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Human ovarian tumors (derived from A2780 cells), growing as subcutaneous xenografts in immunodeficient mice, develop melphalan-resistant cells after a single treatment of the tumor-bearing animal with the drug [Caffrey, Zhang and Frenkel, submitted for publication]. Treatment of the animals with selenite by i.p. injection prevented the development of primary resistance to melphalan as well as cross-resistance to cisplatin. Selenite treatment also prevented the melphalan-induced increase in the cellular level of glutathione. In contrast, selenite administered s.c. or in drinking water had relatively little effect on the development of resistance. The results in this animal model suggest that selenite may be clinically useful in preventing the development of drug resistance during chemotherapy of cancer.

Human ovarian tumors from A2780 cells were grown as xenografts in immunodeficient mice, treated with a single i.p. dose of melphalan and tumor cells were removed and placed into tissue culture. The cells from the treated tumors exhibited an approximately 2-fold resistance to melphalan in vitro compared to cells taken from untreated tumors. This degree of resistance was similar to that of cells from tumors formed from melphalan-resistant A2780-ME cells. The cells from the treated tumors were also resistant to cisplatin but not to doxorubicin. They contained approximately 2-fold higher levels of glutathione than cells from the untreated tumors. Exposure of the cells to buthionine sulfoximine (a specific inhibitor of glutathione biosynthesis) eliminated the difference in glutathione levels as well as the difference in sensitivity to melphalan. When tumor-bearing animals were treated with buthionine sulfoximine in addition to melphalan the resulting tumor cells were not resistant to the drug. Resistance could also be demonstrated in the tumors themselves in vivo: the growth of previously untreated tumors was severely inhibited by a high dose of melphalan (11.7 mg/kg) administered i.p. to the animals, whereas the growth of tumors which had received prior treatment with melphalan was unaffected by the subsequent high dose. The rapid development of drug-resistant tumor cells after a single drug treatment in vivo makes this an excellent system for the investigation of the mechanisms by which resistance develops as well as for use in the screening for agents which can prevent it.

The development of tumor cell drug resistance is considered a major cause for the failure of cancer therapy. In many cases drug resistant tumor cells have been shown to contain high levels of glutathione (GSH). Selenium compounds are known to act as anti-cancer agents and compounds such as selenite have been shown to induce cytotoxic effects in tumor cells. The available evidence suggests that for selenite, in contrast to most xenobiotics, a high level of intracellular GSH is correlated with a high degree of cytotoxicity. Based upon these observations, we hypothesized that tumor cells with high GSH levels, which are resistant to chemotherapeutic agents, will prove to be extremely sensitive to selenite. We examined this hypothesis in vitro by comparing the effects of selenite on drug resistant human ovarian tumor (NIH:OVCAR-3) cells, which have high GSH levels, and non-resistant human ovarian tumor (A2780) cells with low GSH levels. We employed three assays which measure different aspects of cytotoxicity in vitro: cell viability (ability of cells to exclude trypan blue), proliferation of viable cells, and attachment of cells to a solid matrix. In each of these assays the drug resistant cells proved to be significantly more sensitive to selenite than the drug sensitive cells. This study represents the first step in the evaluation of selenite as a treatment for drug-resistant tumors.

Our previous studies on selenite cytotoxicity led us to hypothesize that drug resistant tumor cells with high intracellular glutathione will exhibit a high degree of sensitivity to selenite. To examine this we studied the effects of selenite on drug resistant human ovarian tumor (NIH:OVCAR-3) cells in three assays of cytotoxicity: proliferation; cell viability (trypan blue exclusion); and attachment to a solid matrix. The cells were sensitive to low levels of selenite: concentrations as low as 5 microM inhibited cell proliferation and attachment; and viability was decreased by concentrations as low as 20 microM. In each of these assays the NIH:OVCAR-3 cells were more sensitive to selenite than were drug sensitive human ovarian tumor (A2780) cells. These results suggest the potential for the utilization of selenite in the treatment of some drug resistant tumors.

Selenium is an essential trace element that has been shown to have anticarcinogenic activity. One mechanism that has been proposed for this activity is a cytotoxic effect of selenium on tumor cells. As a means of assessing its cytotoxicity, we have examined the effect of selenite on tumor cell viability, using as an assay the ability of the cells to form colonies. We have found that brief exposure of HeLa cells to micromolar concentrations of selenite resulted in significant inhibition of colony formation, indicating that this is an assay for selenite cytotoxicity that is more sensitive than those that have been employed previously. In order to investigate the involvement of cellular glutathione in selenite cytotoxicity, we treated cells with buthionine sulfoximine (BSO) before selenite exposure. This treatment, which resulted in a 7-fold reduction in the level of intracellular glutathione, also caused a significant decrease in the inhibitory effect of selenite on colony formation. However, when cells were exposed to selenite that had previously been reacted with glutathione, the BSO-induced decrease in cytotoxicity was eliminated. In contrast, reaction of selenite with other sulfhydryl compounds, such as cysteine and mercaptoethylamine, did not restore its potency in BSO-treated cells. The simplest explanation for these results is that, for selenite to exert its inhibitory effect, it must react with intracellular glutathione to form the selenodiglutathione derivative.