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Hepsulfam Sensitivity in Human Breast Cancer Cell Lines: The Role of Glutathione and Glutathione S-Transferase in Resistance¹

The Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Hepsulfam (NSC 329680, 1,7-heptanediol disulfamate) is an alkylating agent that showed excellent activity against mouse and human mammary carcinoma in preclinical studies. We therefore studied the cytotoxicity of this drug in six human breast cancer cell lines (Adr^RMCF7, WTMCF7, Hs578T, MDA-MB-231, T47D, and MDA-MB-468). Clonogenic assays of these cell lines showed a range of sensitivity with the 90% inhibitory concentration ranging from 3.1 µM hepsulfam (MDA-MB-468) to 32.3 µM hepsulfam (Adr^RMCF7) after 24-h exposure to the drug. To evaluate possible mechanisms responsible for this observed variation in sensitivity to hepsulfam, we have studied glutathione S-transferase (GST) activity and glutathione (GSH) in these cell lines. Total cytoplasmic GST activity correlated with sensitivity; the most sensitive cell lines had the lowest GST activity, while the two most resistant cell lines, Adr^RMCF7 and Hs578T, had the highest GST levels of the six cell lines. Western blot analysis showed that the only detectable isoenzyme was GST-. The amount of GST- isoform correlated with hepsulfam sensitivity in the three most resistant cell lines and was undetectable in the three most sensitive cell lines. Cellular concentrations of GSH did not correlate with hepsulfam sensitivity. However, GSH depletion with buthionine sulfoximine increased sensitivity to hepsulfam in a dose-dependent fashion in all six cell lines. Evaluation by mass spectrometry revealed that glutathione can form conjugates with hepsulfam. We conclude that the GST/GSH detoxication system plays a role in the sensitivity of these breast cancer cell lines to hepsulfam.

¹ These studies were supported by NIH Grants CA 49634, CA 44530, and 5 P01 CA 15396 and by the Phil N. Allen Charitable Trust. D. K. A. was the recipient of a Stetler Research Fund Fellowship. N. E. D. was the recipient of American Cancer Society Clinical Oncology Career Development Award 90–128 and a Merck Clinician Scientist Award from The Johns Hopkins University School of Medicine. Mass spectra were obtained in the Middle Atlantic Mass Spectrometry Laboratory, an NSF Shared Instrument Facility supported by NSF: DIR 90-16567.

² To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Center, 422 N. Bond Street, Baltimore, MD 21231.

Received 8/15/91. Accepted 1/ 7/92.

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