Abstract
Aim:
Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule in cells, thus may act as effective vehicles for drug or siRNA delivery. In this study we investigated the DNA aptamers that target human glioblastoma multiforme (GBM) cells overexpressing epidermal growth factor receptor variant III (EGFRvIII), which was linked to radiation and chemotherapeutic resistance of this most aggressive brain tumor.
Methods:
A 73-mer ssDNA library containing molecules with 30 nt of random sequence flanked by two primer hybridization sites was chosen as the initial library. Cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) method was used to select the DNA aptamers that target EGFRvIII. The binding affinity of the aptamers was measured using a cell-based biotin-avidin ELISA.
Results:
After 14 rounds of selection, four DNA aptamers (32, 41, 43, and 47) that specifically bound to the EGFRvIII-overexpressing human glioma U87Δ cells with Kd values of less than 100 nmol/L were discovered. These aptamers were able to distinguish the U87Δ cells from the negative control human glioma U87MG cells and HEK293 cells. Aptamer 32 specifically bound to the EGFRvIII protein with an affinity similar to the EGFR antibody (Kd values of aptamer 32 and the EGFR antibody were 0.62±0.04 and 0.32±0.01 nmol/L, respectively), and this aptamer was localized in the cell nucleus.
Conclusion:
The DNA aptamers are promising molecular probes for the diagnosis and treatment of GBM.
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References
Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev 2001; 15: 1311–33.
Okamoto I, Kenyon LC, Emlet DR, Mori T, Sasaki J, Hirosako S, et al. Expression of constitutively activated EGFRvIII in non-small cell lung cancer. Cancer Sci 2003; 94: 50–6.
Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res 1995; 55: 5536–9.
Kuan CT, Wikstrand CJ, Bigner DD . EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr Relat Cancer 2001; 8: 83–96.
Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, et al. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 2007; 21: 2683–710.
Cai XM, Tao BB, Wang LY, Liang YL, Jin JW, Yang Y, et al. Protein phosphatase activity of PTEN inhibited the invasion of glioma cells with epidermal growth factor receptor mutation type III expression. Int J Cancer 2005; 117: 905–12.
Pelloski CE, Ballman KV, Furth AF, Zhang L, Lin E, Sulman EP, et al. Epidermal growth factor receptor variant III status defines clinically distinct subtypes of glioblastoma. J Clin Oncol 2007; 25: 2288–94.
Yoshimoto K, Dang J, Zhu S, Nathanson D, Huang T, Dumont R, et al. Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. Clin Cancer Res 2008; 14: 488–93.
Reist CJ, Archer GE, Kurpad SN, Wikstrand CJ, Vaidyanathan G, Willingham MC, et al. Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts. Cancer Res 1995; 55: 4375–82.
Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55: 3140–8.
Kang D, Wang J, Zhang W, Song Y, Li X, Zou Y, et al. Selection of DNA aptamers against glioblastoma cells with high affinity and specificity. PloS One 2012; 7: e42731.
Ohuchi SP, Ohtsu T, Nakamura Y . Selection of RNA aptamers against recombinant transforming growth factor-beta type III receptor displayed on cell surface. Biochimie 2006; 88: 897–904.
Esposito CL, Passaro D, Longobardo I, Condorelli G, Marotta P, Affuso A, et al. A neutralizing RNA aptamer against EGFR causes selective apoptotic cell death. PloS One 2011; 6: e24071.
Liu Y, Kuan CT, Mi J, Zhang X, Clary BM, Bigner DD, et al. Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis. Biol Chem 2009; 390: 137–44.
Graham JC, Zarbl H . Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells. PloS One 2012; 7: e36103.
Lau IP, Ngan EK, Loo JF, Suen YK, Ho HP, Kong SK . Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells. Biochem Biophys Res Commun 2010; 395: 560–4.
Low SY, Hill JE, Peccia J . A DNA aptamer recognizes the Asp f 1 allergen of Aspergillus fumigatus. Biochem Biophys Res Commun 2009; 386: 544–8.
Mallikaratchy P, Stahelin RV, Cao Z, Cho W, Tan W . Selection of DNA ligands for protein kinase C-delta. Chem Commun (Camb) 2006; 14: 3229–31.
Mendonsa SD, Bowser MT . In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis. Anal Chem 2004; 76: 5387–92.
Famulok M, Hartig JS, Mayer G . Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev 2007; 107: 3715–43.
Zhan Y, O'Rourke DM . SHP-2-dependent mitogen-activated protein kinase activation regulates EGFRvIII but not wild-type epidermal growth factor receptor phosphorylation and glioblastoma cell survival. Cancer Res 2004; 64: 8292–98.
Li S, Xu H, Ding H, Huang Y, Cao X, Yang G, et al. Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX. J Pathol 2009; 218: 327–36.
Wang C, Zhang M, Yang G, Zhang D, Ding H, Wang H, et al. Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment. J Biotechnol 2003; 102: 15–22.
Cao X, Li S, Chen L, Ding H, Xu H, Huang Y, et al. Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus. Nucleic Acids Res 2009; 37: 4621–28.
Chen CH, Dellamaggiore KR, Ouellette CP, Sedano CD, Lizadjohry M, Chernis GA, et al. Aptamer-based endocytosis of a lysosomal enzyme. Proc Natl Acad Sci U S A 2008; 105: 15908–13.
Bagalkot V, Farokhzad OC, Langer R, Jon S . An aptamer-doxorubicin physical conjugate as a novel targeted drug-delivery platform. Angew Chem Int Ed Engl 2006; 45: 8149–52.
Zhou J, Rossi JJ . Bivalent aptamers deliver the punch. Chem biol 2008; 15: 644–5.
Tombelli S, Minunni M, Mascini M . Aptamers-based assays for diagnostics, environmental and food analysis. Biomol Eng 2007; 24: 191–200.
Bayrac AT, Sefah K, Parekh P, Bayrac C, Gulbakan B, Oktem HA, et al. In vitro selection of DNA aptamers to glioblastoma. ACS Chem Neurosci 2011; 2: 175–81.
McNamara JO 2nd, Andrechek ER, Wang Y, Viles KD, Rempel RE, Gilboa E, et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 2006; 24: 1005–15.
Prow TW, Yu C, Hu Y, Duan J, Yuan W, Wang C, et al. Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro. PloS One 2011; 6: e24077.
Chu TC, Twu KY, Ellington AD, Levy M . Aptamer mediated siRNA delivery. Nucleic Acids Res 2006; 34: e73.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No 30973481 and 81272509) and the Guangdong Natural Science Foundation (No 9151051501000053 and 07005145). We would like to thank Shu-hu LIU, Jie WEI, Jing ZHANG, and Ying FANG for their assistance with the experiments. We would also like to thank Professor Tian-ming GAO for providing us with a new experimental platform as well as Ri-yang ZHOU and Charles DEAK for providing assistance with the language.
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Tan, Y., Shi, Ys., Wu, Xd. et al. DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro. Acta Pharmacol Sin 34, 1491–1498 (2013). https://doi.org/10.1038/aps.2013.137
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DOI: https://doi.org/10.1038/aps.2013.137
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