Background Siah proteins play an important role in cancer progression. of

Background Siah proteins play an important role in cancer progression. of SKBR3 and suppressed Tcf/Lef factor activity. Importantly, Siah1 siRNA demonstrated opposite effects in MCF-7 cells. Siah1 and Siah1L overexpression resulted in inhibition of DNA repair as inferred by increased levels of DNA double-strand breaks in irradiated SKBR3 cells. Conclusion Our results reveal for the first time how overexpression of Siah1L and Siah1 can determine radiosensitivity of breast cancer cells. These findings suggest that development of drugs augmenting Siah1 and Siah1L activity could be a novel approach in improving tumor cell kill. Background Breast Procoxacin cancer is the most common malignancy and the major cause of cancer-related deaths of women in industrialized countries [1]. Radiotherapy consists one of the cornerstones in the treatment of patients with breast cancer and its role has been extensively Sox17 studied during the last decades [2,3]. Clinical studies have demonstrated a major benefit of adjuvant radiotherapy in increasing disease-free survival and overall survival. A profound impact of ionizing radiation in improving local control and reducing disease recurrence has also been shown in patients undergoing breast-conserving therapy [3]. However, radiotherapy is associated with side effects including an increased risk of cardiovascular disease [4]. Finding agents that sensitize malignant cells to radiation would therefore increase tumor response while minimizing toxicity to surrounding organs by lowering effective therapeutic doses. Regulation of protein stability through the ubiquitin-proteasome pathway is now being Procoxacin recognized as a major mechanism of regulating a diverse array of cellular processes [5]. The Drosophila seven in absentia (Sina) protein and its human homolog Siah (seven in absentia homolog) are members of an evolutionarily highly conserved family of E3 ubiquitin ligases [6]. The members of this family (Siah1 and Siah2) contain a N-terminal RING domain that binds E2 proteins, followed by two zinc-finger domains involved in protein-protein interactions Procoxacin [6-8]. Siah ligases regulate the ubiquitination and proteasomal degradation of several proteins including DCC, -catenin, c-Myb, alpha-synuclein, the CDK activator RINGO and BAG-1, a Hsp70/Hsc70-binding protein, suggesting a role for Siah proteins in the regulation of cell proliferation, migration, apoptosis and tumor suppression [7-17]. Recent studies demonstrated that lower expression of Siah2 was associated with resistance to endocrine therapy in breast cancer [18]. Furthermore, Siah2 expression predicated a favourable clinical outcome of breast cancer patients [18,19]. Siah1 expression is upregulated by p53, revealing a link between genotoxic injury and destruction of -catenin [9,10,20] and reduced T-cell factor/lymphoid enhancer factor (Tcf/Lef) activity [11,18]. Furthermore, several Siah1 splicing variants such Siah1L and Siah1 S involved in the degradation of -catenin degradation have been previously described [21,22]. A recent study demonstrated a framework of ATM/ATR and Siah1 through the stabilization of HIPK2, a mediator of DNA-damage induced apoptosis, implicating Siah1 in DNA damage response [22,23]. Although Siah1 presents an attractive target for cancer therapy, its potential radiosensitizing effects have not been previously studied. In search for novel strategies to enhance radiosensitivity of breast cancer, we investigated the role of Siah1 and its related variant Siah1L on the radiation response of SKBR3 and MCF-7 breast cancer cells using different approaches. Furthermore, we analysed the impact of Siah1 overexpression on the biologic behaviour of breast cancer cells by employing invasion and Tcf/Lef reporter studies. Methods Plasmid Construction and Transfection Siah1, Siah-1L and the Siah1 mutant with the RING finger deleted that expresses Siah1R were gifts from Prof. SI Matsuzawa, the Burnham Institute, USA [20]. The Tcf/Lef-responsive luciferase reporter gene (Topflash), the negative control with mutated Tcf/Lef binding site (Fopflash), and the Renilla luciferase reporter plasmid (pRL-TK) as an internal control were obtained from Upstate Biotechnology, USA. The human breast cancer cell lines BT-20, MCF7, MD-MBA231, SKBR3 and ZR75-1 were obtained from the American Type Culture Collection (Manassas, VA). To establish stable cell lines,.

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