Inhibition of Tamoxifen-stimulated increase in intracellular Ca²⁺ and apoptosis by vitamin E in estrogen receptor positive breast cancer cells

Abstract

3712

Background: In addition to their nuclear function, estrogen receptors in the plasma membrane modulate growth and survival signals in breast cancer cells through activation of second messenger pathways. We investigated the activity at the cell membrane of the selective estrogen receptor modulator, Tamoxifen (TAM), in breast cancer cell lines. Methods: The estrogen-receptor positive (ER+) cell lines MCF-7 and T47D, and the ER(-) cell lines MDA-MB-231 and LNCap (prostate) were tested in normal culture medium (10% FCS), steroid-free medium, and medium with 10 microM vitamin E (alpha-tocopherol, AT). Cells were loaded with the calcium-sensitive dye Fluo 4AM. Fluo-4 was excited at 488 nm and emitted fluorescence was filtered with a 535 ± 25 nm bandpass filter and read into a computer running Scanalytics software (Scanalytics Inc., Fairfax, VA). Changes in the intensity of fluorescence (F/Fo) were determined using fluorescence microscopy (Leica, DMIRE2, Plymouth, MN) and plotted against time to evaluate changes in intracellular calcium levels (Ca_i) in the presence of TAM and vitamin E plus TAM. For apoptosis testing, MCF-7 cells were incubated in test media containing TAM and vitamin E plus TAM. Apoptosis was quantified by pan-caspase activity (R&D systems) at 2 and 5 hours. Results: Exposure of TAM (20 microM) stimulated a significant increase in Ca_i in T47D and MCF7 cells. The increase in Ca_i was abolished by pre-incubation with vitamin E (10 microM) and by flowing vitamin E between TAM exposures. The increase in Ca_i was also abolished by removing calcium from the bathing medium and was not reconstituted after depleting intracellular stores with thapsigargin. TAM did not stimulate an increase in Ca_i in ER- negative cell lines MDA-MB-231, LNCap, or MCF-7 cells which were incubated in steroid-free medium for 24 hours. The population of MCF-7 cells with pan-caspase staining at 5 hours decreased from 72% in cells treated with 20 microM TAM alone to 41% in cells treated with TAM plus 10 microM vitamin E. Conclusion: The rapid increase in Ca_i stimulated by TAM in breast cancer cells is dependent on the presence of estrogen receptors. This increase in Ca_i is followed by caspase activation and apoptosis. Vitamin E abolishes the increase in Ca_i and reduces apoptosis in TAM-treated MCF-7 and T47D cells. Block of TAM induced increases in Ca_iby vitamin E may decrease the apoptotic efficacy of TAM in cancer treatment.