Effect of thiol manipulation on chemopotentiation by nitroimidazoles

0360.3016/89 $3.00 + .OO Copyright 0 1989 Pergamon Press plc

Inr. J. Radmlion Oncology Biol. Phys., Vol. 16, pp. 1341-1345 Printed in the U.S.A. All rights revved.

0 Session VII EFFECT

DIETMAR

W.

OF THIOL

SIEMANN,

PH.D.,*

MANIPULATION ON CHEMOPOTENTIATION BY NITROIMIDAZOLES AMY

A. FLAHERTY,

B.S.

AND DAVID

P. PENNEY,

PH.D.~

University of Rochester Cancer Center, 601 Elmwood Avenue, Box 704, Rochester, NY 14642 Highly electron affinic compounds such as the nitroimidazole misonidazole (MISO) have been shown both in vitro and in viva to be effective potentiators of certain conventional chemotherapeutic agents. Mechanistically, the observation that nitroheterocyclics reduce intra-cellular thiols by enhancing the oxidation of glutathione (GSH), has suggested that thiol depletion by MIS0 may be a key factor in this enhancement. The present investigations were undertaken to determine whether the use of buthionine sulfoximine (BSO) to affect GSH metabolism may lead to more effective potentiation of chemotherapeutic agents by sensitizers. KHT/iv cells were treated in exponential phase under hypoxic conditions with variable doses of the activated form of cyclophosphamide (Chydroxy-cyclophosphamide, 40H-CY) administered concominantly with or without MIS0 (2.5 mM) for an exposure time of 4 hr. Inclusion of the sensitizer in the treatment protocol resulted in a dose modifying factor of -2.4. Exposing cells to 1.0 mM BSO for 2 hr prior to treatment reduced intracellular GSH levels to 70-8096 of control and increased the efficacy of 40H-CY -1.2-fold. If BSO was administered prior to the 40H-CY + MIS0 combination, severe tumor cell toxicity resulted. For example, when combined with 40H-CY, similar cell kill could be achieved with 5 to 6-fold lower MIS0 doses in the presence of BSO as in the absence of BSO. Ultrastructural evaluations revealed that in the three agent combination, membrane damage, as reflected by the formation of surface blebs, may play a key role in the mechanism of the observed enhanced cytotoxicity. 4-hydroxy-cyclophosphamide,

Buthionine sulfoximine,

Glutathione manipulation, Chemosensitization.

in chemopotentiation to at least some degree. The aim of the present investigations was to determine whether the use of buthionine sulfoximine (BSO) to affect thiol metabolism could lead to more effective potentiation of chemotherapeutic agents by radiosensitizers or to similar magnitudes of chemopotentiation at lower sensitizer doses.

INTRODUCTION

such as misonidazole (MISO) have been demonstrated to potentiate the action of alkylating anticancer drugs both in vitro and in vivo (3, 12). In addition, such compounds effectively reduce intra-cellular nonprotein sulfhydryl levels in hypoxic cells by enhancing the oxidation of glutathione (GSH) in a manner dependent on the sensitizer exposure dose and time (1, 4). Consequently, it has been suggested that the reduction by MIS0 of cellular GSH levels could be an important factor in the enhancement of chemotherapeutic agent activity by radiosensitizers (3, 10, 12, 13). However, when equivalent intra-cellular concentrations of GSH were achieved by pretreatment with thiol depletors or MISO, MIS0 enhanced melphalan cytotoxicity significantly more (9, 13). It therefore has been concluded that, while depletion of GSH by sensitizers can account for some of the drug potentiation, it is not responsible for the entire effect (3, 10, 12). Nevertheless, intracellular GSH levels are involved Nitroimidazoles

METHODS

AND

MATERIALS

U-IT/iv cells (11) in exponential phase were used in all experiments. For treatment, single cells were suspended at a concentration of 2 X lo5 cells/ml in alpha-MEM supplemented with 10% fetal calf serum in a Type I vial at 37°C. The medium was gassed with 5% COZ:95% N2 or 5% COZ:95% air for 3 hr prior to inoculation of the cells and during the treatment period. For thiol manipulation or nitroimidazole chemopotentiation studies, BSO and MIS0 were dissolved at the desired concentration

* Experimental Therapeutics Division. t Department of Pathology. Acknowledgements-The 40H-CY was a gift from Dr. Richard Borch, Department of Pharmacology, University of Rochester. MIS0 was received from Dr. V. Narayanan of the Drug Synthesis

and Chemistry Branch, NCI. The authors thank R. Stepanik for preparing the manuscript. These studies were supported by PHS grant CA 38637 awarded by the NCI, DHHS. Accepted for publication 16 November 1988.

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directly in the medium prior to the equilibration with the gas phase. In all cases, exposure to 4OH-CY was for a period of 4 hr. In the chemopotentiation studies, MIS0 was administered concomitantly with the chemotherapeutic agent. In the thiol depletion experiments, BSO was present 2 hr prior to as well as during the entire drug treatment period. In the three agent combinations, cells were depleted of GSH to known levels by BSO pretreatment and then exposed to the drug plus sensitizer. At the end of the treatment period, the cells were removed from the vials, rinsed, counted, and various dilutions were prepared. To determine clonogenic cell survival, RI-IT/iv cells were plated into 60 mm plastic dishes containing alphaMEM with 10% fetal calf serum. These dishes were incubated for 12 days at 37’C, harvested, stained with crystal violet and colonies of over 50 cells were counted. Reversephase high performance liquid chromatography (HPLC) technique, as described in detail previously (7), was used to analyze cellular GSH contents. For ultrastructural analysis, cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH = 7.2), dehydrated with increasing concentrations of EtOH and embedded in epoxy resin. Sections were cut on an LKB Nova ultramicrotome at 40-60 nm thickness, stained with lead citrate and uranyl acetate and observed in a Zeiss 10A electron microscope.

RESULTS Figure 1 shows the depletion of GSH in RI-IT/iv cells treated either with 2.5 rg/ml40H-CY or 1 mM BSO for various periods of time. Both agents readily depleted intracellular GSH, but whereas the thiol concentrations continued to fall during the entire BSO exposure period, after treatment with activated CY, GSH levels reached a minimum during the first hr of treatment and then remained constant. Similar GSH depletion patterns were observed for cells maintained under either aerobic or hypoxic conditions. The observation that 40H-CY fails to deplete GSH beyond the level detected after a 1 hr drug exposure, is consistent with our recent results demonstrating similar cytotoxicity for this chemotherapeutic agent when administered in vitro for either 1 or 4 hr (data not shown). As has been the case in a number of other preclinical investigations using a variety of chemotherapeutic agents (2, 5, 7-IO), cellular GSH depletion by BSO potentiated the activity of 40H-CY (Fig. 2b). This thiol depletion and potentiation of drug efficacy was independent of the oxygenation state of the tumor cells (data not shown). In contrast, chemopotentiation by MIS0 is dependent on nitroreduction (12) and thus, when 40H-CY and MIS0 were administered concurrently (co-incubation), a significant increase in cell kill (-2.4-fold), compared to that for the antitumor agent alone, was seen only under hypoxic conditions (Fig. 2a).

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Fig. 1. GSH depletion in KHT/iv cells treated with either (a) 2.5 &ml 40H-CY or (b) 1 mM BSO for various periods of time.

To determine the efficacy of a regimen combining thiol manipulation with chemosensitization, KHT/iv cells were exposed to 1 mM BSO for 2 hr prior to concomitent treatment with 2.5 mM MIS0 and variable doses of 40HCY. However, this combination proved acutely toxic to hypoxic tumor cells. When evaluated ultrastructurally, cells treated with this combination were found to exhibit severe membrane changes, including surface blebbing (Fig. 3d). These effects were not seen in RI-IT cells treated with this three agent combination under aerobic conditions (Fig. 3c), nor in any other aerobic or hypoxic single or two agent combinations. In view of the significant potentiation of 40H-CY activity in the presence of both a thiol depletor and a nitroimidazole, experiments were carried out to determine whether under conditions of reduced intracellular thiols, similar magnitudes of chemopotentiation could be achieved at lower sensitizer doses. KHT/iv cells were treated under hypoxic conditions with or without BSO ( 1 mM) for 2 hr prior to a combination of a fixed dose of 40H-CY ( 1 pg/ml) and variable doses of MISO. The data

Thiol manipulation on chemopotentiation

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40H-CY Fig. 2. Survival of KHT/iv cells treated under with either 2.5 mM MIS0 (0) or 1 mM BSO agent. BSO preceded the 40H-CY treatment curve in b. is the response of cells to 40H-CY + S.E.

SIEMANN ef al.

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anoxic conditions for 4 hr with 40H-CY alone (0) or in combination (m). MIS0 was administered concurrently with the chemotherapeutic by 2 hr and was present during the entire drug exposure. The dashed alone redrawn from a. Data shown are the mean of 4- 11 experiments

show that in the presence of BSO, MIS0 doses >0.3 mM readily potentiated the activity of 40H-CY. In addition, similar levels of cell kill could be achieved with -6 times less sensitizer when 40H-CY was combined with MIS0 in the presence of BSO. (Fig. 4)

DISCUSSION GSH has been shown to play a critical role in protecting cellular macromolecules against a wide variety of reactive intermediates (2). In cancer therapy, recent reports have demonstrated the importance of GSH as a determinant of tumor cell sensitivity to both radiation and chemotherapeutic agents (2, 5,7-10). Further, cellular GSH depletion by BSO, has been shown to potentiate radiation and chemotherapy in a number of preclinical model systems (2, 5, 7-10, 13). In the context of chemopotentiation, the interaction between CY and thiols, on its own or in thiol manipulation studies, may be of particular interest. GSH has been shown to react readily with 40H-CY, preventing the release of active alkylating species and thereby leading to loss of cytotoxic efficacy (6). This deactivation of 40HCY by GSH does however lead to cellular GSH depletion (Fig. la). Consequently, in the presence of sufficient anticancer agent, the loss of GSH available for 40H-CY detoxification, ultimately results in increased generation of the alkylating species phosporamide mustard as well

as acrolein. Since acrolein has been shown to be a potent depletor of intra-cellular GSH (5), its generation in turn leads to a further increase in the cytotoxicity of CY (5). Supporting this view, preliminary studies from our laboratories (Siemann, D. W. and Lee, F. Y. F., unpublished data, May, 1988) suggest that doses of 40H-CY which fail to deplete GSH during the treatment period are also non-cytoxic. Preclinical investigations therefore strongly support an essential role for thiols in the anticancer treatment efficacy of CY and suggest GSH depletion to be a determinant of cellular sensitivity to this chemotherapeutic agent. The efficacy of 4OH-CY can be enhanced by combining this drug with thiol depleting agents such as BSO or nitroimidazoles such as MIS0 (Fig. 2). This enhancement is cellular oxygenation state dependent for one of the agents (MISO), but not the other (BSO). Consequently, the combination of a thiol depleting agent, a nitroimidazole and 40H-CY was found to be extremely toxic only to hypoxic tumor cells (Figs. 3d and 4). Since all three agents impact on the GSH redox cycle (MIS0 and 40HCY by conjugation with GSH, BSO by specifically inhibiting GSH biosynthesis), it is conceivable that potentiation of oxidative stress was responsible for the severe cell injury observed when these three agents were combined under hypoxic conditions. At high sensitizer doses (2.5 mM) this injury was manifested by significant cell membrane changes including the development of surface blebs (Fig.

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Fig. 3. Transmission electron micrographs of KHT/iv cells treated in suspension for 4 hr with A: no treatment, B: 40HCY, C: BSO, MIS0 and 40H-CY under aerobic conditions, D: BSO, MIS0 and 40H-CY under anoxic conditions. Exposure times and doses as in text. All micrographs = X7750.

3d). The formation of surface blebs has previously been shown to be a characteristic, early sign of toxicity in hepatocytes under oxidative stress from agents such as tbutyl hydroperoxide ( 14). This agent can cause GSH depletion and NADPH oxidation leading to impairment of calcium sequestering by the mitochondria and increased cytosolic free calcium concentrations ( 14). These results suggest that intracellular calcium may have played a critical role in the acute cell injury observed in the present studies which combined thiol manipulation and chemopotentiation by hypoxic cell radiosensitizers. In summary, the present investigations have shown

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Fig. 4. Survival of KHT/iv cells treated concurrently with a 1 lg/ml dose of 40H-CY and a range of MIS0 doses. Treatments were under anoxic conditions either with (closed symbols) or without (open symbols) prior exposure to BSO (1 mM for 2 hr). Hatched areas show the survival of cells treated with 40H-CY f BSO without sensitizer. Different symbols represent separate experiments.

that, in the presence of BSO, 5-6-fold lower doses of MIS0 are required to potentiate the activity of 40H-CY. However, it should be recognized that a possible mechanism for enhanced normal tissue toxicity observed in vivo with sensitizer-cytotoxic drug combinations is that chemopotentiation may occur at intermediate oxygen tensions ( 12). Consequently, whether an approach which combines a thiol depletor, sensitizer and chemotherapeutic agent will be an effective in vivo therapy will be critically dependent on the extent of normal tissue toxicity enhancements resulting from such combined treatments.

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3. Brown, J. M. The mechanism of cytotoxicity and chemosensitization by misonidazole and other nitroimidazoles. Int. J. Radiat. Oncol. Biol. Phys. 8:675-682; 1982. 4. Bump, E. A.; Yu, N. Y.; Taylor, Y. C.; Brown, J. M.; Travis, E. L.; Boyd, M. R. Radiosensitization and chemosensiti-

Thiol manipulation on chemopotentiation zation by diethylmaleate. In: Nygaard, 0. F., Simic, M. G., eds. Radioprotectors and anticarcinogens. NY: Academic Press; 1983:297-325. 5. Crook, T. R.; Souhami, R. L.; Whyman, G. D.; McLean, A. E. M. Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide. Cancer Res. 465035-5038; 1986. 6. Draeger, U.; Peter, G.; Hohorst, H.-J. Deactivation of cyclophosphamide (NSC-2627 1) metabolites by sulphydryl compounds. Cancer Treat. Rep. 60:355-359; 1976. Lee, F. Y. F.; Vessey, A. R.; Siemann, D. W. Glutathione as a determinant of cellular response to doxorubicin. NC1 Monogr. 6:211-215; 1988. Louie, K. G.; Behrens, B. C.; Kinsella, T. J.; Hamilton, T. C.; Grotzinger, K. R.; McKay, W. M.; Winker, M. A.; Ozols, R. F. Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine. Cancer Res. 45:21 lo-21 15; 1985. Roizin-Towle, L. Selective enhancement of hypoxic cell killing by melphalan via thiol depletion: in vitro studies with

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13. Taylor, Y. C.; Evans, J. W.; Brown, J. M. Mechanism of sensitization of Chinese hamster ovary cells to melphalan by hypoxic treatment with misonidazole. Cancer Res. 43: 3175-3181; 1983. 14. Thor, H.; Hattzell, P.; Orrenius, S. Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca’+. J. Biol. Chem. lo:66 12-66 15; 1984.