Prediction of tumour sensitivity to 4-hydroperoxycyclophosphamide by a glutathione-targeted assay

Abstract

In an attempt to develop an assay to predict patient tumour response to cyclophosphamide (CP), the feasibility of using a glutathione-targeted assay to assess the in vitro chemosensitivity of tumour cells to 4-hydroperoxycyclophosphamide (4-OOH-CP), an activated congener of CP, was evaluated. A panel of 19 human and three murine tumour cell lines was used. These consisted of three main categories of tumour types, viz. ovarian, lung and squamous cell carcinoma. The major finding was that the occurrence of a significant reduction of tumour cell reproductive capacity was always accompanied by substantial depletion of cellular glutathione (GSH) content, and vice versa. Plots of % GSH depletion versus clonogenic cell survival demonstrated highly significant correlation (r = 0.90-0.91; P less than 0.01). It was determined that for in vitro tumour cell lines, a GSH depletion to 40% of initial content may serve as a cut-off criterion for chemosensitivity to 4-OOH-CP. This degree of GSH depletion is indicative of clonogenic cell survival of approximately 1% (95% confidence limits = 3 x 10(-5)-1.6 x 10(-2)). The relationship between steady state GSH content and intrinsic sensitivity to 4-OOH-CP was also evaluated. The GSH concentration of the tumour cell lines ranged from 1.3-21.2 x 10(-18) moles microns-3; chemosensitivity to 4-OOH-CP, in terms of IC99, was in the range of 5.0-87.1 microM. A good correlation was observed between these two parameters (r = 0.85, P less than 0.02). These results suggest that GSH plays an important role in determining the therapeutic efficacy of 4-OOH-CP in the treatment of cancer. It is uncertain, however, whether a high tumour steady state GSH content in itself is sufficient to cause therapeutic failure in patients.