Abstract 3642: AS1411 causes a specific increase in levels of cell surface nucleolin in responsive cell lines

Abstract

Abstract AS1411 is a 26-mer guanine-rich oligodeoxynucleotide that has antiproliferative activity against many types of cancer cells, but not against normal cells. It is currently being tested as an anticancer agent in Phase II clinical trials. Previously, we reported that this agent has a novel mechanism of action that involves formation of a stable G-quadruplex structure that can bind as an aptamer to nucleolin, a multifunctional protein expressed at high levels by cancer cells. Nucleolin is a predominantly nuclear protein, but can also be present in the cytoplasm and on the surface of cells, where it has been reported to mediate the endocytosis of several ligands. Cell surface nucleolin is believed to play an important role in AS1411 activity, although the precise details have yet to be elucidated. In the present study, we have evaluated the levels of cell surface nucleolin in human cell lines that are responsive or resistant to AS1411, before and after treatment with AS1411. Levels were examined by flow cytometry, with gating to exclude non-viable permeable (PI-positive) cells, and by western blotting of cell surface proteins, isolated using a cell-impermeable biotinylating agent. The amount of cell surface nucleolin appeared to be similar in untreated cells that were either sensitive to AS1411 (DU145 prostate cancer cells and MV-4-11 leukemia cells) or resistant to AS1411 (Hs27 non-malignant skin fibroblasts). However, in the sensitive cell lines, we observed a dramatic and time-dependent increase in levels of cell surface nucleolin in response to AS1411 treatment, whereas there was no significant change in the resistant Hs27 cells. We determined that this AS1411-induced increase in cell surface nucleolin in the cancer cells was not simply a reflection of apoptosis because it was not observed when apoptosis was induced by UV irradiation. We did not observe any changes in the levels of nucleolin in nuclear, cytoplasmic, or membrane extracts, or in total cell lysates, following treatment with AS1411, suggesting a specific re-localization of nucleolin rather than an increase in overall levels. These new results do not support our previous hypothesis that baseline levels of cell surface nucleolin determine responsiveness to AS1411. However, they do clearly demonstrate that “externalization” of nucleolin is a specific response to AS1411 and thereby confirm the important role of cell surface nucleolin in the mechanism of action of AS1411. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3642.