Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines.
One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).
Gaudiano
G
G
Institute of Experimental Medicine, National Research Council, 00133, Rome, Italy.
Koch
T H
TH
Lo Bello
M
M
Nuccetelli
M
M
Ravagnan
G
G
Serafino
A
A
Sinibaldi-Vallebona
P
P
D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism*; D004317:Doxorubicin / Q000378:metabolism* / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D004317:Doxorubicin; D005982:Glutathione Transferase; D005978:Glutathione