glutathione depletion and cytotoxicity of buthionine sulphoximine and sr2508 in rodent and human...

hr. J. Radiafion Oncohgy Biol Phys, Vol. 18, pp. 325-330 0360-3016/90 $3.00 + .oO Printed in the U.S.A. All rights reserved Copyright 0 1990 Pergamon Pres plc

??Original Contribution

GLUTATHIONE DEPLETION AND CYTOTOXICITY OF BUTHIONINE SULPHOXIMINE AND SR2508 IN RODENT AND HUMAN CELLS

C. CLIFTON LING, PH.D., ROSEMARY S. L. WONG, PH.D. AND REDEMPTO D. BASAS, B.Sc.

Physics Division and Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of California, San Francisco, CA 94 143

SR2508 (1 mM) increases the rate of glutathione (GSH) depletion by L-buthionine-S-R-sulphoximine (BSO) in hypoxic V79 rodent and A549 human cells. Specifically, the GSH content for V79 and A549 cells, after incubating for about 6 hr with 50 and 100 NM BSO, respectively, was lower by at least IO-fold when 1 mM SR2508 was present. In addition, 1 mM SR2508 is extremely toxic to hypoxic cells with lower GSH content. Survival probabilities of GSH-depleted V79 and A549 cells are about 10m3 after 10 hr incubation with 1 mM SR2508. By itself, 1 mM SR2508 or 50-100 MM BSO decreased cellular viability by about 50% with a 10 hr treatment period. Both the phenomena described above are preferential towards hypoxic cells with minimal effect on aerobic cells.

GSH depletion, SR2508, Cytotoxicity.

INTRODUCTION

There is much interest in the relation between cellular thiol content and cell killing by radiation. Reduction of endogenous sulfhydryl concentration enhances the radiosensitivity of hypoxic cells (2, 3, 4). Under severe depletion, to levels that alter enzyme activity, even the radiation response of euoxic cells is increased ( 12). Recently, Biag- low et al. reported that prolonged exposure to L-buthionine-S-R-sulphoximine (BSO) caused a continuous in- crease in radiosensitivity for aerobic cells (1). The mechanism of radiosensitization by BSO is as yet not fully understood. The ‘competition model’ may be applicable for hypoxic radioresponse, but its validity for aerobic response is doubtful.

Reduction in cellular thiol content, by BSO or by other means, also enhances the radiosensitizing ability of misonidazole, and to a lesser extent, that of SR2508 (6, 7, 11, 14, 16, 17, 18). Koch et al. demonstrated that radiosensitization of V-79 cells by 0.5 mM misonidazole grad- ually increases as endogenous glutathione (GSH) level is lowered (8).

There are reports in the literature concerning the effects of BSO-induced thiol depletion on cellular response to radiation (1, 2, 3, 4). However, there is a paucity of data on the cytotoxicity of BSO. Biaglow et al. reported that 0.1 mM BSO failed to inactivate A549 human cells, even for prolonged incubation of over 4 days (1). A higher BSO

concentration of 2.0 mM killed about 90% of CHO cells in about 24 hr (1). However, there is little or no published data on BSO toxicity for hypoxic cells. Likewise, aside from the work by Hodgkiss and Middleton (7) with misonidazole, data are scarce concerning the influence of GSH depletion on the cytotoxicity of radiosensitizers towards hypoxic cells.

Investigation is needed in this area, because of the po- tential clinical use of BSO in combination with radiosensitizers such as SR2508. The preferential toxicity of BSO, and BSO + SR2508, towards hypoxic cells relative to aerobic cells, may be another advantage of this approach to circumvent the hypoxia-related radioresistance of human tumors. We investigate the cytotoxicity of BSO and SR2508, singly and in combination, in hypoxic V-79 and A549 cells, respectively. Both V-79 and A549 cells are used to assess the influence of different initial endogenous GSH levels on the experimental outcome. This back- ground knowledge may be important for clinical trials using thiol depleting agents together with hypoxic cell sensitizer.

METHODS AND MATERIALS

Many of the experimental procedures have been previously described (9). The following is a brief summary of the methods and materials used.

Reprint requests to: C. C. Ling, Memorial Sloan Kettering Cancer Ctr., York Ave., NY, NY 10021. Acknowledgement-Supported by Grant CA 42044 from the National Cancer Institute, NIH, DHHS.

Accepted for publication 19 July 1989.

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326 I. J. Radiation Oncology 0 Biology 0 Physics February 1990, Volume 18. Number 2

C’c)ll cultwe and survival assay> Chinese hamster V79 cells are cultured in alpha MEM

medium with 10% fetal bovine serum, and antibiotics penicillin and streptomycin. RPMI-1640 medium, sup- plemented with 10% fetal calf serum, antibiotics and L- glutamine, is used for A549 cells. In the experiments, 15 cm* glass flasks, each containing 5 X lo5 cells in a monolayer under 0.1 cm of medium, are sealed with soft rubber stoppers. To manipulate the oxygen content of the cellular environment, stainless steel 19 gauge needles are pushed through the stoppers, through which humidified gas with the desired p02 level is circulated. Evaporation from the cell samples is negligible because the equilibrating gas was fully humidified. After 1 hr of equilibration, the outlet needle is removed, followed by the inlet needle. The efficacy of the procedure to achieve and maintain any desired ~0, condition within the flask, including extreme hypoxia (15 ppm or 0.0015% O2 in the gas phase) has been previously verified in direct comparison with an all glass and metal system (9). A research grade mixture of 95% N2 and 5% CO2 is used to induce extreme hypoxia. An oxygen analyzer is used to verify the O2 content of the effluent gas. Equilibration of the cell samples is carried out at room temperature.

is substituted immediately before gaseous equilibration. Incubation with drug is always carried out at 37°C.

Cellular glutathione is measured by a modification of the Tietze assay (16, 17). Monolayer cultures are washed twice with phosphate buffered saline (PBS) and trypsin- ized. The resulting cell suspension is washed with PBS twice and then extracted with 0.6% sulfosalycilic acid (_ 1 O6 cells/ml). Insoluble material is separated from the acid-soluble material by centrifugation. The supernatant is assayed for GSH by measuring the change in absorbance at 412 nm of the solution containing glutathione reduc- tase, P-nicotinamide adenine dinucleotide phosphate (NADPH), 5,5’-dithio-bis 2-nitrobenzoic acid (DTNB) and an aliquot of the sample solution. The pellet is sol- ubilized in 0.2 N NaOH and assayed for protein (10). GSH is expressed as nmol/mg protein for each sample. Variation between different experiments was less than 2-fold.

RESULTS

Depletion of cellular glutathione under various conditions

Following experimental treatment, single cell suspension is obtained using 0.05% trypsin with ethylene di- aminetetra-acetic acid (EDTA), counted with an electronic cell counter, diluted, and plated into replicate plastic petri dishes. Two or more dilution chains are used for each data point to bracket the anticipated survival level. The dishes are incubated for 7 days in a humidified atmosphere of 95% air + 5% C02. Colonies of greater than 50 cells are then scored.

Figure 1 shows the kinetics of GSH depletion when V79 cells are incubated at 37°C under various conditions. These data are expressed as a fraction of the GSH content of control cells, which is about 20 nmole per mg of protein. (The molar concentration of GSH in V79 cells is about

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Each experiment is performed two or more times. Data points from individual experiments are presented in the figures. The multiple data points for each treatment time are from different cell samples.

Thiol depletion and assay The cellular level of glutathione is lowered by incu-

bating the cells with buthionine sulphoximine BSO, an inhibitor of glutathione synthesis. Incubation is carried out at 37°C. Preliminary experiments are performed with different BSO concentrations to determine the depletion kinetics of GSH. The final series of experiments used 50 and 100 PM BSO for V79 and A549 cells, respectively. These are the minimum concentrations which induce suf- ficient GSH depletion in a reasonable time period.

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In some experiments, the desired combination of chemicals is added to the cell culture, followed immediately by equilibration with the proper gas mixture and incubation at 37°C. In other experiments, cells are first incubated with BSO at 37°C to lower the level of glutathione, then equilibrated with a gaseous mixture of the desired pOZ concentration, and further incubated with chosen concentrations of SR2508 and/or BSO. For these samples, fresh medium with the proper drug concentration

V79 CELL LINE

ld3 II,,,,,,,,,,,,I,,I, 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920

HOURS AT 37°C Fig. 1. Glutathione level as a function of time when V79 cells are incubated at 37°C under various conditions. 50 pM BSO and 1 mM SR2508 are used.

HYPOXIA t SR2608

HYPOXIA t BSO

GSH depletion and SR2508 cytotoxicity 0 C. C. LING ef al. 327

3-4 mM). Hypoxia by itself causes a modest (30-50%) decrease in glutathione level within the first 10 hr (Fig. l), and a further IO-fold reduction in the next 15 hr (data not shown). Aerated control cells do not show any changes within the same time period. The addition of 1 mM SR2508 to hypoxic cells did not alter the kinetics in the initial 10-l 5 hr (Fig. l), but led to a lower level of glutathione between 20-25 hr (data not shown). As shall be addressed in the discussion section, SR2508 is capable of depleting GSH in hypoxia by nitroreduction, but the process is rather inefficient. For this reason, the depletion kinetics of GSH for cells in hypoxia alone are not significantly altered by the presence of SR2508. The choice of the 1 mM SR2508 is based on what can be achieved in the clinic (5, 13).

50 PM BSO reduced the glutathione level of V79 cells to about 10% in 6 hr, for both hypoxic and aerobic cells. Beyond that time, the rate of decrease in GSH level is slower, to about 3-5% of control in lo-12 hr for aerated cells, and to 7-8% for anoxic cells.

50 PM BSO, together ,with 1 mM SR2508, is extremely effective in lowering the GSH content of hypoxic V79 cells, to about 0.5% in 6 hr. In contrast, the same combination and concentrations of chemical is less effective in aerated cells. The depletion kinetics of GSH in aerobic cells treated in this manner appear as if BSO alone is used; the difference in the two (curves (open and closed triangles) reflects variation between different experiments and is not statistically significant.

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V79 CELL LINE

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 IHOURS AT 37°C

Fig. 2. Recovery of glutathione level in V79 cells after BSO is removed, following a 16 hr incubation period with 50 PM BSO to deplete GSH.

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HOURS AT 37°C Fig. 3. Glutathione level as a function of time when A549 human cells are incubated at 37°C under various conditions. 100 PM BSO and 1 mM SR2508 are used.

Results showing the recovery of GSH level following its depletion in V79 cells are shown in Figure 2. For these experiments cells are incubated with 50 PM BSO for 16 hr at 37°C to lower the GSH content to about 3% of that of control samples. Fresh medium without BSO is then substituted, and the cells are rendered hypoxic. Tietze assay is performed on the cells after periods of incubation at 37“C. Figure 2 shows that the GSH level increases rap- idly, after the cells are released from the influence of BSO, by about 1 O-fold in 8- 10 hr. The GSH level reaches 70- 80% of control by lo- 12 hr, the longest time points tested. This phenomenon is observed whether 1 mM SR2508 is used or not.

A549 cells have a relatively high basal GSH level of 150-200 nmole per mg of protein (14). Thus, a higher concentration of BSO and a longer incubation time are necessary to reduce the glutathione content of A549 cells, relative to V79 cells. For example, incubation in 150 PM BSO for 60 hr is needed to decrease the GSH content of A549 cells to 1% of control (data not shown), as compared to incubation in 50 PM for 18 hr for V79 cells. Figure 3 shows the kinetics of GSH depletion in A549 cells by 100 PM BSO under different experimental conditions. By itself, this BSO concentration is relatively ineffective in lowering the glutathione level, whether the A549 cells are aerobic or hypoxic, only resulting in a decrease of 50% in 12 hr. A similar level is achieved in hypoxia, with or without 1 mM SR2508. The combined use of the two agents at the above stated concentrations, however, yields almost

328 1. J. Radiation Oncology 0 Biology 0 Physics February 1990, Volume 18, Number 2

a 1 OO-fold reduction in GSH by 8- 10 hr. This observation is qualitatively the same as that for V79 cells. Again par- alleling the data for V79 cells, GSH depletion is less in aerobic cells than in anoxic cells when BSO is used in combination with SR2508.

Toxicity of BSO and SR2508 in V79 and A549 cells In the data to be presented, we show that the combined

effect of BSO, SR2508 and hypoxia is extremely toxic to rodent and human cells. Figure 4 shows the surviving fractions of hypoxic V-79 cells, incubated in the presence of different combinations of chemical agents and pOz for various times at 37°C. Hypoxia by itself inactivates about 50% of the cells in a 12-hr period. 50 PM BSO or 1 mM SR2508 did not add to the killing effect of hypoxia. How- ever, when hypoxic cells are incubated with the combination of 50 PM BSO and 1 mM SR2508, there is sub- stantial cell inactivation, to a survival level of 1% at 12 hr (0). In the other experiment, the result of which is shown in Figure 4 (V), aerobic V-79 cells are incubated in 50 PM BSO for 14 hr to lower the GSH level, placed in 50 PM BSO and 1 mM SR2508, equilibrated with the desired gas mixture, and further incubated for the selected time intervals at 37°C. These data show that 50 PM BSO in conjunction with 1 mM SR2508 is extremely toxic to GSH-depleted hypoxic cells (V). A 10 hr exposure decreased cellular viability to about 0.1%. Conversely, the viability of aerobic cells is not affected by the same treatment (0).

1

V79 CELL LINE

5 10 15 20

Time (HR)

Fig. 4. Surviving fractions of V79 cells incubated at 37°C for various times, in the presence of various combinations of chemical agents and pOr (see text). The * symbol indicates that, in addition to the presence of BSO during the actual treatment for toxicity assessment, BSO also was used in a 14 hr pre-treatment to lower the GSH level.

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0 N; + 650

n N2 + sa2508 0 N2 + 5a250a + 653 0 0 N2 + SR2508 + 655. \,

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Fig. 5. Surviving fractions of AS49 cells incubated at 37°C for various times, in the presence of various combinations of chemical agents (see text). The * symbol indicates that, in addition to the presence of BSO during the actual treatment for toxicity assessment, BSO also was used in a 14 hr pretreatment to lower the GSH level.

Results from similar experiments using A549 cells are shown in Figure 5. As in the case for V79 cells, hypoxia alone, hypoxia plus BSO (100 PM), or hypoxia plus SR2508 (1 mM) cause marginal decreases in cell survival. Combined treatment of hypoxic cells with 100 PM BSO and 1 mM SR2508 inactivated about 90% of A549 cells in 10 hr (0). This survival level is higher than that for comparable treatment of V79, perhaps reflecting the higher intrinsic GSH level of the human cells. In another set of experiments, A549 cells are first treated with 100 PM BSO for 48 hr, prior to being rendered hypoxic and incubated at 37°C with 100 PM BSO and 1 mM SR2508 (Cl). For these cells, cellular survival at 10 hr is about 0. l%, similar to that observed for V79 cells that underwent a similar treatment protocol.

DISCUSSION

An impetus for manipulating the cellular level of GSH is the hope that the radiosensitivity of hypoxic and aerobic cells can be differentially altered, so as to minimize hypoxia-related tumor resistance in radiotherapy. BSO, an inhibitor of y-glutamylcysteine synthetase, has been shown to be effective in blocking the pathway of GSH production, resulting in a decrease in cellular GSH as it becomes metabolized (1, 3, 4). Most of the above refer- enced studies involve GSH depletion with BSO in aerobic cells, although for the strategy to be effective, such depletion must occur in hypoxic cells. This study indicates that

GSH depletion and SR2508 cytotoxicity 0 C. C. LING et al. 329

BSO is efficacious in lowering GSH level, for rodent and human cells, whether th!e cells are aerated or not. Previ- ously, Hodgkiss and Middleton arrived at similar conclu- sions with V79-543 cells (7).

The simultaneous application of SR2508 and BSO is most effective in depletmg the GSH content of hypoxic cells, but not aerobic cells (Figs. 1 and 3). If this in vitro result is applicable to the in vivo and human situations, and if preferential depletion of GSH in hypoxic cells is therapeutically beneficial, this suggests the combined use of BSO and SR2508.

The reason for the diEFerence in depletion rates of GSH for aerobic and hypoxic cells, when both BSO and SR2508 are present, is not clearly known. Likely, it is related to the metabolic reduction of the nitro group of SR2508, with the associated depletion of the reducing compound glutathione. It has been postulated that this reduction process is inhibited by oxygen because of the reversal of the first stage of nitroreduction, that is, the nitro-anion radical formed in the initial step is oxidized back to the parent compound (15). By itself and in the absence of BSO, this proposed mechanism consumes SR2508 as well as GSH, and is therefore rather inefficient for GSH depletion. However, with the blockage of the pathway for GSH production by BSO, nitroreduction of SR2508 increases the metabolic depletion of glutathione, enhancing the overall result.

The anaerobic metabolism of nitrocompounds, besides depleting hypoxic cells of reducing agents like GSH, also produces toxic species which cause cytotoxicity and the associated pre-incubation effect (2, 3). Lowering the cellular content of GSH significantly enhances this effect for misonidazole (7, 15). The same phenomenon is observed for SR2508 in this study, although under normal GSH conditions, the cytotoxicity is minimal for this drug. This preferential toxicity of S’R2508 towards hypoxic cells under low GSH conditions may be exploited clinically, as discussed below.

Considering the results of the present study, and those of other reports on this subject, one can conclude that SR2508, when used in conjunction with BSO and radiation, contributes to cell inactivation via several pathways. First, SR2508 adds to the efficacy of BSO in depleting GSH of hypoxic cells. Second, SR2508 is extremely toxic to hypoxic cells with low GSH content. Third, SR2508 sensitizes hypoxic cells to radiation as an oxygen mimetic, and the effect is enhanced for GSH-deprived cells. Im- portantly, these actions are preferential towards hypoxic cells, and appear to be true for human and rodent cells. In addition, these phenomena are operative at relatively low BSO and SR2508 levels which may be clinically achievable. Thus, if similar effects occur in in vivo and clinical situations at a similar magnitude, therapeutic benefit may be derived from combined use of these agents, if such toxicity is either absent or less severe in hypoxic normal tissues.

Concerning clinical feasibility, the data in Figures 4 and 5 indicate that at least 4 hr of exposure to 1 mM SR2508 are required to achieve significant killing of hypoxic cells. Based on clinical data involving a 9-dose sched- ule ( 13) the achieveable quotient of dose and time is about 2 mM-hr per treatment, for a SR2508 dose of 1.5 g/m2. Thus, it appears the 4 mM-hr requirement could be easily met. This is predicated on the assumption that the con- comitant use of BSO does not significantly lower the tol- erance for SR2508. Another consideration is that the use of SR2508 may compromise the ability to administer other chemotherapeutic adjuvants.

Another limitation of this approach is that cytotoxicity is probably limited to the hypoxic cells in the tumor, such that little benefit is expected if the hypoxic fraction is small, or if the hypoxic cells do not remain hypoxic for extended periods of time. However, the possibility of gen- erating diffusible metabolites, toxic to adjacent oxygenated tumor cells may expand the impact on the tumor, except that a higher dose-time level may be required.

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Mitchell, J. B.; Russo, A.; Biaglow, J. E.; McPherson, S. Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio. Radiat. Res. 96:422-428; 1983.

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glutathione depletion and cytotoxicity of buthionine sulphoximine and sr2508 in rodent and human...

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