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

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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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confidence: 98%

Glutathione diminishes the anti-tumour activity of 4-hydroperoxycyclophosphamide by stabilising its spontaneous breakdown to alkylating metabolites

Lee

1991

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“…Of potential importance to cancer therapists is the observation that tumour cells, particularly those of human origin, have been found to contain very high levels of GSH (Allalunis-Turner et Biaglow et al, 1983;Mitchell et al, 1989) which suggests that GSH may be a key factor limiting the therapeutic efficacy of cancer treatment. This view is supported by a number of reports which have shown that resistance to chemotherapeutic agents may be due to elevated cellular GSH concentrations (Hamilton et al, 1985; Lee et al, 1991;Suzukake et al, 1982;Richardson & Siemann, 1992). Consequently there has been considerable interest in developing approaches to overcoming such thiol mediated therapy resistance.…”

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confidence: 58%

“…More recently, Lee and colleagues (Lee et al, 1991) used 21 tumour lines to demonstrate a relationship between steady-state cellular GSH contents and chemosensitivity to 4-hydroperoxycyclophosphamide. Not only is there strong experimental evidence for the role of GSH in at least some forms of drug resistance, but it has been suggested that GSH might be an even greater determining factor when human tumours are treated with chemotherapy.…”

Section: Discussionmentioning

confidence: 99%

“…The potential of BSO as an adjuvant to conventional chemotherapy is supported by a number of recent in vitro and in vivo investigations which have shown that (i) depletion of cellular GSH by BSO can increase the cytotoxicity of a variety of anticancer drugs (Hamilton et al, 1985; Kramer et al, 1987; Lee et al, 1989;1991;Ono et al, 1986;Ozols et al, 1987;Tsutsui et al, 1986;Mitchell et al, 1989;Kramer et al, 1989), and (ii) thiol mediated resistance can be overcome by depletion of cellular GSH prior to drug expsoure (Hamilton et al, 1985;Crook et al, 1986;Richardson & Siemann, 1992 interest in the use of BSO as a chemosensitiser in cancer chemotherapy. Indeed BSO is currently undergoing Phase I clinical trial evaluation.…”

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confidence: 95%

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In vivo therapeutic potential of combination thiol depletion and alkylating chemotherapy

Siemann

Beyers²

1993

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Summary The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-' dose of BSO, tumour GSH reached a nadir of -40% of control 12-16 h after treatment. Bone marrow GSH was depeleted to -45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-' dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of -1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-' doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an -1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.

“…It has previously been demonstrated that the amount of the ultimate active metabolites formed intracellularly is dependent on the intracellular glutathione concentration and its interaction with the toxic metabolites (Lee et al, 1991c). Cyclophosphamide has also been shown to be able to deplete serum glutathione (Carmichael et al, 1986 (Dolan et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Cyclophosphamide decreases O6-alkylguanine-DNA alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation

Lee

Crowther²,

Scarffe³

et al. 1992

Summary 06-alkylguanine-DNA-alkyltransferase (ATase) levels were measured in extracts of peripheral blood lymphocytes taken at various times during chemotherapy from 19 patients with various haematological malignancies. Seven patients with advanced Hodgkin's disease received preparative treatment consisting of cyclophosphamide (1.5 g m-2, daily) administered on days 1 to 4 and BCNU (600 mg m-2) on day 5 prior to autologous bone marrow rescue (ABMR) delivered on day 7. Treatment in the remaining 12 patients consisted of cyclophosphamide (1.8 g m2, daily) given on days 1 and 2 followed at day 4 with total body irradiation (TBI) administered in six fractions over the subsequent 3 days to a total dose of 1200 cGy prior to bone marrow transplantation. In the Hodgkin's group, significant decreases in ATase activity were seen during the cyclophosphamide treatment, and the median ATase nadir was 32% (range 0% to 57%) of pretreatment levels following 4 days of cyclophosphamide. In one patient, no ATase activity was detectable following the 4th cyclophosphamide treatment. ATase activities decreased further after BCNU administration to a median of 19% (range 0% to 32%) of pretreatment levels. Extensive cyclophosphamide-induced reduction of lymphocyte ATase levels was also seen in the other group of 12 patients treated with cyclophosphamide/TBI: postcyclophosphamide median ATase nadir was 35% (range 12% to 78%) of the pretreatment levels. No ATase depletion was seen when cyclophosphamide (up to 1O mM) was incubated for 2 h with pure recombinant human ATase in vitro whereas ATase activity was reduced by 90% on preincubation with 100 tsm acrolein or with > 1 mM phosphoramide mustard. This suggests that a cyclophosphamide-induced decrease in ATase levels in human peripheral lymphocytes in vivo may be due to depletion mediated by the production of intracellular acrolein. Since ATase appears to be a principal mechanism in cellular resistance to the cytotoxic effects of BCNU and related alkylating agents, these observations suggest that a cyclophosphamide-induced reduction in ATase activity may be an additional factor in the effectiveness of the combined sequential therapy.