Adriamycin activation and oxygen free radical formation in human breast tumor cells: protective role of glutathione peroxidase in adriamycin resistance.
Previous studies with Adriamycin-sensitive and -resistant (ADRR) MCF-7 human breast tumor cell lines indicated that Adriamycin formed significantly less hydroxyl radical (.OH) as the result of enhanced detoxification of reactive oxygen intermediates in the ADRR cell line. In order to further define the sites of drug activation and the role of detoxification mechanisms in free radical levels, subcellular fractions were isolated from these two cell lines and free radical formation in the presence of Adriamycin was examined by using electron spin resonance spectroscopy. Studies reported here show that considerable NADPH-cytochrome P-450 reductase and NADH dehydrogenase activities were present in microsomes and mitochondria, respectively, and in nuclei obtained from these cells, and the relative activity of NADH dehydrogenase was 2-fold higher in the mitochondrial fraction of ADRR cells compared to the mitochondrial fraction from the parental wild type cells. In the presence of Adriamycin and a reducing cofactor (NADPH or NADH), Adriamycin semiquinone free radical, superoxide anion, and .OH were detected in all these fractions. Although only a small difference in the relative amount of oxy radical formation was detected in tumor microsomes, both mitochondria and nuclei of ADRR cells showed an overall 2-fold decreased formation of oxy radicals. The formation of the free radicals was significantly inhibited by superoxide dismutase, catalase, and dimethyl sulfoxide, indicating that free .OH generation was both superoxide and hydrogen peroxide dependent. The addition of purified glutathione peroxidase likewise inhibited .OH formation in a dose-dependent fashion. Similarly, when the lysate from ADRR cells, which contains 12- to 14-fold more glutathione peroxidase than Adriamycin-sensitive cells, was added to reaction mixtures containing Adriamycin-sensitive cells and Adriamycin, the .OH formation was diminished. Decreased free radical formation in nuclei and mitochondria, as a result of detoxification of hydrogen peroxide by glutathione peroxidase, may be significant in the protection of ADRR cells from Adriamycin-induced cell killing.
Sinha
B K
BK
Biochemical Pharmacology Section, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Mimnaugh
E G
EG
Rajagopalan
S
S
Myers
C E
CE
D001711:Biotransformation; D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D004317:Doxorubicin / Q000378:metabolism*; D004351:Drug Resistance; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005609:Free Radicals; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D005609:Free Radicals; D004317:Doxorubicin; D005979:Glutathione Peroxidase; D009251:NADPH-Ferrihemoprotein Reductase; D009245:NADH Dehydrogenase