ABSTRACT
This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.
Subject(s)
Breast Neoplasms/genetics , Neoplastic Stem Cells/metabolism , Receptor, Notch1/biosynthesis , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Mice , Receptor, Notch1/genetics , Signal TransductionABSTRACT
RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.
