Inhibitors of protein and RNA synthesis block the cytotoxic effects of non-steroidal antiestrogens

Abstract

Non-steroidal antiestrogens such as tamoxifen are known to exert cytotoxic effects against various cell lines in culture. When the antiestrogens are present at sufficiently high concentrations, their cytotoxicity cannot be reversed by estrogens and is demonstrable even with cell lines which lack the estrogen receptor. The mechanism of this cytotoxicity, which is clearly independent of estrogen antagonism, remains unknown. Using two murine cancer cell lines (the K36 leukemia and the EL4 lymphoma cell line), the human breast cancer cell line MCF7, and two non-steroidal antiestrogens (tamoxifen and clomiphene), our laboratory attempted to determine whether the cytotoxic action of non-steroidal antiestrogens was mediated by a mechanism requiring protein or RNA synthesis. In the case of K36 and EL4 cells, inclusion of tamoxifen or clomiphene in the culture medium regularly caused the viable call count to fall below 20-30% of control in 36-48 h. Under these conditions, the addition of inhibitors of protein or RNA synthesis consistently increased viable cell count in a dose-dependent manner. With cultures of K36 cells grown in the presence of 10 μM tamoxifen, for example, the addition of appropriate concentrations of emetine, cycloheximide, puromycin, or actinomycin D increased the percentage of viable cells to 5.0, 2.4, 4.0, and 4.0 times that of control, respectively. Additional experiments revealed that the macromolecular synthesis inhibitors, while effective in inhibiting protein or RNA synthesis to varying degrees, did not affect the cellular uptake of [ 3H]tamoxifen, suggesting that their ability to protect cells against antiestrogen-induced cell death was not due to an inhibition of cellular uptake of antiestrogens. In the case of MCF7 cells, however, inhibition of protein synthesis did not protect the cells against the cytotoxic effect of tamoxifen. These observations suggest that non-steroidal antiestrogens may exert their cytotoxic effect by at least two different mechanisms; only one of these require de novo protein synthesis. The effect of antiestrogens on K36 and EL4 cells may provide a useful system for the identification of proteins involved in cell death.