Summary Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were 'sensitive' decreased, whereas that of cell lines that were 'resistant' increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment (r=0.92; P<0.01). Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the 'resistant' and 'sensitive' lines was that the 'resistant' lines had higher steady-state rates of GSH synthesis than the 'sensitive' lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.The role of glutathione (GSH) in the development of drug and radiation resistance in cancer cells is a subject of much current research effort. Although the mechanism(s) by which GSH protects cells from the damaging effects of cytotoxic agents is still not fully understood, it is now clear that GSH can greatly influence the intrinsic sensitivity of tumour cells to radiation (Biaglow et al., 1983;Shrieve et al., 1985;Revesz, 1985) and to a variety of cytotoxic drugs (Hamilton et al., 1985;Suzukake et al., 1982;Green et al., 1984;Crook et al., 1986;Lee et al., 1988;Kramer et al., 1987. However, previous studies have fo...
Summary 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-hydroperoxylcyclophosphamide (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<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-i8 moles lm-3; chemosensitivity to 4-OOH-CP, in terms of IC99, was in the range of 5.0 -87.1IM. A good correlation was observed between these two parameters (r = 0.85, P <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.
There is considerable evidence that glutathione (GSH) plays a major role in protecting tumour cells against the cytotoxicity of the oxazaphosphorines, including cyclophosphamide (CP) and its active congener, 4-hydroperoxycyclophosphamide (4-OOH-CP), both in vitro (Russo et al., 1986; Crook et al., 1986a) and in vivo (Gurtoo et al., 1981;Carmichael et al., 1986b; Ono & Shrieve, 1987). Recently, in a study of 17 tumour cell lines, we noted a close correlation between the chemosensitivity of these cell lines to 4-OOH-CP and their steady-state GSH level (Lee et al., 1990). It was further noted that whilst GSH 'detoxifies' 4-OOH-CP, 4-OOH-CP in turn depletes GSH. In fact, 4-OOH-CP is at toxic concentrations a potent depletor of cellular GSH. Most important, the tumour cell GSH depletion and the lethality produced by 4-OOH-CP appear to be linked events. Significant 4-OOH-CP cytotoxicity was invariably associated with GSH depletion, and vice versa. In the present paper, evidence is presented showing that GSH modulates the cytotoxicity of 4-OOH-CP by participating in chemical reactions at three separate locations in the metabolic pathway of 4-OH-CP, its spontaneous breakdown product. (4-OOH-CP gives rise rapidly to 4-OH-CP following dissolution without any enzymic involvement and may be regarded as equivalent to 4-OH-CP pharmacologically (Sladek, 1987). 4-OOH-CP is the preferred 'activated' cyclophosphamide for routine use only because of its higher stability in crystalline state and easier synthesis.) 4-OH-CP, sometimes called 'activated' cyclophosphamide, is formed from the hydroxylation of CP by the hepatic mixed-function oxidases (Figure 1). 4-OH-CP is in reality the 'transport' form of CP since it is in this form that active CP reaches the target tumour cells (Sladek, 1987). Intracellular 4-OH-CP is in equilibrium with its ring-opened tautomer aldophosphamide (AP). The fate of AP may follow one of three main competing metabolic pathways: (1) spontaneous fission to acrolein (AC) and phosphoramide mustard (PM), (2) ring-closure and hydroxylation to produce once again the parent 4-OH-CP. As depicted in Figure 1, glutathione (GSH) can participate in conjugative reactions at three separate locations that may have considerable influence on the eventual cytotoxicity of 4-OH-CP. (1) Reaction with AP as described above which shifts the pseudoequilibrium between 4-OH-CP and AP in favour of the former and thereby curtails the spontaneous degradation of AP to toxic metabolites. GSH also reacts irreversibly with the toxic metabolites AC and PM, but particularly the former (2 and 3) (Gurtoo et al., 1981). In the accompanying paper we demonstrated that when the combined rates of the conjugation reactions exceed the rate of GSH recovery, i.e. when GSH is being depleted, significant cytotoxicity inevitably occurs. The present findings suggest that GSH depletion impacts directly on the cytotoxic potency of 4-OH-CP by destabilising AP (see Figure 1)
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