Abbreviations: DFO, desferrioxamine; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; GAS, interferon-γ activated site; HIF-1, hypoxia-inducible factor-1; PRL, prolactin; ROS, reactive oxygen species; STAT, signal transducers and activators of transcription

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Abstract. Cyclin D1 and insulin-like growth factor 1 receptor (IGF-1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. The purpose of the present study was to investigate the molecular mechanism by which hemin exerts its inhibitory effects on aggressive breast cancer cells. We found that hemin regulates cyclin D1 and IGF-1R proteins and insulin-like growth factor-1 gene expression through STAT5b in breast cancer cells. We confirmed that STAT5b, cyclin D1, and IGF-1R is up-regulated by hypoxia, and the increased STAT5b binds strongly to the STAT5-binding sites contained within the distal 5'-flanking region of IGF-1 gene in breast cancer cells. EMSA studies showed that STAT5 binding activity to the IGF-1 and cyclin D1 promoter was distinctly decreased by hemin in STAT5b-transfected COS-7 or MDA-MB 231 cells. IGF-1 gene expression was also decreased by hemin in mammary epithelial cells. STAT5b expression was inhibited in siRNA experiments and by hemin, leading to decreased levels of IGF-1. These results provide a basis for molecular targets in cancer treatment via the STAT5b/IGF-1 or /cyclin D1 pathway in solid tumor cells.These data indicate that hemin inhibits the cyclin D1 and IGF-1 expression via STAT5b under hypoxia in ER·-negative breast cancer cells. These findings are valuable toward understanding the role of hemin-induced inhibition of cyclin D1 and IGF-1 expression under hypoxia in invasive and metastatic breast cancer. IntroductionHeme (ferroprotoporphyrin IX) is a prosthetic group found in a relatively large number of cellular hemoproteins (hemecontaining proteins) that carry out diverse biological functions. In its oxidized state, when it is known as hemin (ferriprotoporphrin IX), heme is bound less tightly to hemoglobin, and interacts more readily with alternative acceptor proteins and the lipid membranes of cells. Hemin (ferric chloride heme) is, as a natural agent, an essential growth promoter of early hematopoietic progenitors (BFU-Es, CFU-Es), and a potent inducer of globin gene activation. Hemin has therapeutic potential as a natural regulator of hematopoiesis, as well as an agent that protects hematopoietic cells from cytotoxic treatment (drugs, hypoxia, or other stimuli), for treatment of hematological and even neurodegenerative disorders (1).Insulin-like growth factors (IGFs), including IGF-I and IGF-II, are a family of polypeptide growth factors structurally related to proinsulin (2). IGF-1 mediates many of the growthpromoting effects of growth hormones during post-natal life (3,4). Interest in the role of the IGF axis in growth control and carcinogenesis has increased because elevated serum IGF-I levels are associated with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer (5,6). In 1998, Physicians' Health Study researchers reported that men with high IGF-1 blood levels run a greater risk of prostate cancer. Similar results for breast cancer in premenopausal women in the Nurses' Health Study w...