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

doi:10.1016/S0006-2952(00)00521-9

Biochemical Pharmacology

Volume 60, Issue 12, 15 December 2000, Pages 1915-1923

https://doi.org/10.1016/S0006-2952(00)00521-9 Get rights and content

Abstract

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° in the 1–2 μM range, scarcely dependent on their stereochemistry at C(7).

Section snippets

General

Solvents and chemicals were purchased from Janssen with the exception of 2,4-dinitrofluorobenzene and DMSO, obtained from Sigma-Aldrich. DHM-3 dimer was prepared as described earlier [39]. The uv-vis spectra were obtained with a Hewlett-Packard 8452A diode array spectrometer.

Anthracyclines and their GSH conjugates

ADR (1) and DAUN (2) were gifts of Pharmacia-Farmitalia. Menogaril (5) was a gift from Pharmacia-Upjohn. ADRIGLU (3) (Fig. 1) was obtained as a mixture of the epimers 3-I and 3-II as reported in the literature [28]. Epimers

Results and discussion

To address the question, do anthracyclines undergo GSH conjugation in MDR tumor cells, we have chosen the MCF-7/DOX cell line to see if any conjugate with ADR (1) forms in significant amounts. The main reason for the choice was the fact that MCF-7/DOX cells were considered to be among the best candidates for GSH conjugation both because of the very high GST activity and the above-mentioned observations on cellular localization and relative toxicity of anthracyclines. Although 1 does not show

Acknowledgements

We thank Marco Paterno for technical assistance with the HPLC analyses. This study was supported by the Institute of Experimental Medicine, NRC, by grants to the Department of Biology, University of Rome “Tor Vergata” from Ministero della Sanità, and by grants to the University of Colorado from the American Cancer Society (RPG-98–110-01-ROG), the U.S. National Institutes of Health (CA78756), and the U.S. Department of Defense (DAMD17–98-1–8298).

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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

Abstract

Section snippets

General

Anthracyclines and their GSH conjugates

Results and discussion

Acknowledgements

Eur J Cancer

Pharmacol Ther

Pharmacol Ther

Trends Biochem Sci

Biochem Pharmacol

Biochem Biophys Res Commun

J Biol Chem

Protein Expr Purif

J Biol Chem

J Biol Chem

Anal Biochem

Biochem Pharmacol

Multidrug resistance

J Natl Cancer Inst

How cancer cells evade chemotherapySixteenth Richard and Hinda Rosenthal Foundation Award Lecture

Cancer Res

Biochemistry of multidrug resistance mediated by the multidrug transporter

Annu Rev Biochem

Cell biological mechanism of multidrug resistance in tumors

Proc Natl Acad Sci USA

Glutathione-associated enzymes in anticancer drug resistance

Cancer Res

Cell surface P-glycoprotein is associated with multidrug resistance in mammalian cell lines

Science

The biochemistry of P-glycoprotein-mediated multidrug resistance

Annu Rev Biochem

Over-expression of P-glycoprotein and glutathione S-transferase π in MCF-7 cells selected for vincristine resistance in vitro

Int J Cancer

Glutathione transferase and cancer

Crit Rev Biochem Mol Biol

Glutathione Conjugation, Mechanism and Biological Significance

Differential increases in glutathione S-transferase activities in a range of multidrug-resistant human tumor cell lines

Cancer Commun