The Nucleolin Targeting Aptamer AS1411 Destabilizes Bcl-2 Messenger RNA in Human Breast Cancer Cells

Abstract

We sought to determine whether nucleolin, a bcl-2 mRNAbinding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 Mmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 Mmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the halflife of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 Mmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358-65]

Kruppel-like Factor 4 Is a Novel Mediator of Kallistatin in Inhibiting Endothelial Inflammation via Increased Endothelial Nitric-oxide Synthase Expression

“…However, it is also possible that KLF4 may be migrated to cell surface under overexpression conditions. Recent studies showed that nucleolin, a nuclear protein, was present on the surface of various cell types, despite lacking a transmembrane domain or a signal sequence (24,25). Therefore, nucleolin acts as a receptor on plasma membrane and shuttles the ligand from the plasma membrane to the cytoplasm and nucleus (24,26).…”

RNA-binding protein nucleolin in disease

“…133,134 AS1411 may directly inhibit the RNA-binding activity of nucleolin, as AS1411 reduced nucleolin binding to BCL2 mRNA resulting in BCL2 mRNA destabilization, decreased Bcl-2 protein levels, apoptotic death of cultured human breast cancer cells, and inhibition of cancer cell growth in xenografts. 135,136 Other effects of AS1411 include the inhibition of NFκB via the formation of a complex containing nucleolin and the NFκB essential modulator (NEMO). 137 Cell-surface nucleolin mediates the effects of this aptamer through binding and internalization leading to inhibition of cell growth.…”

The Requirement of c-Jun N-terminal Kinase 2 in Regulation of Hypoxia-inducing Factor-1α mRNA Stability

“…The role of nucleolin in these pathological processes roots from its function(s) in DNA metabolism, chromatin re-modeling, ribosome biogenesis and mRNA decay (23). Nucleolin has been found to bind to the mRNA of several important genes, including p53 (24), bcl-2 (25), and bcl-xl (26), leading to regulation on mRNA turnover or translation. Moreover, nucleolin has been shown to have binding activity to a number of G-rich oligonucleotides, like the G-quadruplex structure in the promoter region of c-myc gene (27) and vegf gene (28).…”