The TSP-1Cspecific antibody prevented this proliferation, indicating that the effect can be attributed to the induction of TSP-1 by E2. Open in a separate window Figure 4 TSP-1-induced proliferation of breast cancer cells(A) MCF-7 cells were cultured in DME/F12 with 5% DCC serum. estrogen-responsive region in the human TSP-1 promoter, located between -2200 and -1792 bp upstream of the transcription start site. An antibody against TSP-1 restricted the proliferation of E2-dependent MCF-7 cells in vitro and in vivo. A panel of breast malignancy cells proliferated in the presence of low concentrations of exogenous TSP-1, whereas higher concentrations inhibited proliferation. A real-time PCR analysis showed that E2 also induced TSP-1 mRNA in the normal mammary glands of immature ovariectomized mice in an ER-dependent manner. In summary, we report the novel observation that TSP-1 production is usually directly controlled by estrogens in ER-positive breast malignancy cells, and the released protein has pro-growth regulatory functions. Consequently, we propose that TSP-1 could be a therapeutic target Bitopertin for anti-tumor therapy in early-stage tumors. anti-angiogenic properties of TSP-1 have been exhibited primarily in animal model systems under nonphysiological conditions, for example in tumor-prone TSP-1 knockout mice (10) and in TSP-1 transgenic animals that overexpress TSP-1 from the MMTV promoter and have reduced tumor burden (11). In contrast, when synthesis of endogenously produced TSP-1 is usually blocked, progression of breast tumors is usually reduced (4), a finding that is usually more consistent with a pro-angiogenic, or proliferative, rather than anti-angiogenic role. TSP-1 is present at a higher level in malignant and invasive human breast tumors than in noninvasive tumors or normal human breast tissue (12-14), supporting the notion that TSP-1 is in fact pro-angiogenic or pro-proliferative in its effect on breast tumor development. Other studies have shown pro-angiogenic effects of TSP-1 that are dose-dependent, including an Bitopertin ability to stimulate cell survival and endothelial cell migration (9, 16, 17). As is becoming increasingly apparent, the role of TSP-1 is usually complex and likely to vary depending on cellular context, cell type, hormonal milieu, and TSP-1 receptorCdependent signaling (2, 9, 16, 17). Furthermore, individual regions of the TSP-1 protein may independently stimulate or inhibit angiogenesis (2, 18). This raises the interesting possibility that breast tumors may produce a pro-angiogenic environment in which the estradiol (E2)-induced TSP-1 protein either interacts with other proteins or that TSP-1 is usually cleaved generating angiogenic regions, leading ultimately to tumor cell proliferation and tumor progression. Many breast cancer cells express steroid hormone Rabbit polyclonal to LDH-B receptors (19), including estrogen receptor (ER) and (E2) affects the proliferation of many hormone receptorCpositive breast cancer cells. E2 can function via both ER-alpha and Bitopertin ER-beta, the two isoforms known for ER, though most proliferative functions of E2 are associated with ER-alpha and ER-beta is usually thought to prevent such proliferation (20). We recently observed that E2 downregulates CD36, a TSP-1 receptor in human breast epithelial cells (21). Here we sought to determine whether TSP-1 expression is usually regulated by E2 in such cells, and found that E2 directly controls TSP-1 production in human breast malignancy cells via the alpha type of ER (ER-alpha). Furthermore, Bitopertin E2-induced TSP-1 stimulated breast tumor cell proliferation, both in vitro and in vivo in nude mice. Materials and Methods Cell Culture, Cell Treatments, and RNA collection Cells of the T47-D, MCF-7, and MDA-MB-231 breast malignancy cell lines (ATCC, Manassas, VA) were produced in phenol redCfree DMEM/F12 medium (Invitrogen Corporation & Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS). When the cells were 60%-70% confluent, they were washed once with PBS and incubated for 18-24 h in 5 mL phenol redCfree DMEM/F12 medium supplemented.