1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and its synthetic analog EB1089 induce characteristic morphological features of apoptosis in MCF-7 cells in vitro that coincide with up-regulation of clusterin and cathepsin B, proteins associated with apoptosis in the mammary gland, and with down-regulation of Bcl-2, an antiapoptotic protein. To determine whether vitamin D3 compounds could mediate apoptosis of breast tumors in vivo, we treated nude mice carrying established MCF-7 xenografts with the low calcemic vitamin D3 analog EB1089 via daily injection or sustained release pellets for up to 5 weeks. The volume of tumors from mice treated with 45 pmol/day EB1089 was 4-fold lower than that of tumors from vehicle-treated control mice after 5 weeks. The reduced growth of tumors from EB1089-treated mice was associated with characteristic apoptotic morphology and a marked reduction in the proportion of epithelial cells to stroma. After 5 weeks of treatment with EB1089, MCF-7 tumors exhibited a 6-fold increase in DNA fragmentation (as measured by in situ end labeling) relative to that in control tumors. The enhanced rate of apoptosis in tumors from EB1089-treated mice was coupled to a 2-fold reduction in proliferation (as measured by expression of proliferating cell nuclear antigen) compared with that in tumors from control mice. The antitumor effects of EB1089 were evident at doses that had minimal effects on serum calcium and body weight. EB1089 treatment did not alter the growth of xenografts derived from a vitamin D3-resistant variant of MCF-7 cells (MCF-7(D3Res) cells), which display resistance to EB1089 in vitro, indicating that resistance to EB1089 is maintained in vivo. Tumors derived from both MCF-7 and MCF-7(D3Res) cells underwent apoptotic regression upon estradiol withdrawal, indicating comparable estrogen dependence of tumors with differential sensitivity to vitamin D3 compounds. These are the first studies to demonstrate apoptotic morphology and regression of human breast tumors in response to treatment with a vitamin D3 analog in vivo and support the concept that vitamin D3 compounds can effectively target human breast cancer.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active metabolite of vitamin D3, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have previously demonstrated that 1,25-(OH)2D3 induces morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, and nuclear matrix protein reorganization) consistent with the activation of apoptosis in MCF-7 cells. These morphological changes in 1,25-(OH)2D3-treated cells are associated with up-regulation of TRPM-2/clusterin and cathepsin B (genes associated with mammary gland apoptosis) and down-regulation of bcl-2, an antiapoptotic gene. Thus, the inhibitory effects of 1,25-(OH)2D3 on MCF-7 cell growth involve activation of apoptosis. To investigate the mechanisms by which vitamin D3 activates apoptosis, we have selected a vitamin D3-resistant variant (MCF-7D3Res cells) by continuous culture of MCF-7 cells in 100 nM 1,25-(OH)2D3. The MCF-7D3Res cells represent a stably selected phenotype that grows equally well with or without 100 nM 1,25-(OH)2D3. In contrast to the MCF-7 cells from which they were derived (MCF-7WT cells), MCF-7D3Res cells do not exhibit apoptotic morphology, DNA fragmentation, or up-regulation of apoptosis-related proteins after treatment with 1,25-(OH)2D3. MCF-7D3Res cells exhibit cross-resistance to several vitamin D3 analogs that are potent growth regulators of MCF-7WT cells. MCF-7WT and MCF-7D3Res cells exhibit comparable sensitivity to induction of apoptosis and up-regulation of clusterin in response to the antiestrogen 4-hydroxytamoxifen. MCF-7D3Res cells express comparable levels of the vitamin D receptor (VDR), as assessed by Western blotting or ligand binding, as MCF-7WT cells. In both sensitive and resistant cell lines, 1,25-(OH)2D3 up-regulates whereas 4-hydroxytamoxifen down-regulates VDR protein expression, indicating appropriate homologous and heterologous VDR regulation in MCF-7D3Ras cells. Gel shift analyses indicate that nuclear extracts from MCF-7WT and MCF-7D3Res cells bind equally well to the DR3 consensus vitamin D3 response element. These data suggest that MCF-7D3Res cells have a functional VDR that is uncoupled from a functional apoptotic pathway. MCF-7D3Res cells offer a unique model system for identification of the mechanisms by which vitamin D3 regulates the cell death pathway in breast cancer cells.

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Our studies have identified 1,25(OH)2D3 as a coordinate regulator of proliferation and apoptosis in breast cancer cells. In MCF-7 cells, 1,25(OH)2D3 down regulates the estrogen receptor (ER), suggesting that the effects of 1,25(OH)2D3 may be linked to disruption of estrogen regulated survival signals. Although studies have demonstrated that 1,25(OH)2D3 inhibits growth of ER negative breast cancer cells, previous data were generated by comparison of cell lines derived from heterogeneous human tumors and harboring diverse genetic alterations. To provide more conclusive evidence for independent growth regulatory pathways mediated by antiestrogens and 1,25(OH)2D3, we examined vitamin D3 sensitivity in MCf-7 cells selected for resistance to ICI 182, 780 (Zeneca, Macclesfield, UK). The clones we selected for resistance to ICI 182,780 retain functional VDR and undergo 1,25(OH)2D3 mediated growth arrest and apoptosis, in vitro and in vivo, despite loss of estrogen dependence. Cell cycle data indicate that treatment of parental or anti-estrogen resistant MCF-7 clones with 1,25(OH)2D3, in the presence or absence of ICI 182,780, increases the percentage of cells in G0/G1 while reducing the number of cells in S phase. In addition, 1,25(OH)2D3 induces characteristic features of apoptosis, including DNA fragmentation, in both parental and anti-estrogen resistant MCF-7 cells. Furthermore, we report that cells selected for vitamin D3 resistance retain sensitivity to ICI 182,780 mediated growth arrest and apoptosis. This work emphasizes that vitamin D3 compounds and anti-estrogens trigger growth arrest and apoptosis in breast cancer cells by distinct mechanisms, and that breast cancer cell sensitivity to 1,25(OH)2D3 is not diminished during the progression to estrogen independence.