Molecular Mechanisms of Selenium-Induced Growth Inhibition

Selenium is a nutritional essential trace element. It is required for the activity of a number of selenoproteins which contain one or more selenocysteine residues that are encoded by in-frame UGA codons. Epidemiological studies also show that there is an inverse correlation between the dietary intake of selenium and the incidence of cancer. In animals, supra-nutritional but sub-toxic dietary levels of selenium have also been shown to have anti-carcinogenic activity in many chemical- and viral-induced tumour models and is effective at inhibiting both initiation and post-initiation (promotion) stages of tumour progression suggesting that selenium may act by inhibiting mutagenic events and also the growth of initiated cells. The anti-carcinogenic efficacy of selenium is also highly dependent on chemical form, suggesting metabolism is important for its chemoprotective effect. Selenium at nanomolar concentrations is also essential for the optimum growth of cells in culture. However, at higher concentrations, selenium is a potent inhibitor of cell growth and it is thought that the growth inhibitory effects of selenium in vitro, may be mechanistically relevant to the post-initiation chemopreventive effects of selenium. The mechanism of selenium-induced growth inhibition has been studied in a mouse mammary cell line, C57. Of a number of seleno-compounds that have varying degrees of chemopreventive activity, only sodium selenite had a significant effect on cell growth and caused cell death after a lag period of 48h. This lag period could be abolished if glutathione (GSH) was added simultaneously with selenite, suggesting that selenite needed to be reduced by GSH to exert its effect. Consistent with this, selenodiglutathione (SDG), the primary metabolite from the reaction of selenite and GSH, was found to be far more cytotoxic than selenite with micromolar concentrations of SDG causing a significant decrease in cloning efficiency within 1h. SDG- and H2O2-induced cytotoxicity were compared since the reaction between selenite and GSH has been shown to generate reactive oxygen species. H2O2 also had a rapid effect and reduced the cloning efficiency of C57 cells within 1h. However, SDG and H2O2 appeared to be inducing cell death by distinct mechanisms as judged by the following observations: (1) SDG was found to reduce the mRNA levels of phospholipid hydroperoxide glutathione peroxidase (PHGPX), glutathione peroxidase (GPX) and glutathione S-transferase Ya subunit (GST Ya) whereas H2O2 had no effect; and (2) SDG induces both 560Kb and 50Kb DNA fragments whereas H2O2 only induces the formation of 560Kb fragments. The cleavage of DNA into high molecular weight fragments has been suggested to be indicative of apoptosis. However, key morphological markers of apoptosis were not observed with treatment of either agent. To further investigate the mechanism of selenium-induced growth inhibition, a genetic approach was adopted. Using a one-step selection strategy, an SDG-resistant cell line (B19) was generated from C57 cells. B19 cells were found to be cross-resistant to selenite but equally sensitive to H2O2 as C57 cells which is consistent with the conclusions that selenite is acting through the production of SDG and that SDG and H2O2 induce cell death by distinct mechanisms. C57 and B19 cells were compared to gain some insight into the possible mechanisms of selenium-resistance. Selenium-uptake and GSH concentrations were eliminated as possible modes of resistance since these two parameters were unchanged in the B19 cells. However, the levels of a number of mRNAs were found to be different between the two cell lines. GST Ya and GST Yc were 2-fold higher in B19 cells whereas PHGPX and Bcl-xs were 2-fold lower in B19 cells. Additionally, the selenium-labelling protein complement of the two cell lines were compared. Both increases and decreases in the labelling or levels of a number of proteins were found to be altered in the B19 cells. The most striking differences were the absence in C57 cells of two 72KDa selenium-labelling proteins.