9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3502-0129 $02.00

Selenite-lnduced Inhibition of Colony Formation by Buthionine Sulfoximine-Sensitive and Resistant Cell Lines PAULA B. CAFFREY AND GERALD D. FRENKEL*

Department of Biological Sciences, Rutgers University, Newark, NJ Received November 20, 1991; Accepted February 14, 1992

ABSTRACT We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must undergo the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite. Index Entries: Selenite; colony formation; glutathione; buthionine sulfoximine; cytotoxicity.

*Author to whom all correspondence and reprint requests should be addressed.

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INTRODUCTION Selenium is known to act as an anticarcinogen, both in intact animals and in cellular systems (1,2). In addition, epidemiological evidence has suggested a correlation between high dietary selenium intake and low cancer risk (3). The chemopreventive action of selenium compounds has been suggested to result from inhibitory effects on carcinogen activity, potentiation of the immune system, and the inhibition of tumor cell proliferation (1). In addition, selenium compounds have been shown to induce a number of cytotoxic effects in tumor cells in vitro (4-6), which could explain the inhibition of tumor development in vivo (although these effects generally occur at somewhat higher concentrations). An important question is whether selenite itself exerts these cytotoxic effects, or whether metabolism is required either to produce an active metabolite (7) or to induce changes in the cellular oxidation state (8,9). Ganther has shown (10) that selenite reacts with sulfhydryl (SH) compounds to form the selenotrisulfides: H2SeO3 + 4 RSH

~ RSSeSR + RSSR + 3 H20

Several lines of evidence have indicated that this reaction is required for selenite cytotoxicity. SH compounds have been shown to enhance the cytotoxic effects of selenite (11,12), and selenotrisulfides have been found to have cytotoxic activity in vivo and in vitro (13,14). Direct evidence has come from several studies demonstrating that, in order for selenite to exert its cytotoxic effects, there must be a sufficiently high level of intracellular SH compounds (15-19). For example, we have shown that treatment of HeLa cells with buthionine sulfoximine (BSO), which results in a significant decrease in the level of intracellular glutathione (the predominant cellular SH compound), causes a decrease in the inhibitory effect of selenite on cell colony formation (19). To investigate further the requirement for metabolism in selenite cytotoxicity, we have examined the relationship between intracellular SH level and selenite cytotoxicity in two additional cell types. One of these (CCL-210) is a line of normal human lung fibroblasts that has been shown to be sensitive to BSO-induced glutathione depletion (20). The second (A549) is a line of human lung adenocarcinoma cells that have been shown to be relatively insensitive to BSO (20). In this article, we report our finding that BSO treatment causes a large decrease in selenite cytotoxicity in the CCL-210 cells, but not in the A549 cells.

MATERIALS AND METHODS Chemicals and Cells Sodium selenite and buthionine sulfoximine (BSO) were obtained from Sigma, St. Louis, MO. Lung adenocarcinoma (A549) and lung Biological Trace Element Research

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fibroblast (CCL-210) cells were obtained from the American Type Culture Collection, Rockville, MD. They were grown in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 with 10% fetal calf serum (non-heat-inactivated) (GIBCO, Grand Island, NY) at 37~ in a 5% CO2 atmosphere.

ColonyAssay Approximately 2 x 105 cells were seeded in 100-ram dishes. After incubation for 2 d, the cells were exposed to selenite as indicated in the individual experiments. Duplicate cultures were exposed to each dose of selenite. Where indicated, BSO (final concentration 1 mM) was added to the culture 24 h prior to the addition of the selenite and removed immediately before the latter was added. After incubation for I h the cells were trypsinized and counted, and either 200 cells (A549) or 400 cells (CCL-210) were seeded in 60-mm dishes for determination of colony formation. At least four replicate colony determinations were carried out for each culture. After incubation for 10 d, the cells were washed with phosphate-buffered saline, fixed with methanol, stained with Giemsa, and the number of macroscopic colonies per dish was counted. For each replicate experiment, the mean n u m b e r of colonies formed by cells in each exposure treatment was calculated as a percent of the mean number of colonies formed by control cells, i.e., in the absence of selenite.

SH Compounds The level of intracellular nonprotein SH compounds was determined by the Ellman reaction, as described previously (13).

RESULTS A N D DISCUSSION We previously demonstrated that in the case of HeLa cells, the ability of cells to form colonies is a sensitive assay of selenium cytotoxicity (19). We have now examined the effect of selenite on another cell type, CCL-210 cells, and have observed a similar degree of sensitivity. As shown in Fig. 1, a 1-h exposure of these cells to selenite resulted in a dose-dependent decrease in colony formation, with 50% inhibition at approx 5 laM selenite. In our previous studies of HeLa cells (19), we also observed that treatment of the cells with BSO resulted in a significant decrease in the level of intracellular SH compounds, as well as in the potency of selenite in inhibiting colony formation. Furthermore, the potency of selenotrisulfides was not decreased by BSO treatment, indicating that the BSO effect on selenite cytotoxicity was the result of the requirement for selenotrisulfide formation. We have now obtained similar results with CCL-210 cells: BSO treatment caused a 24-fold decrease in the level of Biological Trace Element Research

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100

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Fig. 1. Effect of selenite on colony formation by CCL-210 cells. Cells were exposed to the indicated concentrations of selenite for 1 h, after which the number of cells that formed colonies was determined as described in Materials and Methods. The results are presented as a percentage of the number of colonies formed in the absence of selenite. @, Control cells; O, BSO-treated cells.

intracellular SH c o m p o u n d s (Table 1) and also resulted in a decrease in the inhibitory effect of selenite on their ability to form colonies (Fig. 1). Thus, exposure to 5 laM selenite, which resulted in a 50% inhibition in control cells, had no significant effect on colony formation by BSOtreated cells. (BSO itself had no significant effect on colony formation.) Russo et al. (20) have s h o w n that A549 lung adenocarcinoma cells are relatively resistant to BSO compared to CCL-210 cells. As s h o w n in Table 1, BSO treatment results in a 20-fold decrease in the level of SH c o m p o u n d s in CCL-210 cells, but only a twofold decrease in A549 cells. We would anticipate that, because of its relatively small effect on cellular SH level, BSO w o u l d also have a relatively small effect on the cytotoxicity of selenite. As s h o w n in Fig. 2, although selenite inhibited colony formation by A549 cells, as predicted, BSO treatment of the cells did not decrease its potency. These results provide further support for our hypothesis that the reaction of selenite with a cellular SH c o m p o u n d is necessary for its cytotoxicity. It is important to realize, however, that the formation of the Biological Trace Element Research

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Table 1 Effect of BSO on A549 and CCL-210 Cells nmol SH/106 cells - BSO + BSO A549 cells CCL-210 cells A549/CCL-210

31.5 4.8 6.6

Decrease two-fold 24-fold

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Fig. 2. Effect of selenite on colony formation by A549 cells. The experiment was carried out as in Fig. 1. 0 , Control cells; O, BSO-treated cells. selenotrisulfide is only one part of selenite metabolism (10). There has been a great deal of interest in identifying the metabolic species that are critical for the anticarcinogenic activity of selenite. For example, Ganther has shown that methylated selenides are also intermediates in selenite metabolism (7), and Ip and Ganther (21) have recently suggested that some of these c o m p o u n d s may be the active species. Other researchers have suggested that incorporation of selenium into selenoproteins (which occurs via a hydrogen selenide intermediate) is essential for at least some of the anticarcinogenic activities of selenite (22). Although our results demonstrate the requirement for the formation of the selenotrisulfide for selenite cytotoxicity, they do not address the question of Biological Trace Element Research

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whether this metabolite is itself the active species. Further metabolism to other selenium compounds or incorporation into selenoproteins may be required for this cytotoxic activity to occur. Furthermore, our results are also consistent with the concept that it is the intracellular occurrence of these oxidation-reduction reactions, which results in many of the cellular effects of selenite (8,9). The A549 cells normally contain approximately six times as much SH compounds as the CCL-210 cells. However, as a result of their differential sensitivity, after treatment with BSO the A549 cells contain 70 times as much SH compounds as the CCL-210 cells (Table 1). A comparison of the effect of selenite on colony formation demonstrates that without BSO treatment the two cell types exhibited approximately the same sensitivity to selenite (Fig. 3A), but that after BSO treatment the A549 cells were considerably more sensitive than the CCL-210 cells (Fig. 3B). Thus, a sixfold difference in SH content was not sufficient to generate a difference in selenite sensitivity in this assay, whereas a 70-fold difference was. It should be noted that, in another assay of selenite cytotoxicity, inhibition of DNA and RNA synthesis, the A549 cells did exhibit a higher level of sensitivity even without BSO treatment (Abdullaev, MacVicar and Frenkel, Cancer Letters, in press). We are investigating the reason for this difference between the two assays. In any case, it is clear that in colony formation, where there was no difference between the normal and tumor cells in their sensitivity to selenite, BSO treatment in effect induced a differential sensitivity of the tumor cells. With most anticancer agents, a problem arises in chemotherapy in achieving a preferential effect on tumor cells. Without such selectivity, normal and malignant cells could be equally sensitive to the effects of such agents, and toxic side effects would result. This has led to an interest in approaches to achieving differential sensitivity of tumor cells where none exists. In particular, the manipulation of glutathione levels has been considered as a major strategy. Since glutathione has a protective effect against most exogenous agents, its depletion from cells usually tends to increase their sensitivity to cytotoxic drugs (23-26). However, one might expect the opposite to be true for those cytotoxic agents that require glutathione for their activity. In those cases, glutathione depletion would be expected to decrease the sensitivity of the cell to the agent, and hence, preferential depletion of normal cells would create a selective toxicity of those agents for tumor cells. Thus, Russo and coworkers (20) have shown that differential depletion of CCL-210 and A549 cells with BSO results in a selective sensitivity of the tumor cells for the chemotherapeutic agent neocarzinostatin. Our results have shown that BSO treatment of these cells leads to a similar selective sensitivity of the tumor cells to selenite. Because BSO is nontoxic at effective concentrations (relative to other SH depleting agents [27]), it has become a practical tool for enhancing the selectivity of chemotherapeutic agents in animal models (28,29), and its potential clinical applicability has been suggested (30). Biological Trace Element Research

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Fig. 3. Effect of BSO on the inhibition of colony formation by selenite. The data of Figs. 1 and 3 were replotted to facilitate comparison between the cell types. A: Control cells; B: BSO-treated cells. 9 CCL-210 cells; 0 , A549 cells.

Thus, where malignant cells are more resistant to glutathione depletion than surrounding normal cells, BSO treatment may provide a method for selectively sensitizing the tumor to the cytotoxic effects of selenite.

ACKNOWI~D~NTS We thank Drs. F. Abdullaev and L. Yan for helpful discussions and C. MacVicar for excellent technical assistance. This work was supported by grant number ES~04087 from the National Institutes of Health and a grant from the American Institute for Cancer Research. P. C. is a recipient of a Postdoctoral Fellowship from the National Institute of Environmental Health Sciences. This is publication number 100 from the Department of Biological Sciences, Rutgers-Newark. Biological Trace Element Research

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

D. Medina and D. Morrison, Pathol. Immunopathol. Res. 7, 187-199 (1988). L. Vernie, Biochim. Biophys. Acta 738, 203-217 (1984). L. Clark, K. Cantor, and W. Allaway, Arch. Environ. Health 46, 37-42 (1991). R. LeBoeuf, B. Laishes, and W. Hoekstra, Cancer Res. 45, 5496--5504 (1985). D. Medina and C. Oborn, Cancer Lett. 13, 333, 334 (1981). J. Milner and C. Hsu, Cancer Res. 41, 1652-1656 (1981). H. Ganther, J. Am. Coll. Toxicol. 5, 1-5 (1986). W. Rhead and G. Schrauzer, Bioinorganic Chem. 3, 225-242 (1974). G. Schrauzer, in J. Neve and A. Favier, eds., Selenium in Medicine and Biology, De Gruyter, Berlin, 1989, pp. 251-261. H. Ganther, Biochemistry, 7, 2898-2905 (1968). L. Vernie, M. DeVries, L. Karreman, R. Topp, and W. Bont, Biochim. Biophys. Acta 739, 1-7 (1983). L. Vernie, J. Collard, A. Eker, A. DeWildt, and I. Wilders, Biochem. J. 180, 213-218 (1979). G. Frenkel and D. Falvey, Mol. Pharmacol. 34, 573-577 (1988). G. Frenkel and D. Falvey, Biochem. Pharmacol. 38, 2849-2852 (1989). G. Frenkel, A. Walcott, and C. Middleton, Mol. Pharmacol. 31, 112-116 (1987). G. Batist, A. Katki, G. Klecker, and C. Myers, Cancer Res. 46, 5482-5485 (1986). K. Poirier and J. Milner, J. Nutr. 113, 2147-2154 (1983). L. Vernie, C. Homberg, and W. Bont, Cancer Lett. 14, 303-308 (1981). P. Caffrey and G. Frenkel, Mol. Pharmacol. 39, 281-284 (1991). A. Russo, W. DeGraff, N. Friedman, and J. Mitchell, Cancer Res. 46, 28452848 (1986).. C. Ip and H. Ganther, Cancer Res. 50, 1206--1211 (1990). D. Morrison, R. Berdan, D. Pauly, D. Turner, C. Oborn, and D. Medina, Anticancer Res. 8, 51-64 (1988). J. Biaglow, M. Varnes, E. Clark, and E. Epp, in Biochemical Modulation of Anticancer Agents: Experimental and Clinical Approaches, F. Valeriote and L. Baker, eds., Martinus Nijhoff, Boston, MA, 1986, pp. 205-244. C. Wolf, A. Lewis, J. Carmichael, D. Adams, S. Allan, and D. Ansel, Biochem. Soc. Trans. 15, 728-730 (1987). L. Hosking, R. Whelan, S. Shellard, P. Bedford, and B. Hill. Biochem. Pharmacol. 40, 1833-1842 (1990). S. Barranco, K. Weintraub, E. Beasley, V. Jenkins, and C. Townsend, Investigational New Drugs 9, 29-36 (1991). O. Griffith and A. Meister, J. Biol. Chem. 257, 13,704-13,712 (1982). J. Ford, J. Yang, and W. Halt, Cancer Res. 51, 67-72 (1991). R. Ozols, K. Louie, J. Plowman, B. Behrens, R. Fine, D. Dykes, and T. Hamilton, Biochem. Pharmacol. 36, 147-153 (1987). R. Ozols, T. Hamilton, H. Masuda, and R. Young, in Mechanisms of Drug Resistance in Neoplastic Cells, P. Wooley and K. Tew, eds., Academic, San Diego, CA, 1988, pp. 289-306.

Biological Trace Element Research

VoL 35, 1992

Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines.

We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted...
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