Abstract 4450: A new paradigm for AS1411 activity: Uptake by macropinocytosis and induction of macropinocytosis by a nucleolin-dependent mechanism

Abstract

Abstract AS1411 is an experimental drug that is currently in Phase II clinical trials as a novel therapy for cancer. It is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. We have shown previously that AS1411 can efficiently inhibit proliferation and induce cell death in many types of cancer cells, but has little effect on normal cells. Here, we report on the uptake of fluorescently labeled AS1411 (FL-AS1411) in a variety of cancer cell lines (sensitive to AS1411) and non-malignant cells (resistant to AS1411), which we studied using flow cytometry and confocal microscopy. In both cancer and non-malignant cells, uptake of FL-AS1411 was much higher that that of an inactive control oligonucleotide and was rapid (clearly visible in cytoplasmic foci by 15 min). Uptake of FL-AS1411 (evaluated at 2 h) was independent of the presence of serum, but strongly temperature-dependent, indicating an active process. In cancer cells, FL-AS1411 uptake was significantly suppressed by macropinocytosis inhibitor, amiloride, but not by dynamin inhibitor, dynasore, and confocal microscopy showed that AS1411 co-localized mainly with the macropinocytic tracker, dextran, but not with transferrin (a marker of receptor-mediated endocytosis). In contrast, in non-malignant cells, FL-AS1411 uptake was significantly suppressed by dynasore, but not by amiloride. These results suggest that the major mechanism of internalization of AS1411 occurs via macropinocytosis in cancer cells, but via clathrin-dependent and/or caveolae-dependent pathways in normal cells. Additionally, we found that treatment of cancer cells with an oligonucleotide containing the AS1411 sequence caused hyperstimulation of macropinocytosis, provoking an increment in its own uptake. No stimulation of macropinocytosis was observed in non-malignant cells, which are insensitive to AS1411. By using siRNA approaches, we showed that nucleolin is not required for the initial phase of FL-AS1411 uptake (at 2 h), but it is necessary for the induced macropinocytosis and consequently for the second phase of FL-AS1411 uptake (at 24 h). These data confirm the important role of nucleolin in mediating the activity of AS1411 and provide a new model for the mechanism of action of AS1411 in cancer cells. 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 4450.