Effect of Glutathione Depletion by L-Buthionine Sulfoximine on the Cytotoxicity of Cyclophosphamide in Single and Fractionated Doses to EMT6/SF Mouse Tumors and Bone Marrow2

Abstract

The combined cytotoxicity of cyclophosphamide [(CYC) CAS: 50-18-0] and glutathione [(GSH) CAS: 70-18-8] depletion by buthionine sulfoximine (BSO) toward EMT6/SF tumors and mouse bone marrow was studied in male BALB/c mice. Tumor GSH was depleted to 28.7 and 7.8% of control by 1 or 2 doses of BSO (5 mmol/kg), respectively, administered ip at 12-hour intervals prior to assay. Tumor GSH could be maintained at a level 8% of control by daily dosing with BSO for 3 days. The same BSO administration schedule lowered bone marrow cell GSH to 31% of control for the 3-day period studied. Tumor growth and bone marrow cell counts were unaffected by all BSO treatments studied. However, BSO pretreatment enhanced the cytotoxicity of CYC to tumor cells, as measured by an in vitro colony-forming assay, but it did not enhance the depletion of bone marrow cells in CYC-treated mice. The magnitude of the enhancement of CYC cytotoxicity by BSO depended on the extent of GSH depletion, tumor size, and the CYC dosing schedule: Single and double doses of BSO enhanced the cytotoxicity of a single dose of CYC by factors of 1.5 and 2.0, respectively, in 7-day-old tumors, based on the dose of CYC required to produce a surviving fraction of 4 X 10(-3). The response of 9-day-old tumors to CYC suggested the presence of a subpopulation of cells that were relatively resistant to CYC. There was no evidence for this subpopulation in BSO-pretreated tumors. Multiple doses of BSO and CYC also combined to give enhanced (1.65-fold) tumor cell killing compared to tumors treated with CYC alone. The data suggest a possible role for BSO as a clinical chemosensitizer, which could be combined with fractionated doses of CYC and may be effective on small cancerous lesions.