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 t umor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arreste d cell proliferation. Purpose: Our aim was to investigate the effect o f a new antiestrogen, toremifene, on human breast cancer cells grown e ither in culture or as tumors in nude mice. Methods: The growth and mo rphology of in vitro cultured cells of the human breast cancer cell li ne MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 h uman breast cancer cells were grown as tumors in nude mice and subsequ ently examined by electron microscopy. The integrity of DNA isolated f rom these cells was determined by standard gel electrophoretic techniq ues. Northern blot hybridization analysis was used to determine the st eady-state levels of the mRNAs for testosterone-repressed prostatic me ssage-2 (TRPM-2), tumor growth factor beta-1 (TGFbeta1), and pS2 (a sm all, cysteine-rich protein of unknown function). Results: Time-lapse v ideo microscopy of the cell cultures indicated that treatment with 7.5 muM toremifene for 3 days caused approximately 60% of the cells to ex hibit morphologic characteristics typical of cells undergoing programm ed death, or apoptosis. The number of mitoses gradually decreased to z ero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mi toses. These changes were not associated with the pattern of DNA fragm entation, detectable as ladders in agarose gels, that is characteristi c 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 ce lls treated with 5-10 muM toremifene. Elevated levels of TRPM-2, but n ot TGFbeta1, mRNA were observed in the tumor cells after estrogen with drawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. Con clusion: Toremifene causes growth inhibition of estrogen-sensitive bre ast cancer cells by inducing some cells to undergo apoptosis and by in hibiting other cells from entering mitosis. The higher than normal amo unts of TRPM-2 and TGFbeta1 protein that would likely result from the elevated levels of TRPM-2 and TGFbeta1 mRNAs measured in these cells a fter toremifene treatment may have an important role in the growth inh ibition process. Implication: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast can cer.