Antioxidant and xenobiotic-metabolizing enzyme gene expression in doxorubicin-resistant MCF-7 breast cancer cells.
We investigated the expression of the genes for several antioxidant and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is greater than 500-fold resistant to doxorubicin by clonogenic assay. Enzyme activity determinations in the cytoplasmic compartment of MCF-7/Dox revealed a 25-fold increase in glutathione peroxidase level compared to the parent line (mean +/- SD, 10 +/- 2.8 versus 0.4 +/- 0.24 nmol/min/mg; P less than 0.005). The activity of the other major hydrogen peroxide-detoxifying enzyme, catalase, was diminished in MCF-7/Dox (2.0 +/- 0.4 versus 4.8 +/- 1.4 mumol/min/mg; P less than 0.025 compared to MCF-7). Superoxide dismutase activity did not differ between the two cell lines. The specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was 4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 +/- 45 versus 509 +/- 123 nmol/min/mg; P less than 0.005). Daunorubicinol-producing carbonyl reductase activity was equal in the two lines. Northern blot analysis demonstrated a 0.9-kilobase band of glutathione peroxidase mRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase superoxide dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When normalized to 28S RNA, no difference in the mRNA levels of catalase and superoxide dismutase in MCF-7/Dox and MCF-7 could be determined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous to the putative carbonyl reductase cDNA was also easily detectable in both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobiotic-detoxifying enzymes was decreased 2- to 4-fold in the doxorubicin-resistant cells. Southern blot analysis of PstI- and MspI-restricted genomic DNA revealed no evidence for amplification or rearrangement of the glutathione peroxidase gene. These results indicate that, in addition to the previously described overexpression of anionic glutathione S-transferase in MCF-7/Dox cells, an augmented glutathione peroxidase mRNA level is the major alteration in antioxidant and xenobiotic-detoxifying enzyme expression that could contribute to doxorubicin insensitivity in these multidrug-resistant breast cancer cells.
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010.
S A
SA
Akman
G
G
Forrest
F F
FF
Chu
R S
RS
Esworthy
J H
JH
Doroshow
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics*; D004317:Doxorubicin / Q000378:metabolism*; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011808:Quinone Reductases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004317:Doxorubicin; D000429:Alcohol Oxidoreductases; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D011808:Quinone Reductases