Abstract
Background:Doxorubicin (Dox) inhibits DNA replication and causes DNA damage resulting in cell death. It is a common drug for treatment of many cancers. Treatment efficacy and side effects of Dox are critical issues in using it because the drug lacks of specificity. The objective of this study was to improve the specificity of Dox by the incorporation of this drug with AS1411 aptamer (ASA). Methods:Dox was intercalated into the duplex sites of ASA, a recognition molecule for a number of cancer cells, and formed Dox-loaded ASA. The recognition ability proceeded through specific binding between the aptamer and nucleolin overexpressed in the cancer cells. The tested cells were human colorectal adenocarcinoma cell line (SW480) and human normal colon cell CCD841 CoN (CCD841). Binding of ASA to the cells was tested using flow cytometer and fluorescence microscope. Intercalation of Dox into DNA duplex was confirmed by fluorescence spectrometry. Effect of ASA, Dox, and Dox-loaded ASA on cell viability was examined by cell proliferation assay. Caspase-3 activation was analyzed by western blotting. Results:ASA bound specifically to SW480 cells via interaction between the aptamer and nucleolin because the nucleolin was highly expressed in SW480 cells. ASA decreased the viability of SW480 cells in a dose-dependent manner. Dox was more toxic than ASA. Fluorescence quenching revealed that Dox was able to intercalate in base pairing sites of the aptamer. Dox-loaded ASA inhibited the proliferation of SW480 cells, because the aptamer facilitated the Dox uptake into these cells which caused the cell apoptosis, indicated by the significant decrease in procaspase-3, apoptosis marker protein. Conclusion:This study succeeded to prepare Dox-loaded ASA by intercalation of the drug that inherited the binding function from the aptamer and anti-cancer activity from Dox. Dox-loaded ASA showed promise for effective cancer treatment with lower level of side effects.
Highlights
Doxorubicin (Dox), commercially named as Adriamycin, was extracted from Streptomyces peucetius and was first used in clinical trials in the 1960s
Binding ability of AS1411 aptamer on SW480 cells To investigate the binding ability of ASA, SW480 cells treated with the aptamer and non-binding sequence were characterized by flow cytometry
The expression of nucleolin mRNA was significantly lower in CCD 841 CoN (CCD841) than that in SW480 cells (Figure 4). These results indicated that ASA bound to SW480 cells via the interaction between this aptamer and the nucleolin highly expressed on the cell surface (Soundararajan et al, 2009)
Summary
Doxorubicin (Dox), commercially named as Adriamycin, was extracted from Streptomyces peucetius and was first used in clinical trials in the 1960s. Doxorubicin (Dox) inhibits DNA replication and causes DNA damage resulting in cell death. It is a common drug for treatment of many cancers. Methods: Dox was intercalated into the duplex sites of ASA, a recognition molecule for a number of cancer cells, and formed Dox-loaded ASA. Dox-loaded ASA inhibited the proliferation of SW480 cells, because the aptamer facilitated the Dox uptake into these cells which caused the cell apoptosis, indicated by the significant decrease in procaspase-3, apoptosis marker protein. Conclusion: This study succeeded to prepare Dox-loaded ASA by intercalation of the drug that inherited the binding function from the aptamer and anti-cancer activity from Dox. Dox-loaded ASA showed promise for effective cancer treatment with lower level of side effects
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