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Updated: Jun 21, 2026
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells
Sridharan Soundararajan1, Li Wang, Vijayalakshmi Sridharan
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Abstract:
AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [(32)P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [(32)P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 +/- 10% SE (P < 0.01) compared with cells incubated with FITC-AS1411 only. Cellular uptake of [(32)P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.
Insights
AS1411, a DNA aptamer, binds to cell surface nucleolin, a protein overexpressed in some tumors. This study confirms nucleolin acts as a functional receptor for AS1411 in acute myeloid leukemia cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- AS1411 is a DNA aptamer investigated for acute myeloid leukemia and renal cell carcinoma.
- Nucleolin is overexpressed in certain tumor cells, making it a potential therapeutic target.
- The role of cell surface nucleolin as a receptor for AS1411 was previously unclear.
Purpose of the Study:
- To investigate whether cell surface nucleolin functions as a receptor for the DNA aptamer AS1411.
- To compare AS1411 binding and uptake in cell lines with differing nucleolin expression levels.
Main Methods:
- Biotinylation and immunoblotting to detect cell surface nucleolin.
- Radiolabeled AS1411 incubation and immunoprecipitation to identify AS1411-nucleolin complexes.
- Inhibition assays using anti-nucleolin antibodies and excess unlabeled AS1411 to assess binding and uptake specificity.
Main Results:
- Full-length and truncated nucleolin were detected on the surface of MV4-11 acute myeloid leukemia cells.
- K-562 cells, less sensitive to AS1411, showed significantly lower levels of cell surface nucleolin.
- AS1411 binding and cellular uptake were demonstrated and correlated with nucleolin expression, with uptake blocked by excess unlabeled AS1411.
- Knockdown of nucleolin reduced AS1411 uptake, confirming its role as a receptor.
Conclusions:
- Cell surface nucleolin acts as a functional receptor for the DNA aptamer AS1411.
- Nucleolin expression levels on the cell surface correlate with AS1411 sensitivity and uptake.
- These findings support the therapeutic potential of AS1411 by elucidating its mechanism of cellular entry.
