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A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus

Binding affinity of AS1411 towards HCC cell lines and tissues.(a) Nucleolin is detected on cell surface of Huh7 (left) but not on Hu767 (primary hepatocyte, right). 1 million cells of each cell line were incubated with 2ug/ml Nucleolin Antibody and 1ug/ml secondary antibody of mouse-FITC for 1h at 4°C, which was followed by flow cytometry. (b) AS1411 can stain membrane nucleolin on Huh7 and HCO2. Live cells were incubated with 200 nM of AS1411-FITC for 30 min at room temperature and examined under a confocal microscope. (c) AS1411 stains membrane nucleolin in tumor tissues, but only nuclear nucleolin of non-tumor tissue. Paraffin embedded tissue samples from patients were incubated with 200 nM of AS1411-Biotin for 1 h at room temperature. Signal was developed using HRP-conjugated streptavidin and DAB peroxidase substrate (Dako).
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pone.0136673.g001: Binding affinity of AS1411 towards HCC cell lines and tissues.(a) Nucleolin is detected on cell surface of Huh7 (left) but not on Hu767 (primary hepatocyte, right). 1 million cells of each cell line were incubated with 2ug/ml Nucleolin Antibody and 1ug/ml secondary antibody of mouse-FITC for 1h at 4°C, which was followed by flow cytometry. (b) AS1411 can stain membrane nucleolin on Huh7 and HCO2. Live cells were incubated with 200 nM of AS1411-FITC for 30 min at room temperature and examined under a confocal microscope. (c) AS1411 stains membrane nucleolin in tumor tissues, but only nuclear nucleolin of non-tumor tissue. Paraffin embedded tissue samples from patients were incubated with 200 nM of AS1411-Biotin for 1 h at room temperature. Signal was developed using HRP-conjugated streptavidin and DAB peroxidase substrate (Dako).

Mentions: Nucleolin is aberrantly expressed on the cell membrane of many tumor types including glioma, prostate and liver cancer [21–24]. As AS1411 works by first binding to cell membrane nucleolin [19], we sought to determine the presence of cell-surface nucleolin expression in the HCC cell line Huh7 and in a culture of human primary hepatocytes Hu767. By flow cytometry analysis of immunostained cells, we observed that nucleolin was expressed on the cell membrane of Huh7 cells but not in Hu767 cells (Fig 1A). To determine whether AS1411 could be useful as a marker for HCC tumor imaging, we conjugated AS1411 to FITC and performed staining on two HCC cell lines; Huh7 cells and HCO2 cells, a novel HCC cell line developed in our lab (Fig 1B). Likewise, AS1411-Biotin was used to develop immunohistochemistry staining of paraffin embedded HCC patients tumor samples. 21 HCC tumor tissue samples (formalin fixed and paraffin embedded) from HCC patients were analyzed, and in 15 samples AS1411 were found to preferentially bind in tumor regions compared with adjacent non-tumor tissue. AS1411 showed the strongest staining around nucleus, where nucleolin is the most abundant. However, AS1411 also stained the membrane and the cytoplasm of liver cancer cells clearly distinguishing tumor tissue, indicating aberrant transport of nucleolin throughout HCC tumor cells (Fig 1C left panel). Signal detection on non-tumor tissue was confined to the nucleus (Fig 1C right panel). Additional data confirm that AS1411 stains the membrane of tumor cells and preferentially stains liver tumor tissue, compared with the nucleolin antibody which often only stains the nuclear nucleolin (S1 and S2 Figs). The results indicate that AS1411 binds preferentially to liver cancer cells and can distinguish tumor and non-tumor tissue by the difference in staining intensity. The data show that AS1411 is a suitable candidate for further development as a Drug-DNA adduct for the specific targeting of liver cancer.


A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

Binding affinity of AS1411 towards HCC cell lines and tissues.(a) Nucleolin is detected on cell surface of Huh7 (left) but not on Hu767 (primary hepatocyte, right). 1 million cells of each cell line were incubated with 2ug/ml Nucleolin Antibody and 1ug/ml secondary antibody of mouse-FITC for 1h at 4°C, which was followed by flow cytometry. (b) AS1411 can stain membrane nucleolin on Huh7 and HCO2. Live cells were incubated with 200 nM of AS1411-FITC for 30 min at room temperature and examined under a confocal microscope. (c) AS1411 stains membrane nucleolin in tumor tissues, but only nuclear nucleolin of non-tumor tissue. Paraffin embedded tissue samples from patients were incubated with 200 nM of AS1411-Biotin for 1 h at room temperature. Signal was developed using HRP-conjugated streptavidin and DAB peroxidase substrate (Dako).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4629891&req=5

pone.0136673.g001: Binding affinity of AS1411 towards HCC cell lines and tissues.(a) Nucleolin is detected on cell surface of Huh7 (left) but not on Hu767 (primary hepatocyte, right). 1 million cells of each cell line were incubated with 2ug/ml Nucleolin Antibody and 1ug/ml secondary antibody of mouse-FITC for 1h at 4°C, which was followed by flow cytometry. (b) AS1411 can stain membrane nucleolin on Huh7 and HCO2. Live cells were incubated with 200 nM of AS1411-FITC for 30 min at room temperature and examined under a confocal microscope. (c) AS1411 stains membrane nucleolin in tumor tissues, but only nuclear nucleolin of non-tumor tissue. Paraffin embedded tissue samples from patients were incubated with 200 nM of AS1411-Biotin for 1 h at room temperature. Signal was developed using HRP-conjugated streptavidin and DAB peroxidase substrate (Dako).
Mentions: Nucleolin is aberrantly expressed on the cell membrane of many tumor types including glioma, prostate and liver cancer [21–24]. As AS1411 works by first binding to cell membrane nucleolin [19], we sought to determine the presence of cell-surface nucleolin expression in the HCC cell line Huh7 and in a culture of human primary hepatocytes Hu767. By flow cytometry analysis of immunostained cells, we observed that nucleolin was expressed on the cell membrane of Huh7 cells but not in Hu767 cells (Fig 1A). To determine whether AS1411 could be useful as a marker for HCC tumor imaging, we conjugated AS1411 to FITC and performed staining on two HCC cell lines; Huh7 cells and HCO2 cells, a novel HCC cell line developed in our lab (Fig 1B). Likewise, AS1411-Biotin was used to develop immunohistochemistry staining of paraffin embedded HCC patients tumor samples. 21 HCC tumor tissue samples (formalin fixed and paraffin embedded) from HCC patients were analyzed, and in 15 samples AS1411 were found to preferentially bind in tumor regions compared with adjacent non-tumor tissue. AS1411 showed the strongest staining around nucleus, where nucleolin is the most abundant. However, AS1411 also stained the membrane and the cytoplasm of liver cancer cells clearly distinguishing tumor tissue, indicating aberrant transport of nucleolin throughout HCC tumor cells (Fig 1C left panel). Signal detection on non-tumor tissue was confined to the nucleus (Fig 1C right panel). Additional data confirm that AS1411 stains the membrane of tumor cells and preferentially stains liver tumor tissue, compared with the nucleolin antibody which often only stains the nuclear nucleolin (S1 and S2 Figs). The results indicate that AS1411 binds preferentially to liver cancer cells and can distinguish tumor and non-tumor tissue by the difference in staining intensity. The data show that AS1411 is a suitable candidate for further development as a Drug-DNA adduct for the specific targeting of liver cancer.

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus