Novel HER2 Aptamer Selectively Delivers Cytotoxic Drug to HER2-positive Breast Cancer Cells in Vitro
© Liu et al.; licensee BioMed Central Ltd. 2012
Received: 27 February 2012
Accepted: 20 July 2012
Published: 20 July 2012
Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers.
In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro.
The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a K d of 316 nM , and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells.
The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells .
Breast cancer is the most common malignancy among women worldwide. The overexpression of HER2 (human epithelial growth factor receptor 2) is found in approximately 20-30% of breast cancer . HER2-positive breast cancer is associated with more malignant behaviors, including increased invasiveness, higher recurrence, and reduced overall survival [2–4], comparing to other types of breast cancer. Moreover, more than half of the HER2-positive women are ER/PR-negative, suggesting that a fair proportion of HER2-positive breast cancers do not respond well to endocrine therapies [5, 6]. For those patients only accepting the conventional treatments of surgical resection followed by chemo- and endocrine therapies, the median survival time of HER2-positive patients was about half of that of HER2-negative cases . In view of the above situation, the development of novel therapy for HER2-positive breast cancer, such as tumor-targeted therapy, is an inevitable trend. Targeted therapy is characterized by selective killing of tumor cells with minimal influence to normal cells, and may improve therapeutic efficacy and reduce adverse effect. Trastuzumab (Herceptin), a humanized HER2 monoclonal antibody, has been approved as a first line targeted treatment of HER2-positive metastatic breast cancer. However, drug resistance is developed rapidly with trastuzumab treatment in virtually all patients [2, 3, 8–11]. Therefore, it is necessary to develop novel targeting therapeutic strategies for treatment of HER2-positive breast cancer.
Tumor-targeted therapy requires ligands that can bind to cancer cells. In addition to antibodies, other types of novel tumor-targeting ligands have emerged in the past decades . One new class of ligands are aptamers, which are single-stranded oligonucleotides that may serve as targeting molecules in therapeutic and diagnostic applications . Aptamers have certain advantages as ligands, such as high affinity, excellent specificity, and low immunogenicity. Aptamers are also easy to synthesize and modify chemically . Based on these advantages, aptamers have broad prospect in therapeutic applications. The first aptamer drug, Mucagen, was approved for clinical use years ago. Other aptamer-based agents are being actively tested in clinical trials [14, 15]. Moreover, aptamers could also be employed as tumor-targeting ligands to significantly enhance the treatment outcome in animal tumor models [16–19]. A specially designed aptamer–siRNA chimera that significantly enhanced the inhibition of prostate cancer in mice when administered systemically . It was also reported that aptamer-conjugated to docetaxel-containing nanoparticles could increase the cancer-specific cytotoxicity both in vitro and in vivo. These studies suggest that aptamers can serve as excellent tumor-targeting ligands in targeted therapeutic systems.
So far, however, no aptamer has been explored as tumor-targeting ligand for delivering chemotherapeutic agent to HER2-positive breast cancer, and there are limited published studies on HER2 aptamers [20, 21]. In the present study, we developed a new DNA aptamer (HB5), which was found capable of binding to both the HER2 protein and the HER2-positive breast cancer cells. To evaluate whether the aptamer could be employed to selectively carry cytotoxic drug to HER2-positive breast cancer cells, we also constructed an aptamer-doxirubicin complex (Apt-Dox). We now report that Apt-Dox can selectively deliver doxorubicin to HER2-positive breast cancer cells in vitro.
Cell lines and cultures
The Cell lines, SK-BR-3 (human breast cancer), MDA-MB-231 (human breast cancer), MCF-7 (human breast adenocarcinoma) were obtained from the Cell Resource Center of Chinese Academy of Medical Sciences (Beijing, China). All cell lines were incubated in DMEM medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (Gibico) in 5% CO2 and humid atmosphere at 37°C. All experiments were performed on cells in the exponential growth phase.
Random DNA library and primers
A starting ssDNA library consisted of 86 nucleotides, containing a 40-base random region flanked by two fixed sequences for the amplification reaction. The library sequence was 5’-AACCGCCCAAATCCCTAAGAGTC-N40-CACAGACACACTACACACGCACA-3’. A FITC- labeled 5’ Primer P1 (5’-FITC-AACCGCCCAAATCCCTAAGAGTC-3’) was used in the PCR to monitor the binding of aptamer with target agents during the selection process. A biotinylated 3’ primer P2 (5’-biotin-TGTGCGTGTGTAGTGTGTCTGTG-3’) was used to amplify double strand DNA (dsDNA) and enable this double labeled dsDNA binding to streptavidin-coated magnetic beads. The FITC-conjugated sense single-strand DNA (ssDNA) was separated from the biotin- and FITC- labeled dsDNA by denaturing in alkaline condition (0.1 M NaOH) and applying a magnetic field. The FITC-labeled ssDNA were used for aptamer selection. Unlabeled P1 and P2 primers were used for cloning the enriched library after the end of selection. All primers and the initial random DNA library were synthesized and purified by Invitrogen China. Streptavidin-coated magnetic beads were purchased from Promega (USA).
HER2 Peptide of at least 95% purity was synthesized by SBS Genetech (Beijing, China). Magnetic monodispersed microspheres embedded in SiO2 (Affimag SLE, 3 μm- 4 μm) were purchased from BaseLine ChromTech (Tianjin, China). 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Sigma. Bovine serum albumin (BSA) and trypsin were purchased from Amresco Inc. (USA). The extracellular domain of HER2 protein (HER2 ECD) was purchased from Sino Biological Inc. (Beijing, China).
A peptide from the juxtamembrane region of HER2 ECD was used as the target for selection, with the sequence of INCTHSCVDLDDKGCPAEQR. The HER2 peptide was immobilized to carboxylated magnetic beads through cross-linking of –COOH and –NH2. The carboxylated SLE beads (1x105, 200 μL) were washed twice with 200 μL of PBS. The beads were resuspended in 200 μL deionized water dissolving with HER2 peptide (2 μg) and EDC (40 mM), and then incubated at room temperature with gentle stirring for 2 h. The beads were then washed for three times with PBS, and stored at 4°C in PBS. The same method was utilized to conjugate the beads with other substances, including HER2 ECD protein, bovine serum albumin (BSA), trypsin and HER2 ECD, for specificity assays of the selected aptamer.
To reduce background interference, 0.1 mg/mL salmon sperm DNA and 1 mg/mL of BSA were added to the binding buffer (Hanks buffer). The procedures of selection were as follows: in the initial selection round, the ssDNA pool (200 pmol) was first heated at 95°C for 5 min and then cooled immediately on ice for 15 min. The HER2-peptide coated beads were suspended in 200 μL of binding buffer containing 200 pmol of random ssDNA. After incubating the mixture at 37°C for 30 min with gentle shaking, the unbound oligonucleotides were removed by washing four times with 500 μL of binding buffer. Subsequently, target-binding oligonucleotides on beads were amplified by PCR with FITC- or biotin-labeled primers (25 cycles of 40 s at 94°C, 30 s at 65°C, 40 s at 72°C, followed by 10 min at 72°C, the Taq polymerase and dNTPs were obtained from Takara). The selected sense ssDNA were separated from the biotinylated antisense ssDNA strand by streptavidin-coated magnetic beads and used for the next round of SELEX. After multiple rounds of selection, the selected ssDNA pool was PCR-amplified using unmodified primers and cloned into Escherichia coli with the TA cloning kit for DNA sequencing.
Flow cytometric analysis
To monitor the enrichment of aptamer pool each round, the FITC-labeled ssDNA pool were incubated with HER2-peptide coated magnetic beads in 200 μL of selection buffer containing 10% FBS at 37°C for 30 min. The beads were washed twice with 0.5 mL of binding buffer, suspended in 0.2 ml of binding buffer, and then analyzed by Flow cytometry (Accuri® C6 Flow Cytometer, BD). Peptide-coated beads stained with FITC-labeled random ssDNA were used as control.
To evaluate the binding specificity of the aptamers, FITC-labeled aptamers were separately incubated with HER2 ECD, BSA, or trypsin-coated magnetic beads. The beads were washed twice with 0.5 mL of binding buffer, suspended in 0.2 mL of binding buffer, and analyzed by Flow Cytometry. For assessing aptamer binding on cells, the cells were scraped off the culture bottle and washed twice with Hanks buffer. The FITC-labeled aptamers were incubated separately with 5 × 105 of SK-BR-3, MDA-MB-231, or MCF-7 in binding buffer at 37°C for 30 min. Cells were then washed twice with Hanks buffer and analyzed by flow cytometry.
To evaluate the binding affinity of the aptamer to HER2 structure, we incubated HER2-peptide coated magnetic beads with varying concentrations of the FITC-labeled aptamer HB5 in 200 μL binding buffer at 37°C for 30 min. The beads were washed twice with 0.5 mL of binding buffer, suspended in 0.2 mL of binding buffer, and then analyzed by Flow Cytometry. The peptide-coated beads stained with FITC-labeled random ssDNA were used as a control. All of the experiments for binding assay were repeated for at least three times. The mean fluorescence intensity of the aptamer bound to the target was used to calculate the specific binding by subtracting the mean fluorescence intensity of nonspecific binding from unselected library DNA. The equilibrium dissociation constant (Kd) of the aptamer was obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the aptamers to the equation: Y = B max X/ ( Kd + X) .
The aptamer were first heated at 95°C for 5 min and then cooled immediately on ice for 15 min. Next, a fixed concentration of Dox (3 nM) was incubated for 1 h with various concentrations of aptamer HB5, at aptamer/Dox molar ratios of 1, 0.1, 0.01, 0.003, 0.0001, and 0, respectively. The fluorescence spectrum of doxorubicin was examined in 96-well black plate by a Synergy4 analyzer (λEx = 488 nm, λEm = 520–720 nm).
Cellular uptake studies
The cellular uptake of Apt-Dox or free Dox by cells was studied by confocal microscopy (Perkin Elmer Ultraview, US) and flow cytometry. Cells were allowed to adhere to a glass cover slip for 24 h. The cells were then incubated with 1.5 μM of Dox or Apt-Dox complex for 2 h at 37°C. After washing twice with Hanks buffer, the cells were fixed with 4% formaldehyde for 10 min and analyzed by confocal fluorescence scanning microscopy.
For flow cytometry analysis, cells were scraped off from the culture bottle and washed twice with Hanks buffer. The cells were incubated with 1.5 μM Dox or Apt-Dox complex for 4 h at 37°C, and washed twice with Hanks buffer. The cells were then fixed with 4% formaldehyde for 10 min and analyzed by Flow Cytometry.
In vitro cytotoxicity assays
To evaluate the cytotoxicity of Apt-Dox or Dox against SK-BR-3 and MDA-MB-231 cells, both cell lines were first grown in 96-well plates, and then co-incubated with Apt-Dox, Dox, or aptamer separately at the concentration of 2 μM for 4 h at 37°C. The cells were washed with Hanks buffer twice, and cultured for a further 40 h. Afterwards, MTS viability assay was performed according to the standard protocol outlined by the manufacture (Promega, US).
Statistical analysis was performed with the statistical SPSS 13.0 software. The nonparametric test was used to calculate the probability of significant differences among the groups. Statistical significance was defined as p < 0.05.
Binding property of the aptamer HB5
To evaluate whether the aptamer HB5 had a relatively preferred binding to the HER2 structure, we directly compared, under the same experimental conditions, the bindings of the aptamer to HER2 peptide, HER2 ECD protein, albumin (BSA), or trypsin. Albumin was tested because it is the most abundant protein in blood; and trypsin was commonly used for testing the binding specificity of the aptamers. The results are presented in Figure2. While aptamer HB5 showed relatively strong bindings to HER2 peptide and HER2 ECD protein, its binding to BSA or trypsin was weak ( Figure 2A). In addition, random DNA showed no binding preference to all four structures ( Figure 2B). The data suggested that the aptamer HB5 had a targeting preference towards HER2, and tended not to bind the other proteins such as BSA or trypsin.
Aptamer HB5 selectively recognized HER2-positive breast cancer cells
Formation of aptamer-doxorubicin complex
The Apt-Dox complex was selectively uptaken into HER2-positive breast cancer cells
Next we performed flow cytometry experiments to further evaluate the uptake of Apt-Dox and free Dox by HER2-positive and -negative breast cancer cells (Figure 6E&F). For HER2-positive SK-BR-3 cells, the fluorescent signals from free Dox or Apt-Dox were similar (Figure 6E); whereas for HER2-negative MDA-MB-231 cells, the fluorescent signal from Apt-Dox was lower than that from free Dox (Figure 6F). Taken together, both the confocal and flow cytometry studies suggested that Apt-Dox conjugate mainly entered HER2-positive breast cancer cells, while free Dox entered both HER2-positive and -negative cells.
The Apt-Dox complex selectively reduced cytotoxity to HER2-negative breast cancer cells
HER2 over-expression is found not only in 20–30% of breast cancers, but also in gastric, ovarian, bladder, and lung cancers [22, 31–36]. Therefore, HER2 may serve as a potential target for targeted tumor therapy against many malignancies. Aptamers can bind to target with high affinity and specificity, and may be employed as targeting ligand in selective drug delivery system. Here in this study, using the SELEX technique with a HER2 peptide as the target, we developed a novel HER2 aptamer (HB5) with a Kd of 18.9 nM for binding with the target peptide (Figure 1 & 3A). In addition to binding the HER2 peptide, HB5 could also bind to HER2 ECD protein with a K d of 316 nM (Figure 2 & 3B), and with minimal cross-reactivity to trypsin and albumin (Figure 2), suggesting that the aptamer had a binding preference for HER2 structures. Moreover, the aptamer HB5 could selectively bind to HER2-positive breast cancer cells with weak cross-reaction to HER2-negative cells (Figure 4). An aptamer-doxorubicin complex (Apt-Dox) was formed by intercalating Dox into the DNA structure of the aptamer (Figure 5). While retaining the efficacy of doxorubicin against HER2-positive breast cancer cells, the Apt-Dox complex reduced the drug intake and the toxicity to HER2-negative cells (FigureS 6&7). The results suggested that Apt-Dox could discern target and non-target cells, and that HER2 aptamer might serve as targeting ligand for selective drug delivery to HER2-positive breast cancer cells.
Monoclonal antibodies have been the main approach for HER2-targeted cancer therapy, and trastuzumab (Herceptin), a humanized HER2 monoclonal antibody, has been approved for treatment of HER2-positive breast cancer. However, drug resistance is commonly and rapidly developed with trastuzumab treatment [2, 3, 8–11], making it necessary to explore other HER2-targeting therapeutic strategies. Recent studies have identified some RNA or DNA HER2 aptamers [20, 21]. Kim et al. developed an RNA aptamer using HER2 protein as the target, and proposed that the selected aptamer could potentially be utilized in constructing novel imaging agents for HER2-positive cancers . Kazem et al. also obtained a library of DNA aptamers by using HER2-positive cells as the target for aptamer selection . So far, however, HER2 aptamer has not been explored as the tumor-targeting ligand for selective drug delivery to HER2-positive breast cancer cells. To investigate whether HER2 aptamer can be utilized in targeted HER2 therapy, we developed an aptamer-based doxorubicin delivery system in this study, and found that the aptamer could be employed to selectively deliver cytotoxic agent to HER2-postive breast cancer cells in vitro. The results suggest that, in addition to monoclonal antibodies, HER2 aptamers may also have application potentials in construction of novel HER2-targeted therapeutic systems.
There are multiple technical approaches to develop HER2 aptamers. One of them is termed Cell-SELEX, which uses the entire HER2-positive cell as the target during the aptamer selection process . One potential drawback of this approach is that the selected aptamers may or may not bind to the HER2 structure, since these aptamers also may bind to the other proteins in the cell membrane of the target cell . In order to select out aptamers that could truly bind to HER2 structure, here in this study, we chose an epitope sequence (peptide) of HER2 protein as the target in our SELEX process. The peptide sequence was a part of the extra-cellular domain (ECD) of HER2 protein, and contained 20 AAs. The advantage of this target peptide was that it could be synthesized with high purity, allowing us to select a population of aptamers that mostly bound to the target. Interestingly, some of the aptamers thus generated could also bind to the ECD of HER2 protein and the HER2-positive breast cancer cells (Figure 2&4). We postulated that this might be related to the properties of the peptide and the structure of the HER2 protein. First, the peptide is a confirmed B cell epitope that is found close to the cell membrane of the ECD of HER2 protein . B cell epitope usually locates at a flexible and exposed part of the protein [30, 39]. Second, HER2 crystal structure reveals that the extra-cellular domain of this protein is always in an extended conformation [40–43]. Thus, the target epitope is probably exposed on the surface of the HER2 protein, and the aptamer recognizing this epitope may consequently bind to the HER2 protein and the HER2-positive cells as well. It is interesting that the K d for binding with the HER2 ECD protein was higher than that for binding with the HER2 peptide. One factor possibly contributing to the difference is that the original aptamer binding site on the HER2 peptide might be partially masked by the more complex structure of the HER2 ECD protein. It is also possible that the binding affinity of the aptamer might be weakened by the influences from the protein structures adjacent to the aptamer binding site.
The binding preference of an aptamer is important for its application as a targeting ligand. An ideal tumor-targeting aptamer should bind to the target molecule (in this case, the HER2 structure), with minimal binding to other proteins. Since albumin is the most abundant protein in blood, we added BSA to the selection solution as a background component, in order to reduce the aptamer’s binding to albumin. As a result, the HER2 aptamer HB5 developed here had a weak binding to BSA, while maintaining a good binding profile to HER2 structures. Interestingly, the HER2 aptamer also had minimal reactivity to trypsin (Figure 2), which was commonly used for testing the binding specificity of the aptamers. Moreover, the HER2-aptamer exhibited a relatively strong binding to HER2-positive breast cancer cells, but minimal binding towards the HER2-negative cells (Figure 4). These results indicated that the HER2 aptamer HB5 exhibited certain degrees of targeting preference in vitro. Nevertheless, the aptamer’s in vivo HER2 preference still needs to be verified with future animal experiments in the follow-up studies.
Prior studies have shown that aptamers can deliver doxorubicin to tumor cells that bind the aptamer . Similarly, in this study, the confocal microscopy demonstrated that the Apt-Dox selectively delivered doxorubicin to HER2-positive breast cancer cells (Figure 6C). Interestingly, in HER2-positive breast cancer cells, Apt-Dox stained both the nuclei and the cytoplasm, while the free Dox exclusively stained the nuclei. This observation may reflect a difference in the drug uptake mechanisms between Apt-Dox and the free Dox. We hypothesized that the lipophilic free Dox entered cells via passive diffusion, while the hydrophilic aptamer prevented the Apt-Dox from freely defusing into the lipid cell membrane. The Apt-Dox possibly entered HER2-positive cells via a receptor-mediated endocytosis. In other words, the aptamer in the Apt-Dox recognized and bound to the HER2 structure on HER2-positive cells. Since the binding site was very close to the cell membrane, the binding might induce a conformational change of HER2 protein and disturb the adjacent cell membrane, resulting in the endocytosis of the Apt-Dox complex. Obviously, extensive future research is needed to clearly unveil the mechanism by which the Apt-Dox entered the HER2-positive cancer cells. It should be noted that the Apt-Dox produced weak staining in the HER2-negatives cells, a finding consistent with the lack of HER2 expression in these cells (Figure 6D). The results indicated that the Apt-Dox had the capability to selectively deliver doxorubicin to HER2-positive breast cancer cells. This presumably would decrease the adverse effects of Dox against non-tumor tissues with low HER2 expression. Future research may concentrate on improving the binding kinetics, targeting efficacy, drug-loading capacity, and the in vivo stability of the targeted drug delivery system based on HER2 aptamers. In addition, extensive animal studies are also needed to evaluate the pharmacokinetics and the in vivo efficacy of the HER2-targeted drug-delivery system.
In this study, a newly developed DNA aptamer (HB5) was found capable of binding to HER2 protein and HER2-positive breast cancer cells, with minimal binding to HER2-negative cells. A complex of the aptamer and doxorubicin (Apt-Dox) could selectively deliver doxorubicin to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells. The results suggest that HER2 aptamers may have application potentials in targeted therapy against HER2-positive breast cancer cells.
We acknowledge the funding support from the Chinese Ministry of Science and Technology (2011CB933504, 2011CB911003, 2011CB911004) and the Natural Science Foundation of China (81071870).
- King CR, Kraus MH, Aaronson SA: Amplification of a novel v-erbB-related gene in a human mammary carcinoma. Science. 1985, 229: 974-976.View ArticlePubMedGoogle Scholar
- Garrett JT, Arteaga CL: Resistance to HER2-directed antibodies and tyrosine kinase inhibitors: mechanisms and clinical implications. Cancer Biol Ther. 2011, 11: 793-800.View ArticlePubMedPubMed CentralGoogle Scholar
- Nahta R, O'Regan RM: Evolving strategies for overcoming resistance to HER2-directed therapy: targeting the PI3K/Akt/mTOR pathway. Clin Breast Cancer. 2010, 10 (Suppl 3): S72-S78.View ArticlePubMedGoogle Scholar
- Cooke T, Reeves J, Lanigan A, Stanton P: HER2 as a prognostic and predictive marker for breast cancer. Ann Oncol. 2001, 12 (Suppl 1): S23-S28.View ArticlePubMedGoogle Scholar
- Wright C, Nicholson S, Angus B, Sainsbury JR, Farndon J, Cairns J, Harris AL, Horne CH: Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br J Cancer. 1992, 65: 118-121.View ArticlePubMedPubMed CentralGoogle Scholar
- Ariazi EA, Clark GM, Mertz JE: Estrogen-related receptor alpha and estrogen-related receptor gamma associate with unfavorable and favorable biomarkers, respectively, in human breast cancer. Cancer Res. 2002, 62: 6510-6518.PubMedGoogle Scholar
- Press MF, Bernstein L, Thomas PA, Meisner LF, Zhou JY, Ma Y, Hung G, Robinson RA, Harris C, El-Naggar A: HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas. J Clin Oncol. 1997, 15: 2894-2904.PubMedGoogle Scholar
- Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, Paton V, Shak S, Lieberman G, Slamon DJ: Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol. 1999, 17: 2639-2648.PubMedGoogle Scholar
- Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002, 20: 719-726.View ArticlePubMedGoogle Scholar
- Raguz S, Yague E: Resistance to chemotherapy: new treatments and novel insights into an old problem. Br J Cancer. 2008, 99: 387-391.View ArticlePubMedPubMed CentralGoogle Scholar
- Shattuck DL, Miller JK, Carraway KL, Sweeney C: Met receptor contributes to trastuzumab resistance of Her2-overexpressing breast cancer cells. Cancer Res. 2008, 68: 1471-1477.View ArticlePubMedGoogle Scholar
- Barbas AS, Mi J, Clary BM, White RR: Aptamer applications for targeted cancer therapy. Future Oncol. 2010, 6: 1117-1126.View ArticlePubMedGoogle Scholar
- Famulok M, Hartig JS, Mayer G: Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev. 2007, 107: 3715-3743.View ArticlePubMedGoogle Scholar
- Bates PJ, Laber DA, Miller DM, Thomas SD, Trent JO: Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp Mol Pathol. 2009, 86: 151-164.View ArticlePubMedPubMed CentralGoogle Scholar
- Ng EW, Shima DT, Calias P, Cunningham ET, Guyer DR, Adamis AP: Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006, 5: 123-132.View ArticlePubMedGoogle Scholar
- Dhar S, Kolishetti N, Lippard SJ, Farokhzad OC: Targeted delivery of a cisplatin prodrug for safer and more effective prostate cancer therapy in vivo. Proc Natl Acad Sci U S A. 2011, 108: 1850-1855.View ArticlePubMedPubMed CentralGoogle Scholar
- Dhar S, Gu FX, Langer R, Farokhzad OC, Lippard SJ: Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA-PEG nanoparticles. Proc Natl Acad Sci U S A. 2008, 105: 17356-17361.View ArticlePubMedPubMed CentralGoogle Scholar
- Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, Richie JP, Langer R: Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc Natl Acad Sci U S A. 2006, 103: 6315-6320.View ArticlePubMedPubMed CentralGoogle Scholar
- Dassie JP, Liu XY, Thomas GS, Whitaker RM, Thiel KW, Stockdale KR, Meyerholz DK, McCaffrey AP, McNamara JO, Giangrande PH: Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors. Nat Biotechnol. 2009, 27: 839-849.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim MY, Jeong S: In vitro selection of RNA aptamer and specific targeting of ErbB2 in breast cancer cells. Nucleic Acid Ther. 2011, 21: 173-178.View ArticlePubMedPubMed CentralGoogle Scholar
- Dastjerdi K, Tabar GH, Dehghani H, Haghparast A: Generation of an enriched pool of DNA aptamers for an HER2-overexpressing cell line selected by Cell SELEX. Biotechnol Appl Biochem. 2011, 58: 226-230.View ArticlePubMedGoogle Scholar
- Sefah K, Meng L, Lopez-Colon D, Jimenez E, Liu C, Tan W: DNA aptamers as molecular probes for colorectal cancer study. PLoS One. 2010, 5: e14269-View ArticlePubMedPubMed CentralGoogle Scholar
- Wilson KS, Roberts H, Leek R, Harris AL, Geradts J: Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues. Am J Pathol. 2002, 161: 1171-1185.View ArticlePubMedPubMed CentralGoogle Scholar
- Cuello M, Ettenberg SA, Clark AS, Keane MM, Posner RH, Nau MM, Dennis PA, Lipkowitz S: Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res. 2001, 61: 4892-4900.PubMedGoogle Scholar
- Govindarajan S, Sivakumar J, Garimidi P, Rangaraj N, Kumar JM, Rao NM, Gopal V: Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate. Biomaterials. 2011, 33: 2570-2582.View ArticlePubMedGoogle Scholar
- Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, Shepard HM: Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother. 1993, 37: 255-263.View ArticlePubMedGoogle Scholar
- Magnifico A, Albano L, Campaner S, Delia D, Castiglioni F, Gasparini P, Sozzi G, Fontanella E, Menard S, Tagliabue E: Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab. Clin Cancer Res. 2009, 15: 2010-2021.View ArticlePubMedGoogle Scholar
- 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: 8149-8152.View ArticlePubMedGoogle Scholar
- Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A: p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol. 1989, 9: 1165-1172.View ArticlePubMedPubMed CentralGoogle Scholar
- Xu W, Ellington AD: Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope. Proc Natl Acad Sci U S A. 1996, 93: 7475-7480.View ArticlePubMedPubMed CentralGoogle Scholar
- Yoshino I, Goedegebuure PS, Peoples GE, Parikh AS, DiMaio JM, Lyerly HK, Gazdar AF, Eberlein TJ: HER2/neu-derived peptides are shared antigens among human non-small cell lung cancer and ovarian cancer. Cancer Res. 1994, 54: 3387-3390.PubMedGoogle Scholar
- Brabender J, Danenberg KD, Metzger R, Schneider PM, Park J, Salonga D, Holscher AH, Danenberg PV: Epidermal growth factor receptor and HER2-neu mRNA expression in non-small cell lung cancer Is correlated with survival. Clin Cancer Res. 2001, 7: 1850-1855.PubMedGoogle Scholar
- Coombs LM, Pigott DA, Sweeney E, Proctor AJ, Eydmann ME, Parkinson C, Knowles MA: Amplification and over-expression of c-erbB-2 in transitional cell carcinoma of the urinary bladder. Br J Cancer. 1991, 63: 601-608.View ArticlePubMedPubMed CentralGoogle Scholar
- Berchuck A, Kamel A, Whitaker R, Kerns B, Olt G, Kinney R, Soper JT, Dodge R, Clarke-Pearson DL, Marks P: Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res. 1990, 50: 4087-4091.PubMedGoogle Scholar
- Gravalos C, Gomez-Martin C, Rivera F, Ales I, Queralt B, Marquez A, Jimenez U, Alonso V, Garcia-Carbonero R, Sastre J: Phase II study of trastuzumab and cisplatin as first-line therapy in patients with HER2-positive advanced gastric or gastroesophageal junction cancer. Clin Transl Oncol. 2011, 13: 179-184.View ArticlePubMedGoogle Scholar
- Jorgensen JT: Targeted HER2 treatment in advanced gastric cancer. Oncology. 2010, 78: 26-33.View ArticlePubMedGoogle Scholar
- Tang Z, Shangguan D, Wang K, Shi H, Sefah K, Mallikratchy P, Chen HW, Li Y, Tan W: Selection of aptamers for molecular recognition and characterization of cancer cells. Anal Chem. 2007, 79: 4900-4907.View ArticlePubMedGoogle Scholar
- Dakappagari NK, Lute KD, Rawale S, Steele JT, Allen SD, Phillips G, Reilly RT, Kaumaya PT: Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities. J Biol Chem. 2005, 280: 54-63.View ArticlePubMedGoogle Scholar
- Novotny J, Handschumacher M, Haber E, Bruccoleri RE, Carlson WB, Fanning DW, Smith JA, Rose GD: Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains). Proc Natl Acad Sci U S A. 1986, 83: 226-230.View ArticlePubMedPubMed CentralGoogle Scholar
- Franklin MC, Carey KD, Vajdos FF, Leahy DJ, de Vos AM, Sliwkowski MX: Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. Cancer Cell. 2004, 5: 317-328.View ArticlePubMedGoogle Scholar
- Vicente-Alique E, Nunez-Ramirez R, Vega JF, Hu P, Martinez-Salazar J: Size and conformational features of ErbB2 and ErbB3 receptors: a TEM and DLS comparative study. Eur Biophys J. 2011, 40: 835-842.View ArticlePubMedGoogle Scholar
- Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW, Leahy DJ: Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 2003, 421: 756-760.View ArticlePubMedGoogle Scholar
- Eigenbrot C, Ultsch M, Dubnovitsky A, Abrahmsen L, Hard T: Structural basis for high-affinity HER2 receptor binding by an engineered protein. Proc Natl Acad Sci U S A. 2010, 107: 15039-15044.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.