Paula B. Caffrey

Sensitivity of melphalan-resistant tumors to selenite in vivo.

Previous studies have demonstrated that melphalan-resistant human ovarian tumor cells exhibit a higher degree of sensitivity to the cytotoxic effects of selenite in vitro than comparable drug-sensitive cells (P.B. Caffrey, G.D. Frenkel, Selenite cytotoxicity in drug resistant and non-resistant human ovarian tumor cells, Cancer Res. 52 (1992) 4812-4816; P.B. Caffrey, G.D. Frenkel, The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite, Cancer Lett. 81 (1994) 59-65). We have now examined the sensitivity of drug-resistant tumors to selenite in vivo. A2780 human ovarian tumor cells, or their melphalan-resistant derivative (A2780ME) cells were injected subcutaneously into nude mice and the resulting tumors were found to be melphalan-sensitive and -resistant, respectively, in vivo. Treatment with selenite (2 mg/kg Se s.c.), which had no overt toxic effect on the animals, resulted in a significant decrease in the rate of growth of the melphalan-resistant tumors, but not on the rate of growth of the drug-sensitive tumors. Thus, melphalan-resistant ovarian tumors are also more sensitive to selenite treatment in vivo.

A Prevention Strategy for Circumventing Drug Resistance in Cancer Chemotherapy

The development of drug resistance is considered to be a major cause for the failure of chemotherapy in a number of types of cancer, including ovarian, breast and lung. Most previous research has focused on approaches to reverse drug resistance once it has arisen, that is, on the use of agents which can make drug-resistant tumors more sensitive to chemotherapy. Unfortunately, this approach has thus far met with only limited clinical success. Because of the prevalence of drug resistance in cases of advanced cancer, there exists an urgent need to develop new approaches to dealing with this problem. We have hypothesized the feasibility of an alternative approach: the use of specific agents to prevent the development of resistance before it arises. Our initial studies to examine this hypothesis have focused on ovarian cancer. We have designed both in vitro and in vivo systems in which resistance develops rapidly after exposure of tumor cells or xenografts to melphalan or cisplatin. Using these systems we have shown that two selenium compounds, selenite and selenomethionine are able to prevent the induction of resistance. Furthermore, inclusion of selenite in a chemotherapeutic protocol can result in a significant enhancement of the efficacy of cisplatin in suppressing the growth of human ovarian tumor xenografts. These results have supported the idea that prevention may be a useful new approach to the problem of drug resistance in cancer chemotherapy.

The development of drug resistance by tumor cells in vitro is accompanied by the development of sensitivity to selenite

The effects of selenite on cell viability and proliferation in a line of drug-sensitive human ovarian tumor (A2780) cells were compared with its effects on a melphalan-resistant derivative of these cells (A2780-ME) which had been developed in vitro (Hamilton et al. (1985) Biochemical Pharmacol., 34, 2583-2586). With the A2780-ME cells there was a 50% decrease in the number of viable cells (i.e. which exclude Trypan Blue dye) after exposure to less than 100 microM selenite for 6 h. In contrast, exposure to more than 300 microM selenite was required to achieve the same effect in the parent line. Similarly, exposure to 10 microM selenite resulted in a 50% decrease in A2780-ME cell proliferation, whereas this treatment had only a small inhibitory effect on proliferation of the parent cells. Thus, the development of melphalan resistance in vitro was accompanied by the development of selenite sensitivity. Pre-exposure of the two cell types to buthionine sulfoximine eliminated the difference in their intracellular glutathione levels, as well as most of their differential sensitivity to selenite. Furthermore, the two cell types did not exhibit a difference in sensitivity to selenodiglutathione, the product of the reaction of selenite with glutathione. Thus, the increase in intracellular glutathione, which has been shown to be responsible for the development of drug resistance in these cells is also responsible for the development of selenite sensitivity.

Rapid development of glutathione-S-transferase-dependent drug resistance in vitro and its prevention by ethacrynic acid

Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 days resulted in the development of melphalan resistance. This resistance was not a stable characteristic of the cells since it was lost after 2 weeks in culture in the absence of drug. The melphalan-resistant cells exhibited significant cross-resistance to cisplatin but only minor cross-resistance to doxorubicin. The resistant cells had elevated levels of glutathione-S-transferase activity and mRNA. Exposure of the cells to the ethacrynic acid resulted in a decrease in enzyme activity as well as a reversal of their drug-resistant phenotype, indicating that the enzyme is involved in the resistance. When ethacrynic acid was present during the 7-day exposure of the cells to melphalan, the development of drug resistance was prevented. This system may serve as a useful preliminary step in screening for agents which can prevent the development of chemotherapy-induced drug resistance in human cancer.

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 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 under-go 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.

PREVENTION OF THE DEVELOPMENT OF MELPHALAN RESISTANCE IN VITRO BY SELENITE

Exposure of A2780 human ovarian tumor cells to a low concentration of melphalan in vitro for 7 d results in the development of melphalan resistance, which is dependent on elevated cellular levels of glutathione and glutathioneS-transferase. The inclusion of selenite (at concentrations as low as 0.2 ΜM) during the exposure to melphalan completely prevented the development of resistance. Selenite did not prevent the melphalan-induced increase in glutathione, but it did prevent the increase in the activity of glutathioneS-transferase. It also prevented the increase in the expression of the glutathioneS-transferase gene, suggesting that this may be the mechanism by which it prevents the development of melphalan resistance. The results of this in vitro study suggest that selenite may prove to be useful in preventing the development of drug resistance in vivo.

Phase I trial of selenium plus chemotherapy in gynecologic cancers

PURPOSE Preclinical studies performed in our laboratory have shown that high-dose selenium inhibits the development of carboplatin drug resistance in an ovarian cancer mouse xenograft model. Based on these data, as well as the potential serious toxicities of supranutritional doses of selenium, a phase I trial of a combination of selenium/carboplatin/paclitaxel was designed to determine the maximum tolerated dose, safety, and effects of selenium on carboplatin pharmacokinetics in the treatment of chemo-naive women with gynecologic cancers. Correlative studies were performed to identify gene targets of selenium. METHODS Chemo-naïve patients with gynecologic malignancy received selenious acid IV on day 1 followed by carboplatin IV and paclitaxel IV on day 3. A standard 3 + 3 dose-escalating design was used for addition of selenium to standard dose chemotherapy. Concentrations of selenium in plasma and carboplatin in plasma ultrafiltrate were analyzed. RESULTS Forty-five patients were enrolled and 291 treatment cycles were administered. Selenium was administered as selenious acid to 9 cohorts of patients with selenium doses ranging from 50 μg to 5000 μg. Grade 3/4 toxicities included neutropenia (66.7%), febrile neutropenia (2.2%), pain (20.0%), infection (13.3%), neurologic (11.1%), and pulmonary adverse effects (11.1%). The maximum tolerated dose of selenium was not reached. Selenium had no effect on carboplatin pharmacokinetics. Correlative studies showed post-treatment downregulation of RAD51AP1, a protein involved in DNA repair, in both cancer cell lines and patient tumors. CONCLUSION Overall, the addition of selenium to carboplatin/paclitaxel chemotherapy is safe and well tolerated, and does not alter carboplatin pharmacokinetics. A 5000 μg dose of elemental selenium as selenious acid is suggested as the dose to be evaluated in a phase II trial.