APOPTOSIS IN TOREMIFENE-INDUCED GROWTH-INHIBITION OF HUMAN BREAST-CANCER CELLS IN-VIVO AND IN-VITRO

Background: Antiestrogens inhibit the stimulative effects of estrogens on breast cancer growth, but the mechanism(s) by which they trigger tumor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arrested cell proliferation. Purpose: Our aim was to investigate the effect of a new antiestrogen, toremifene, on human breast cancer cells grown either in culture or as tumors in nude mice. Methods: The growth and morphology of in vitro cultured cells of the human breast cancer cell line MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 human breast cancer cells were grown as tumors in nude mice and subsequently examined by electron microscopy. The integrity of DNA isolated from these cells was determined by standard gel electrophoretic techniques. Northern blot hybridization analysis was used to determine the steady-state levels of the mRNAs for testosterone-repressed prostatic message-2 (TRPM-2), tumor growth factor beta-1 (TGFbeta1), and pS2 (a small, cysteine-rich protein of unknown function). Results: Time-lapse video microscopy of the cell cultures indicated that treatment with 7.5 muM toremifene for 3 days caused approximately 60% of the cells to exhibit morphologic characteristics typical of cells undergoing programmed death, or apoptosis. The number of mitoses gradually decreased to zero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mitoses. These changes were not associated with the pattern of DNA fragmentation, detectable as ladders in agarose gels, that is characteristic of the DNA of cells undergoing apoptosis. Elevated levels of TRPM-2 and TGFbeta1 mRNAs were observed in in vitro or in vivo grown tumor cells treated with 5-10 muM toremifene. Elevated levels of TRPM-2, but not TGFbeta1, mRNA were observed in the tumor cells after estrogen withdrawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. Conclusion: Toremifene causes growth inhibition of estrogen-sensitive breast cancer cells by inducing some cells to undergo apoptosis and by inhibiting other cells from entering mitosis. The higher than normal amounts of TRPM-2 and TGFbeta1 protein that would likely result from the elevated levels of TRPM-2 and TGFbeta1 mRNAs measured in these cells after toremifene treatment may have an important role in the growth inhibition process. Implication: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.