as-1411 and Breast-Neoplasms

This article focuses on nanomaterials, nanomedicine and photothermal therapy (PTT) to treat breast cancer. Nanomaterials refer to materials with at least one dimension in nanometer size (1–100 nm) or materials composed as basic units in a 3D space. Nanomedicine is the application of nanomaterials in medicine. Nanoparticles can deliver drugs to areas that are difficult for the drugs themselves to reach. PTT is a noninvasive tumor therapy that uses photothermal conversion agents to convert light energy into heat energy to kill tumor cells under the irradiation of external near-infrared (NIR) light. In recent years, combination therapy for cancers has drawn more and more attention. In the current study, we investigated the

Topics: Aptamers, Nucleotide; Breast Neoplasms; Doxorubicin; Female; Humans; Photothermal Therapy; Silver

Herein, a dual-targeting delivery system using mesoporous silica nanoparticles with hollow structures (HMSNs) was developed for the specific delivery of epirubicin (EPI) to cancer cells and introducing a H. The collected data illustrated that BGNS-EPI-HA-Apt complex controlled the release of EPI in a sustained method. Afterward, receptor-mediated internalization via nucleolin and CD44 was verified in 4T1 and MCF-7 cells using fluorescence microscopy assay and flow cytometry analysis. The results of tumor inhibitory effect study exhibited that BGNS-EPI-HA-Apt complex decreased off-target effect and improved on-target effects because of its targeting ability.. The data acquired substantiates that HA-surface modified HMSNs functionalized with aptamers possess significant potential as a focused platform for efficient transportation of anticancer agents to neoplastic tissues.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Drug Delivery Systems; Epirubicin; Female; Humans; Hyaluronic Acid; MCF-7 Cells; Mice; Nanoparticles; Silicon Dioxide

Herein, we presented a novel DOX-loaded multi-storey DNA nanostructure, including AS1411 aptamer as a targeting agent for treatment of target cells (MCF-7 and 4T1). Gel retardation test and fluorometric analysis were used to examine the construction of DNA nanostructure and loading of DOX in the complex. At pH 5.5 and 7.4, the release patterns of DOX from the prepared formulation were studied. Cell viability test was conducted to analyse the cell cytotoxicity ability of the DOX loaded multi-storey DNA nanostructure compared to free DOX in 4T1, MCF-7 (target) and CHO cells (non-target). Flow cytometry analysis was used to examine the DOX-loaded DNA nanostructure internalisation. Finally, the developed DOX-loaded multi-storey DNA nanostructure was tested

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cricetinae; Cricetulus; DNA; Doxorubicin; Drug Delivery Systems; Female; Humans; MCF-7 Cells; Nanostructures

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Mice; Neoplastic Stem Cells; Oligodeoxyribonucleotides; Photothermal Therapy; Silver

In this study, a catalytic polydopamine-remodeling gold nanoparticle sensitized with an antinucleolin AS1411 probe (pAu nanoprobe) is synthesized, where the surface of the gold nanoparticles (AuNPs) is modified with a spontaneous self-polymerization of a polydopamine coating that imparts the probe functionalization ability and antispecific protein binding while the intrinsic catalytic property of the AuNPs is preserved. The functionalized AS1411 probe exerts specific recognition with nucleolin protein that is found to be overexpressed on the surface of breast cancer cells (MDA-MB-231). Scanning electron microscopy (SEM) confirms that the specific binding of the pAu nanoprobe occurs at the cancer cell surface. Taking advantage of the catalytic ability of the pAu nanoprobe in reducing blue-colored methylene blue (MB) to colorless leuco-MB, a colorimetric biosensing platform is established based on the accessible catalytic active sites on the pAu nanoprobe toward MB. The specific binding inhibits the pAu nanoprobe from efficiently catalyzing the reduction of MB, resulting in a "

Topics: Animals; Aptamers, Nucleotide; Biocompatible Materials; Biosensing Techniques; Breast Neoplasms; Catalysis; Cattle; Cells, Cultured; Gold; Humans; Indoles; Materials Testing; Metal Nanoparticles; Molecular Structure; Nucleolin; Oligodeoxyribonucleotides; Particle Size; Phosphoproteins; Polymers; RNA-Binding Proteins; Serum Albumin, Bovine; Single-Cell Analysis

Gold nanoparticles (GNPs) have been used to sensitize cancer cells and enhance the absorbed dose delivered to such cells. Active targeting can provide specific effect and higher uptake of the GNPs in the tumor cells, while having small effect on healthy cells. The aim of this study was to assess the possible radiosensitiazation effect of GNPs conjugated with AS1411 aptamer (AS1411/GNPs) on cancer cells treated with 4 MeV electron beams.. Cytotoxicity studies of the GNPs and AS1411/GNPs were carried out with MTT and MTS assay in different cancer cell lines of MCF-7, MDA-MB-231 and mammospheres of MCF-7 cells. Atomic absorption spectroscopy confirmed the cellular uptake of the gold particles. Radiosensitizing effect of the GNPs and AS1411/GNPs on the cancer cells was assessed by clonogenic assay.. AS1411 aptamer increased the Au uptake in MCF-7 and MDA-MB-231 cells. Clonogenic survival data revealed that AS1411/GNPs at 12.5 mg/L could result in radiosensitization of the breast cancer cells and lead to a sensitizer enhancement ratio of 1.35 and 1.66 and 1.91 for MCf-7, MDA-MB-231 and mammosphere cells.. Gold nanoparticles delivery to the cancer cells was enhanced by AS1411 aptamer and led to enhanced radiation induced cancer cells death. The combination of our clonogenic assay and Au cell uptake results suggested that AS1411 aptamer has enhanced the radiation-induced cell death by increasing Au uptake. This enhanced sensitization contributed to cancer stem cell-like cells to 4 MeV electron beams. This is particularly important for future preclinical testing to open a new insight for the treatment of cancers.

Topics: Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Breast Neoplasms; Cell Proliferation; Electrons; Female; Gold; Humans; Metal Nanoparticles; Oligodeoxyribonucleotides; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Development of proteolysis targeting chimeras (PROTACs) is emerging as a promising strategy for targeted protein degradation. However, the drug development using the heterobifunctional PROTAC molecules is generally limited by poor membrane permeability, low in vivo efficacy and indiscriminate distribution. Herein an aptamer-PROTAC conjugation approach was developed as a novel strategy to improve the tumor-specific targeting ability and in vivo antitumor potency of conventional PROTACs. As proof of concept, the first aptamer-PROTAC conjugate (APC) was designed by conjugating a BET-targeting PROTAC to the nucleic acid aptamer AS1411 (AS) via a cleavable linker. Compared with the unmodified BET PROTAC, the designed molecule (APR) showed improved tumor targeting ability in a MCF-7 xenograft model, leading to enhanced in vivo BET degradation and antitumor potency and decreased toxicity. Thus, the APC strategy may pave the way for the design of tumor-specific targeting PROTACs and have broad applications in the development of PROTAC-based drugs.

Topics: Animals; Antineoplastic Agents; Aptamers, Nucleotide; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Disulfides; Heterocyclic Compounds, 3-Ring; Humans; Mice; Oligodeoxyribonucleotides; Proof of Concept Study; Proteolysis; Pyrrolidines; Transcription Factors; Xenograft Model Antitumor Assays

MicroRNAs are key factors for many biological functions. These regulatory molecules affect various gene networks and involve the subsequent signaling pathways. Therefore, disrupting the expression of these molecules is associated with multiple anomalies in the cells and body. One of the most important related abnormalities is the incidence of cancer. Thus, targeting microRNAs (miRNAs) is an effective approach for cancer gene therapy. Various factors are used for this purpose, including the antagomir nucleotide structure. There are some obstacles in the delivery of nucleotide therapeutics to the target cells, however, the use of nanoparticles could partly overcome these defeciencies. On the other hand, targeted delivery of antagomirs using aptamers, reduces nonspecific effects on nontarget cells. Considering the above, in this study, we designed and fabricated a nanocarrier composed of gold nanoparticles (GNPs), antagomir-155, and nucleolin specific aptamer for breast cancer study and therapy. Here, GNPs were synthesized using citrate reduction and were modified by polyA sequences, AS1411 aptamer, and antagomir-155. Attachment of molecules were confirmed using gel electrophoresis, atomic force microscopy imaging and electrochemical test. The specific entry of modified nanoparticles was investigated by fluorescence microscopy. The efficacy of modified nanoparticles was evaluated using a quantitative polymerase chain reaction (q-PCR) for miR-155 and its target gene. Efficient and specific delivery of AuNP-Apt-anti-miR-155 to target cells was confirmed in comparison with the control cell. The q-PCR analysis showed not only a significant decrease in mir-155 levels but also an elevated TP53INP1 mRNA, direct target of miR-155. The proposed structure inhibits proliferation and stimulates apoptosis by increasing the expression of TP53INP1. Our results suggest that AuNP-Apt-anti-miR-155 could be a promising nano constructor for breast cancer treatment.

Topics: Animals; Antagomirs; Apoptosis; Aptamers, Nucleotide; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetulus; Drug Delivery Systems; Female; Gold; Humans; MCF-7 Cells; Metal Nanoparticles; MicroRNAs; Oligodeoxyribonucleotides

An aptamer-conjugated gold nanostar (dsDDA-AuNS) has been developed for targeting nucleolin present in both tumor cells and tumor vasculature for conducting a drug-resistant cancer therapy. AuNS with its strong absorption in the near-infrared (NIR) region was assembled with a layer of the anti-nucleolin aptamer AS1411. An anticancer drug, namely doxorubicin (DOX), was specifically conjugated on deoxyguanosine residues employing heat and acid labile methylene linkages. In response to NIR irradiation, dsDDA-AuNS allowed on-demand therapeutics. AS1411 played an active role in drug cargo-nucleus interactions, enhancing drug accumulation in the nuclei of drug-resistant breast cancer cells. The intravenous injection of dsDDA-AuNS allowed higher drug accumulation in drug-resistant tumors over naked drugs, leading to greater therapeutic efficacy even at a 54-fold less equivalent drug dose. The in vivo triggered release of DOX from dsDDA-AuNS was achieved by NIR irradiation, resulting in simultaneous photothermal and chemotherapeutic actions, yielding superior tumor growth inhibition than those obtained from either type of monotherapy for overcoming drug resistance in cancers.

Topics: Administration, Intravenous; Animals; Antineoplastic Agents; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Drug Resistance, Neoplasm; Female; Gold; Humans; MCF-7 Cells; Metal Nanoparticles; Mice; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; Xenograft Model Antitumor Assays

A multi-functional nanoplatform with diagnostic imaging and targeted treatment functions has aroused much interest in the nanomedical research field and has been paid more attention in the field of tumor diagnosis and treatment. However, some existing nano-contrast agents have encountered difficulties in different aspects during clinical promotion, such as complicated preparation process and low specificity. Therefore, it is urgent to find a nanocomplex with good targeting effect, high biocompatibility and significant therapeutic effect for the integration of diagnosis and treatment and clinical transformation.. Nanoparticles (NPs) targeting breast cancer were synthesized by phacoemulsification which had liquid fluorocarbon perfluoropentane(PFP) in the core and were loaded with Iron(II) phthalocyanine (FePc) on the shell. The aptamer (APT) AS1411 was outside the shell used as a molecular probe. Basic characterization and targeting abilities of the NPs were tested, and their cytotoxicity and biological safety in vivo were evaluated through CCK-8 assay and blood bio-chemical analysis. The photoacoustic (PA) and ultrasound (US) imaging system were used to assess the effects of AS1411-PLGA@FePc@PFP (A-FP NPs) as dual modal contrast agent in vitro and in vivo. The effects of photothermal therapy (PTT) in vitro and in vivo were evaluated through MCF-7 cells and tumor-bearing nude mouse models.. A-FP NPs, with good stability, great biocompatibility and low toxicity, were of 201.87 ± 1.60 nm in diameter, and have an active targeting effect on breast cancer cells and tissues. With the help of PA/US imaging, it was proved to be an excellent dual modal contrast agent for diagnosis and guidance of targeted therapy. Meanwhile, it can heat up under near-infrared (NIR) laser irradiation and has achieved obvious antitumor effect both in vitro and in vivo experiments.. As a kind of nanomedicine, A-FP NPs can be used in the integration of diagnosis and treatment. The treatment effects and biocompatibility in vivo may provide new thoughts in the clinical transformation of nanomedicine and early diagnosis and treatment of breast cancer.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Contrast Media; Female; Fluorocarbons; Humans; Indoles; Iron; Isoindoles; MCF-7 Cells; Mice, Inbred BALB C; Multifunctional Nanoparticles; Oligodeoxyribonucleotides; Phototherapy; Polylactic Acid-Polyglycolic Acid Copolymer; Ultrasonography; Xenograft Model Antitumor Assays

Recently, a DNA tetrahedron has been reported to be a novel nanomedicine and promising drug vector because of its compactness, biocompatibility, biosafety, and editability. Here, we modified the DNA tetrahedron with a DNA aptamer (AS1411) as a DNA-based delivery system, which could bind to nucleolin for its cancer cell selectivity. Nucleolin is a specific biomarker protein overexpressed on membranes of malignant cancer cells and its deregulation is implicated in cell proliferation. The antimetabolite drug 5-fluorouracil (5-FU) is an extensively used anticancer agent; however, its major limitation is the lack of target specificity. Cyanine 5 (Cy5), a fluorescent probe, can be used to label DNA tetrahedron and enhance photostability with minimal effects on its basic functions. In this study, we additionally attached 5-FU to the DNA-based delivery system as a new tumor-targeting nanomedicine (AS1411-T-5-FU) to enhance the therapeutic efficacy and targeting of breast cancer. We examined the difference of the cellular uptake of AS1411-T-5-FU between breast cancer cells and normal breast cells and concluded that AS1411-T-5-FU had a better targeting ability to kill breast cancer cells than 5-FU. We further evaluated the expressions of cell apoptosis-related proteins and genes, which are associated with the mitochondrial apoptotic pathway. Ultimately, our results suggest the potential of DNA tetrahedron in cancer therapies, and we develop a novel approach to endow 5-FU with targeting property.

Topics: Antimetabolites; Apoptosis; Aptamers, Nucleotide; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Drug Carriers; Female; Fluorouracil; Humans; Mitochondria; Nanomedicine; Oligodeoxyribonucleotides; Proto-Oncogene Proteins c-bcl-2

Topics: Aptamers, Nucleotide; Breast Neoplasms; Delayed-Action Preparations; Female; Fluorouracil; Humans; MCF-7 Cells; Nanocapsules; Oligodeoxyribonucleotides; Particle Size

To enhance efficacy of chemotherapy and achieve real-time imaging of cancer cells, it is crucial to develop nanocarriers with targeted drug delivery capacity and fluorescence property for cancer theranostics. Herein, a dual-targeting DNA tetrahedron nanocarrier (MUC1-Td-AS1411) was constructed for breast cancer cell imaging and targeted drug delivery. This nanocarrier consisted of three components: (i) DNA tetrahedron core for multivalent conjugation of function ligands and loading doxorubicin (Dox); (ii) activatable MUC1 aptamer probe (MUC1-probe), formed by the hybridization of MUC1 aptamer sequence with fluorophore extended from one vertex and complementary sequence with quencher, for targeting and imaging MUC1 protein on cytomembrane; (iii) AS1411 aptamer, which was hybridized to the overhang on three vertexes via prolonged sequence, for binding to nucleolin. Firstly, MUC1-probe of this nanocarrier targeted MUC1 protein of MUC1-positive cells, causing a conformational reorganization of MUC1 aptamer, releasing complementary sequence with quencher and leading to fluorescence recovery. Subsequently, after internalizing into cells, AS1411 aptamer moiety of nanocarrier bound to nucleolin selectively, then the whole nanocarrier targeted nucleus and released Dox into nucleus. MUC1-positive cells and MUC1-negative cells could be differentiated by means of fluorescence imaging. Versus free Dox, Dox-loaded MUC1-Td-AS1411 showed lower cytotoxicity to MUC1-negative HL-7702 cells (P < 0.01), approximately equal lethality to sensitive MCF-7 cells (P > 0.05) whereas more effective to doxorubicin-resistant MCF-7 cells (P < 0.01). Therefore, this nanocarrier could be used as a promising candidate for cancer theranostics.

Topics: Antibiotics, Antineoplastic; Aptamers, Nucleotide; Breast Neoplasms; Cell Nucleus; Diagnostic Imaging; DNA; Doxorubicin; Drug Carriers; Female; Gene Expression; Hep G2 Cells; Humans; MCF-7 Cells; Molecular Targeted Therapy; Mucin-1; Nanostructures; Neoplasm Proteins; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Protein Binding; RNA-Binding Proteins; Theranostic Nanomedicine

ErbB2, a member of the ErbB family of receptor tyrosine kinases, is an essential player in the cell's growth and proliferation signaling pathways. Amplification or overexpression of ErbB2 is observed in ∼30% of breast cancer patients, and often drives cellular transformation and cancer development. Recently, we have shown that ErbB2 interacts with the nuclear-cytoplasmic shuttling protein nucleolin, an interaction which enhances cell transformation in vitro, and increases mortality risk and disease progression rate in human breast cancer patients. Given these results, and since acquired resistance to anti-ErbB2-targeted therapy is a major obstacle in treatment of breast cancer, we have examined the therapeutic potential of targeting the ErbB2-nucleolin complex. The effect of the nucleolin-specific inhibitor GroA (AS1411) on ErbB2-positive breast cancer was tested in vivo, in a mouse xenograft model for breast cancer; as well as in vitro, alone and in combination with the ErbB2 kinase-inhibitor tyrphostin AG-825. Here, we show that in vivo treatment of ErbB2-positive breast tumor xenografts with GroA reduces tumor size and leads to decreased ErbB2-mediated signaling. Moreover, we found that co-treatment of breast cancer cell lines with GroA and the ErbB2 kinase-inhibitor tyrphostin AG-825 enhances the anti-cancer effects exerted by GroA alone in terms of cell viability, mortality, migration, and invasiveness. We, therefore, suggest a novel therapeutic approach, consisting of combined inhibition of ErbB2 and nucleolin, which has the potential to improve breast cancer treatment efficacy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aptamers, Nucleotide; Benzothiazoles; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Drug Synergism; Female; Humans; MCF-7 Cells; Mice; Mice, Nude; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Receptor, ErbB-2; RNA-Binding Proteins; Signal Transduction; Tyrphostins; Xenograft Model Antitumor Assays

Active targeting of nanostructures containing chemotherapeutic agents can improve cancer treatment. Here, a three-way junction pocket DNA nanostructure was developed for efficient doxorubicin (Dox) delivery into cancer cells. The three-way junction pocket DNA nanostructure is composed of three strands of AS1411 aptamer as both a therapeutic aptamer and nucleolin target, the potential biomarker of prostate (PC-3 cells) and breast (4T1 cells) cancers. The properties of the Dox-loaded three-way junction pocket DNA nanostructure were characterized and verified to have several advantages, including high serum stability and a pH-responsive property. Cellular uptake studies showed that the Dox-loaded DNA nanostructure was preferably internalized into target cancer cells (PC-3 and 4T1 cells). MTT cell viability assay demonstrated that the Dox-loaded DNA nanostructure had significantly higher cytotoxicity for PC-3 and 4T1 cells compared to that of nontarget cells (CHO cells, Chinese hamster ovary cell). The in vivo antitumor effect showed that the Dox-loaded DNA nanostructure was more effective in prohibition of the tumor growth compared to free Dox. These findings showed that the Dox-loaded three-way junction pocket DNA nanostructure could significantly reduce the cytotoxic effects of Dox against nontarget cells.

Topics: Animals; Aptamers, Nucleotide; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Survival; CHO Cells; Cricetulus; DNA; DNA Adducts; Doxorubicin; Drug Carriers; Drug Delivery Systems; Female; Humans; Male; Mice; Mice, Inbred BALB C; Nanoparticles; Nanostructures; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Prostatic Neoplasms; RNA-Binding Proteins

Herein, the AS1411 aptamer-targeted ultrasmall gold nanoclusters (GNCs) were assessed at different aspects as a radiosensitizer.. AS1411 aptamer-conjugated gold nanoclusters (Apt-GNCs) efficacy was evaluated at cancer cells targeting, radiosensitizing effect, tumor targeting, and biocompatibility in breast tumor-bearing mice.. Flow cytometry and fluorescence microscopy exhibited more cellular uptake for Apt-GNCs in comparison with GNCs. In addition, inductively coupled plasma optical emission spectrometry results demonstrated its effective tumor targeting as the tumors' gold content for GNCs and Apt-GNCs were 8.53 and 15.33 μg/g, respectively. Apt-GNCs significantly enhanced radiotherapy efficacy as mean tumors' volume decreased about 39% and 9 days increase in the mice survival was observed. Both GNCs and Apt-GNCs were biocompatible.. The Apt-GNCs is a novel and efficient  radiosensitizer.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Female; Flow Cytometry; Humans; Metal Nanoparticles; Mice; Oligodeoxyribonucleotides; Radiation-Sensitizing Agents

Small interfering RNAs (siRNA)/microRNAs (miRNA) have promising therapeutic potential, yet their clinical application has been hampered by the lack of appropriate delivery systems. Herein, we employed extracellular vesicles (EVs) as a targeted delivery system for small RNAs. EVs are cell-derived small vesicles that participate in cell-to-cell communication for protein and RNA delivery. We used the aptamer AS1411-modified EVs for targeted delivery of siRNA/microRNA to breast cancer tissues. Tumor targeting was facilitated via AS1411 binding to nucleolin, which is highly expressed on the surface membrane of breast cancer cells. This delivery vesicle targeted let-7 miRNA delivery to MDA-MB-231 cells

Topics: Animals; Antineoplastic Agents; Aptamers, Nucleotide; Biological Products; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Drug Carriers; Drug-Related Side Effects and Adverse Reactions; Extracellular Vesicles; Mice; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; RNA, Small Untranslated; Treatment Outcome

Aptamers are single-stranded RNA or DNA ligands that can specifically bind to various molecular targets with high affinity. Owing to this unique character, they have become increasingly attractive in the field of drug delivery. In this study, we developed a multifunctional composite micelle (CM) with surface modification of aptamer AS1411 (Ap) for targeted delivery of doxorubicin (DOX) to human breast tumors. This binary mixed system consisting of AS1411 modified Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-b-polylactide could enhance DOX-loading capacity and increase micelle stability. Cellular uptake of CM-Ap was found to be higher than that of untargeted CM due to the nucleolin-mediated endocytosis effect. In vivo study in MCF-7 tumor-bearing mice demonstrated that the AS1411-functionalized composite micelles showed prolonged circulation time in blood, enhanced accumulation in tumor, improved antitumor activity, and decreased cardiotoxicity. In conclusion, aptamer-conjugated multifunctional composite micelles could be a potential delivery vehicle for cancer therapy.

Topics: Animals; Antineoplastic Agents; Aptamers, Nucleotide; beta-Cyclodextrins; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclodextrins; Doxorubicin; Drug Delivery Systems; Female; Humans; Mice; Micelles; Nanomedicine; Neoplasm Transplantation; Oligodeoxyribonucleotides; Poloxamer; Polyethylene Glycols

AS1411 is a quadruplex-forming DNA oligonucleotide that functions as an aptamer to target nucleolin, a protein present on the surface of cancer cells. Clinical trials of AS1411 have indicated it is well tolerated with evidence of therapeutic activity, but improved pharmacology and potency may be required for optimal efficacy. In this report, we describe how conjugating AS1411 to 5 nm gold nanospheres influences its activities in vitro and in vivo. We find that the AS1411-linked gold nanospheres (AS1411-GNS) are stable in aqueous and serum-containing solutions. Compared to unconjugated AS1411 or GNS linked to control oligonucleotides, AS1411-GNS have superior cellular uptake and markedly increased antiproliferative/cytotoxic effects. Similar to AS1411, AS1411-GNS show selectivity for cancer cells compared to non-malignant cells. In a mouse model of breast cancer, systemic administration of AS1411-GNS could completely inhibit tumor growth with no signs of toxicity. These results suggest AS1411-GNS are promising candidates for clinical translation.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Female; Gold; Humans; MCF-7 Cells; Metal Nanoparticles; Mice; Mice, Nude; Nanospheres; Oligodeoxyribonucleotides; Random Allocation; Xenograft Model Antitumor Assays

Targeted uptake of therapeutic nanoparticles in cell- or tissue-specific manner is an attractive technology since they can offer greater efficacy and reduce cytotoxicity on peripheral healthy tissues. In this study, AS1411 (AP), a DNA aptamer specifically binding to nucleolin that is overexpressed on the plasma membrane of breast cancer (BC) cells, was exploited as the targeting ligand of a nanoparticle-based drug delivery system. Vinorelbine (VRL) loaded PLGA-PEG nanoparticles (NP) were formulated by an emulsion/solvent evaporation method, and AP was conjugated to the particle surface using the EDC/NHS technique. The drug-loading efficiency and in vitro drug release studies were measured using HPLC. The resulting AP-NP/VRL formed spherical nanoparticles (<200 nm) with drug loading of about 7% and a stable in vitro drug release profile. Fluorescence microscopy was used to confirm the cellular uptake of the particles and targeted drug delivery. Moreover, cytotoxicity studies were carried out in two different cell lines, MDA-MB-231 BC cells and MCF-10A normal epithelial cells. AP-nucleolin interaction significantly enhanced in vitro cytotoxicity to nucleolin overexpressed cells, as compared with non-targeted nanoparticles, while there was no significant difference in cytotoxicity of the two types of nanoparticles on the nucleolin negative cells. The results further support that AS1411-functionalized nanoparticles are potential carrier candidates for targeted drug delivery towards BC.

Topics: Antineoplastic Agents; Aptamers, Nucleotide; Breast Neoplasms; Cell Line; Cell Line, Tumor; Drug Delivery Systems; Female; Humans; Lactic Acid; Nanoparticles; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; RNA-Binding Proteins; Vinblastine; Vinorelbine

Numerous studies have described the altered expression and the causal role of microRNAs (miRNAs) in human cancer. However, to date, efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here we find that nucleolin (NCL), a major nucleolar protein, posttranscriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, that are causally involved in breast cancer initiation, progression, and drug resistance. We also show that NCL is commonly overexpressed in human breast tumors and that its expression correlates with that of NCL-dependent miRNAs. Finally, inhibition of NCL using guanosine-rich aptamers reduces the levels of NCL-dependent miRNAs and their target genes, thus reducing breast cancer cell aggressiveness both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estradiol; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Genes, Neoplasm; Guanine; HEK293 Cells; Humans; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; Transcription, Genetic; Up-Regulation

As an emerging category of fluorescent metal nanoclusters, oligonucleotide-templated silver nanoclusters (Ag NCs) have attracted a lot of interest and have shown wide application in biorelated disciplines. However, the weak fluorescence emission and poor permeability to cell membranes tethered further intracellular applications of Ag NCs. AS1411 is an antiproliferative G-rich phosphodiester oligonucleotide and currently an anticancer agent under phase II clinical trials. Herein, we present a strategy to synthesize AS1411-functionalized Ag NCs with excellent fluorescence through a facile one-pot process. Confocal laser scanning microscopy and Z-axis scanning confirmed that the AS1411-functionalized Ag NCs could be internalized into MCF-7 human breast cancer cells and were able to specifically stain nuclei with red color. To our surprise, 3-[4,5-dimethylthiazol-z-yl]-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated the Ag NCs were cytocompatible and showed better inhibition effects than pure AS1411 on MCF-7 human breast cancer cells. In addition, a universal design of the oligonucleotide scaffold for synthesis of Ag NCs was extended to other aptamers, such as Sgc8c and mucin 1 aptamer. Due to the facile synthesis procedure and capability of specific target recognition, this fluorescent platform will potentially broaden the applications of Ag NCs in biosensing and biological imaging.

Topics: Animals; Antineoplastic Agents; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Female; Fluorescent Dyes; Humans; Mice; Microscopy, Confocal; Nanostructures; NIH 3T3 Cells; Oligodeoxyribonucleotides; Silver

The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.. FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.. Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; COUP Transcription Factor II; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Neoplasm Grading; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, Retinoic Acid; Repetitive Sequences, Nucleic Acid; RNA-Binding Proteins; Tissue Array Analysis; Transcriptional Activation; Tretinoin

AS1411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-forming oligodeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completely understood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to novel targeted therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for AS1411, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS1411 (FL-AS1411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblasts (resistant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much more efficient than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower in cancer cells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macropinocytosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment of various cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in its own uptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required for initial FL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411 uptake at later times. Our results are inconsistent with the previous mechanistic model but confirm that nucleolin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drug delivery.

Topics: Antineoplastic Agents; Aptamers, Nucleotide; Blotting, Western; Breast; Breast Neoplasms; Cells, Cultured; Drug Delivery Systems; Female; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Humans; Male; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Pinocytosis; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; RNA, Small Interfering; Skin

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.

Topics: Apoptosis; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Genes, bcl-2; Humans; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Proteins; RNA, Messenger