Abstract
NSC3852 (5-nitroso-8-quinolinol) has cell differentiation and antiproliferative activity in human breast cancer cells in tissue culture and antitumor activity in mice bearing P388 and L1210 leukemic cells. We investigated the mechanism of NSC3852 action in MCF-7 human breast cancer cells using electron spin resonance (ESR). Reactive oxygen species (ROS) were detected in MCF-7 cell suspensions incubated with NSC3852 using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Formation of the DMPO-OH adduct was quenched by the addition of superoxide dismutase but not by catalase, and we concluded that superoxide was generated in the NSC3852-treated cells. The flavoprotein inhibitor diphenylene iodonium suppressed ROS production, providing evidence for the involvement of a flavin-dependent enzyme system in the ROS response to NSC3852. A biologically significant oxidative response to NSC3852 occurred in MCF-7 cells. An early marker of oxidative stress was a decrease in the [glutathione]/[glutathione disulfide] ratio 1 h after NSC3852 addition. Oxidative DNA damage, marked by the presence of 8-oxoguanine, and DNA-strand breakage occurred in cells exposed to NSC3852 for 24 h. Apoptosis peaked 48 h after exposure to NSC3852. Pretreatment with the glutathione precursor N-acetyl-l-cysteine (NAC) prevented DNA-strand breakage and apoptosis. Pretreatment with NAC also reversed NSC3852 decreases in E2F1, Myc, and phosphorylated retinoblastoma protein, indicative of redox-sensitive pathway(s) in MCF-7 cells during G1 phase of the cell cycle. We conclude that ROS formation is involved in the apoptotic and cell differentiation responses to NSC3852 in MCF-7 cells.
Footnotes
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This work was funded by DAMD 17-00-1-500.
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Portions of this work were presented in abstract form: Strobl J, Martirosyan A, Rahim-Bata R, Freeman A, and Clarke C (2004). Experimental breast cancer differentiation agents NSC3852, NSC69603, NSC10010, and NSC305819, in 1st ISC International Conference on Cancer Therapeutics, Molecular Targets, Pharmacology and Clinical Applications; 2004 February 19–24, International Society of Chemotherapy and Società Italiana di Chemioterapia, Florence, Italy.
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doi:10.1124/jpet.105.096891.
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ABBREVIATIONS: NSC3852, 5-nitroso-8-quinolinol; DMPO, 5, 5-dimethyl-1-pyrroline-N-oxide; DPI, diphenylene iodonium; DTNB, 5′,5-dithiobis-2-nitrobenzoic acid; DMEM, Dulbecco's modified Eagle's medium; ESR, electron spin resonance; GSH, glutathione; GSSG, glutathione disulfide; HDI, histone deacetylase inhibitor(s); MS-275, N-(2-aminophenyl)-4-[N-pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide; MTS, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfonphenyl)-2H-tetrazolium; PBS, phosphate-buffered saline, pH 7.4; NAC, N-acetyl-l-cysteine; NSC2039, 8-quinolinol; Rb, retinoblastoma protein; ROS, reactive oxygen species; SAHA, suberoylanilide hydroxamic acid; SBHA, suberic bishydroxamate; SOD, superoxide dismutase; TSA, trichostatin A, [R-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; FBS, fetal bovine serum.
- Received October 16, 2005.
- Accepted February 22, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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