NO-Donor, in Human Ovarian Cancer Cells

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism

Hydroxyl radical production and DNA damage induced by anthracycline-iron complex

AbstractAdriamycin-Fe3+ complex catalyzes the formation of hydroxyl radical from hydrogen peroxide but the DNA-adriamycin-iron ternary complex is much more effective. 11-Deoxyadriamycin, which shows no spectral evidence of complex formation with iron, was ineffective. The generation of hydroxyl radical by adriamycin-Fe3+ complex in the presence of DNA correlates with its ability to cleave DNA. Hydroxyl radicals are thus implicated as the reactive oxygen species involved in the DNA damage caused by the adriamycin-Fe3+ complex

Gene Expression Profiling Elucidates Cellular Responses to NCX4040 in Human Ovarian Tumor Cells: Implications in the Mechanisms of Action of NCX4040

The nitric oxide donor, NCX4040 is a non-steroidal anti-inflammatory-NO donor and has been shown to be extremely cytotoxic to a number of human tumors, including ovarian tumors cells. We have found that NCX4040 is cytotoxic against both OVCAR-8 and its adriamycin-selected OVCAR-8 variant (NCI/ADR-RES) tumor cell lines. While the mechanism of action of NCX4040 is not entirely clear, we as well as others have shown that NCX4040 generates reactive oxygen species (ROS) and induces DNA damage in tumor cells. Recently, we have reported that NCX4040 treatment resulted in a significant depletion of cellular glutathione, and formation of both reactive oxygen and nitrogen species (ROS/RNS), resulting in oxidative stress in these tumor cells. Furthermore, our results indicated that more ROS/RNS were generated in OVCAR-8 cells than in NCI/ADR-RES cells due to increased activities of superoxide dismutase (SOD), glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. Further studies suggested that NCX4040-induced cell death may be mediated by peroxynitrite formed from NCX4040 in cells. In this study we used microarray analysis following NCX4040 treatment of both OVCAR-8 and its ADR-resistant variant to identify various molecular pathways involved in NCX4040-induced cell death. Here, we report that NCX4040 treatment resulted in the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes. A mechanism of tumor cell death is proposed based on our findings where oxidative stress is induced by NCX4040 from simultaneous induction of NOX4, TNF-&alpha; and CHAC1 in tumor cell death

Gene Expression Profiling Elucidates Cellular Responses to NCX4040 in Human Ovarian Tumor Cells: Implications in the Mechanisms of Action of NCX4040

The nitric oxide donor, NCX4040 is a non-steroidal anti-inflammatory-NO donor and has been shown to be extremely cytotoxic to a number of human tumors, including ovarian tumors cells. We have found that NCX4040 is cytotoxic against both OVCAR-8 and its adriamycin-selected OVCAR-8 variant (NCI/ADR-RES) tumor cell lines. While the mechanism of action of NCX4040 is not entirely clear, we as well as others have shown that NCX4040 generates reactive oxygen species (ROS) and induces DNA damage in tumor cells. Recently, we have reported that NCX4040 treatment resulted in a significant depletion of cellular glutathione, and formation of both reactive oxygen and nitrogen species (ROS/RNS), resulting in oxidative stress in these tumor cells. Furthermore, our results indicated that more ROS/RNS were generated in OVCAR-8 cells than in NCI/ADR-RES cells due to increased activities of superoxide dismutase (SOD), glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. Further studies suggested that NCX4040-induced cell death may be mediated by peroxynitrite formed from NCX4040 in cells. In this study we used microarray analysis following NCX4040 treatment of both OVCAR-8 and its ADR-resistant variant to identify various molecular pathways involved in NCX4040-induced cell death. Here, we report that NCX4040 treatment resulted in the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes. A mechanism of tumor cell death is proposed based on our findings where oxidative stress is induced by NCX4040 from simultaneous induction of NOX4, TNF-α and CHAC1 in tumor cell death