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Modulating effects of zinc on the efficacy of tamoxifen in human breast cancer cells Tsukada, Yoko Ann

Abstract

Growth can be viewed as the net balance between cell proliferation and cell death. Zinc is essential to cell proliferation and functions as a regulator in apoptosis (genedirected cell death). Thus zinc status can critically influence overall growth. We hypothesized that modulating medium zinc concentration will alter the availability of metabolically active intracellular zinc, which in turn affects the overall growth of human cancer cells. The overall objective of my thesis project was to investigate the modulating effects of zinc on the efficacy of tamoxifen in human breast cancer cells. The cells were first cultured in DMEM (Dublecco's Modified Eagle Media; 10% fetal bovine serum (FBS)) until approximately 70% confluence. Then the cells were cultured in a low zinc medium supplemented with 0, 5, 50, or 150 μmol/L zinc for 72h. Upon zinc treatment, the cells were treated with a combination of zinc and tamoxifen (0, 1, 5, or 10 μmol /L) for 2, 24, or 48 h followed by a 24 h recovery period. At the end of the recovery period, cells were assessed for overall cell growth by counting cell numbers, cell viability by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, and the profile of live cell and cell death (both necrotic and apoptotic cell death) using flow cytometry. To explore the possible mechanisms involved, the mRNA levels of targeted apoptotic regulatory proteins (p53, gadd45, Bax, and Bcl-2) were determined in human breast cancer MDA-MB-231 cells using reverse transcription-polymerase chain reaction. Either zinc supplementation (e.g. 150 μmol/L) or tamoxifen alone suppressed overall cell growth, but had little effect on cell survival and cell death. In contrast, a combination of zinc and tamoxifen suppressed overall cell growth, reduced cell viability and cell survival, and increased both necrotic and apoptotic cell death. In addition, a combination of zinc and tamoxifen also elevated Bax and gadd45 mRNA levels in human breast cancer MDA-MB-231 cells. Since Bax is a well-known pro-apoptotic protein and gadd45 is involved in both negative growth control and the induction of apoptosis, zinc and tamoxifen-induced apoptosis in the above cell line appeared to be Bax- and gadd45-dependent. Overall, these observations suggested that zinc increased the efficacy of tamoxifen as indicated by decreased overall cell growth in breast cancer cells.

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