Anti-proliferation and induction of mitochondria-mediated apoptosis by Garcinia hanburyi resin in colorectal cancer cells.

Introduction: Several parts of Garcinia hanburyi are used in traditional medicine for many purposes. In this study, Garcinia hanburyi resin (GHR) was explored for possible anti-proliferative effects and the underlying mechanism on colorectal cancer (CRC) cells. Methods: Gambogic acid (GA) content in GHR was analyzed by HPLC method. The cytotoxicities of GA and GHR were assessed in human CRC cell lines (SW480 and Caco-2) and normal colon cells (CCD841 CoN) using a trypan blue exclusion assay, MTS assay, and cell morphology analysis. Cell cycle and apoptosis at its half maximal inhibitory concentration (IC50) were analyzed using flow cytometry. And, the levels of intrinsic apoptosis-related proteins were measured by Western blot analysis. Results: GA was the major compound as 71.26% of the GHR. The cell viability of CRC cells was decreased in a time- and dose-dependent manner after exposure to GHR. The selectivity index indicated that GHR had a high degree of selectivity against CRC cells. The same result was obtained for GA treatment. In addition, GHR markedly induced typical apoptotic morphology of CRC cells, but had no obvious effect on normal colon cells. GHR induced apoptosis with the cell cycle arrest at the G2/M phase. An increase in Bax/Bcl-2 ratio and a decrease in procaspase-3 proteins indicated that GHR promoted apoptosis by disrupting the mitochondrial outer membrane permeability and the subsequent activation of caspase-3. Conclusion: GHR, which contained GA as an active compound, significantly inhibited CRC cell proliferation via the induction of intrinsic apoptosis, while having low toxicity on normal colon cells. Therefore, GHR could be proposed as a potent candidate for the treatment of CRC.

A molecular hybrid comprising AS1411 and PDGF-BB aptamer, cholesterol, and doxorubicin for inhibiting proliferation of SW480 cells.

Cancer treatment commonly relies on chemotherapy. This treatment faces many challenges, including treatment specificity and undesired side effects. To address these, a Dox-loaded Chol-aptamer molecular hybrid (Dox-CAH) was developed. This multivalent interaction system combines the key function of each integrated species: doxorubicin, cholesterol, and two aptamers binding to nucleolin and platelet-derived growth factor BB (PDGF-BB). The study has four stages: preparation of CAH via oligonucleotide hybridization, intercalation of doxorubicin into CAH, verification of CAH binding on SW480 by fluorescence microscopy and flow cytometry, and investigation of effect of Dox-CAH on SW480 proliferation. CAH was successfully prepared, as confirmed by electrophoresis. Flow cytometry and fluorescence microscopy demonstrated CAH binding to SW480, due to the presence of the AS1411 aptamer. This molecular hybrid exhibited specific binding because it did not bind to CCD 841 CoN. CAH binding to PDGF-BB compromises its function, as shown by enzyme-linked immunosorbent assay (ELISA) and cell assay. The DNA duplex in this molecular hybrid reduces the cytotoxicity of the Dox-CAH. Binding and the reduction of Dox-CAH toxicity may improve treatment specificity and minimize side effects. Dox-CAH is a model for more effective anticancer therapy, allowing incorporation of chemotherapeutic drugs and recognition elements.

Anti-Proliferative Effect of Doxorubicin-Loaded AS1411 Aptamer on Colorectal Cancer Cell.

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.

Inhibition of Colorectal Cancer Cell Proliferation by Regulating Platelet-Derived Growth Factor B Signaling with a DNA Aptamer

Background: Overexpression of platelet-derived growth factor-BB (PDGF-BB) is associated with colorectal carcinogenesis. PDGF-BB plays a role in the autocrine growth stimulation of cancer cells. Aptamers are short single-stranded oligonucleotides that can bind to cellular targets with high affinity and specificity and offer the advantage of non-immunogenicity, non-toxicity and high stability. Thus, they receive interest as potential therapeutic agents. Methods: The endogenous level of PDGF-BB in Caco-2 and SW480, colorectal cancer (CRC) cells, was evaluated using ELISA. The effect of the PDGF-BB aptamer on cell proliferation was investigated in two CRC cell lines and CCD841 CoN, normal colon cells. The effective molar ratio between PDGF-BB and PDGF-BB aptamer was further explored. Cell viability in all experiments was analyzed using MTS assay. Western blotting was performed to examine the alteration of relevant signaling pathways. Results: Caco-2 and SW480 cells endogenously synthesized and secreted PDGF-BB to stimulate their growth. Cells treated with the PDGF-BB aptamer proliferated at a slower rate, but CCD841 CoN did not. Pre-incubation of PDGF-BB with the corresponding aptamer at the molar ratio 1:1 could significantly silence its proliferative effect on CRC cells. Western blot analysis revealed that the phosphorylation level of ERK1/2, a key component in PDGF downstream signaling pathway, was down-regulated by the aptamer, indicating the underlying mechanism of inhibition of CRC cell proliferation. Conclusions: This study demonstrated that using a DNA aptamer to interfere with the binding of PDGF-BB to its receptor suppressed CRC cell proliferation in part via down-regulation of the Ras/Raf/MEK/ERK signaling pathway. It raised the possibility that the PDGF-BB-specific aptamer could be a promising therapeutic agent for CRC targeted therapy.

Effect of PDGF-B aptamer on PDGFRß/PDGF-B interaction: Molecular dynamics study.

PDGFRß/PDGF-B interaction plays a role in angiogenesis, and is mandatory in wound healing and cancer treatment. It has been reported that the PDGF-B aptamer was able to bind to PDGF-B, thus regulating the angiogenesis. However, the binding interaction between the aptamer and the growth factor, including the binding sites, has not been well investigated. This study applied a molecular dynamics (MD) simulation to investigate the aptamer-growth factor interaction in the presence or absence of a receptor (PDGFRß). Characterization of the structure of an aptamer-growth factor complex revealed binding sites from each section in the complex. Upon the complex formation, PDGF-B and its aptamer exhibited less flexibility in their molecular movement, as indicated by the minimum values of RMSD, RMSF, loop-to-loop distance, and the summation of PCA eigenvalues. Our study of residue pairwise interaction demonstrated that the binding interaction was mainly contributed by electrostatic interaction between the positively-charged amino acid and the negatively-charged phosphate backbone. The role of the PDGF-B aptamer in PDGFRß/PDGF-B interaction was also investigated. We demonstrated that the stability of the Apt-PDGF-B complex could prevent the presence of a competitor, of PDGFRß, interrupting the binding process. Because the aptamer was capable of binding with PDGF-B, and blocking the growth factor from the PDGFRß, it could down regulate the consequent signaling pathway. We provide evidence that the PDGF-BB aptamer is a promising molecule for regulation of angiogenesis. The MD study provides a molecular understanding to modification of the aptamer binding interaction, which could be used in a number of medical applications.

Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer.

Truncation can enhance the affinity of aptamers for their targets by limiting nonessential segments and therefore limiting the molecular degrees of freedom that must be overcome in the binding process. This study demonstrated a truncation protocol relying on competitive antibody binding and the hybridization of complementary oligonucleotides, using platelet derived growth factor BB (PDGF-BB) as the model target. On the basis of the immunoassay results, an initial long aptamer was truncated to a number of sequences with lengths of 36-40 nucleotides (nt). These sequences showed apparent KD values in the picomolar range, with the best case being a 36-nt truncated aptamer with a 150-fold increase in affinity over the full-length aptamer. The observed binding energies correlated well with relative energies calculated by molecular dynamics simulations. The effect of the truncated aptamer on PDGF-BB-stimulated fibroblasts was found to be equivalent to that of the full-length aptamer.

Aptamer-gelatin composite for a trigger release system mediated by oligonucleotide hybridization.

Nucleic acid aptamers not only specifically bind to their target proteins with high affinity but also form intermolecular hybridization with their complementary oligonucleotides (CO). The hybridization can interrupt aptamer/protein interaction due to the changes of aptamer secondary structure which rely on hybridization length and base-pairing positions. Herein we aim to use this unique property of the aptamers, when combined with gelatin to develop a novel composite with desirable protein release profiles. Platelet-derived growth factor-BB (PDGF-BB) and its aptamer were used as target molecules. Prior to performing the release study, the effects of CO on aptamer-protein interaction were observed by surface plasmon resonance (SPR). The SPR sensorgram indicated that the aptamer dissociated from the bounded proteins when it hybridized with the CO. The aptamer was then immobilized onto streptavidin coated polystyrene particles via biotin/streptavidin interaction. Then, PDGF-BB and aptamer functionalized particles were mixed with gelatin solution and cast as small pieces of composite. The success of the composite preparation was confirmed by flow cytometry and microscopy. PDGF-BB release at several time points was quantified by ELISA. The results showed that the aptamer-gelatin composite could slow the release rate of the proteins from the composite due to strong binding of proteins and aptamers. Once the CO was added to the system, the release rate was significantly enhanced because the aptamer hybridized with the CO and lost its active secondary structure. Therefore, the proteins were triggered to release out from the composite. This work suggests a promising strategy for controlling the release of bioactive molecules in medical treatments.

Programmable release of multiple protein drugs from aptamer-functionalized hydrogels via nucleic acid hybridization.

Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer-protein interactions and aptamer-CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

Enhanced loading and controlled release of antibiotics using nucleic acids as an antibiotic-binding effector in hydrogels.

Antibiotic delivery is important to treat bacterial infections, one of the most challenging health problems globally. This study explored the application of nucleic acids as an antibiotic-binding effector for antibiotic loading and release. The data showed that the partition coefficient of tetracycline increased proportionally to the oligonucleotide concentration ranging from 0 to 1 mM. Resultantly, the incorporation of the oligonucleotides led to enhanced tetracycline loading in the hydrogels. In addition to the enhanced drug loading, the oligonucleotides could slow the release of tetracycline from the hydrogels. Experiments were further carried out to examine the capability of oligonucleotide-functionalized hydrogels in the inhibition of bacterial growth. The results showed that the oligonucleotide-functionalized hydrogels had higher antibacterial efficiency. Moreover, after tetracycline release, the oligonucleotide-functionalized hydrogels could be refilled with fresh tetracycline to reproduce the capability of inhibiting bacterial growth. Therefore, nucleic acid oligonucleotides are a promising antibiotic-binding effector for hydrogel functionalization in antibiotic delivery.

Cell adhesion on an artificial extracellular matrix using aptamer-functionalized PEG hydrogels.

The development of an artificial extracellular matrix (ECM) is important to regenerative medicine because the ECM plays complex and dynamic roles in the regulation of cell behavior. In this study, nucleic acid aptamers were applied to functionalize hydrogels for mimicking the adhesion sites of the ECM. The results showed that nucleic acid aptamers could be incorporated into polyethylene glycol (PEG) hydrogels via free radical polymerization. The incorporation of the aptamers produced only a moderate effect on the mechanical properties of the PEG hydrogels. Importantly, the results also showed that the aptamers effectively induced cell type-specific adhesion to the PEG hydrogels without affecting cell viability. The cell adhesion was a function of the aptamer concentration, the spacer length and the cell seeding time. In addition, cell adhesion to the aptamer-functionalized hydrogel could be attenuated by means of aptamer inactivation in a physiological condition. Thus, aptamer-functionalized hydrogels are promising biomaterials for the development of artificial ECMs.

Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

Structural prediction and binding analysis of hybridized aptamers.

Few studies were performed to investigate the molecular recognition capabilities of hybridized aptamers. This study is aimed at applying both theoretical algorithms and experimental assays to examine the effects of hybridization length and region on the secondary structures and binding functionality of hybridized aptamers. The experimental results were significantly different from the structural predictions in many hybridization conditions. To explain this difference, we further proposed a novel equilibrium reaction model that can explicitly analyze the molecular interactions between hybridized aptamers and their targets. We believe that the research findings and the novel model can be used to guide numerous hybridized aptamer-based applications.

Nucleic acid aptamers for clinical diagnosis: cell detection and molecular imaging.

Nucleic acid aptamers have recently attracted significant attention in the field of clinical diagnosis because they have numerous merits, such as high affinity, high specificity, small size, little immunogenicity, stable structures, and ease of synthesis. This review focuses on discussing the potential applications of aptamers in cell detection and molecular imaging. For the ex vivo cell detection, this review discusses the status of five strategies: endogenous nucleic acid analysis, flow cytometry analysis, nanoparticle-based cell sensing, microfluidic cell separation, and histological examination. This review also discusses in vivo molecular and cell imaging by introducing aptamer-based molecular imaging, cell imaging, and integrated imaging and therapy. On the basis of the status of these promising studies, this review summarizes several challenging issues and unmet needs that may require more effort or attention in the future.

Aptamer-functionalized in situ injectable hydrogel for controlled protein release.

Various in situ injectable hydrogels have been developed for protein delivery in treating human diseases. However, most hydrogels are highly permeable, which can lead to the rapid release of loaded proteins. The purpose of this study is to apply nucleic acid aptamers to functionalize an in situ injectable hydrogel model to control the release of proteins. The aptamers were studied using secondary structural predictions and binding analyses. The results showed that the structural predictions were different from the experimental measurements in numerous cases. The affinity of the aptamer was significantly affected by the mutations of the essential nucleotides, whereas it was not significantly affected by the variations of the nonessential nucleotides. The mutated aptamers were then used to functionalize the injectable hydrogel model. The results showed that the aptamer-functionalized hydrogel could prolong protein release. Moreover, the release rates could be controlled by adjusting the affinity of the aptamer.

A temperature-responsive antibody-like nanostructure.

Antibodies play an essential role in various applications. However, antibodies exhibit considerable challenges in applications that require tunable binding capabilities and exposure to nonphysiological conditions such as chemical conjugation. This study is aimed to develop a novel antibody-like nanostructure with special features. The key components of the nanostructure are two DNA aptamers and a dendrimer. The aptamers are used to mimic the antigen-binding sites of an antibody; the dendrimer is used to provide a defined conjugation site for carrying molecules of interest. The results showed that the bivalent nanostructure exhibited a high binding affinity and specificity. Moreover, a temperature shift from 0 to 37 degrees C would trigger its rapid dissociation from the bound target cells, which is not possible in antibody-antigen complexes. Thus, an antibody-like nanostructure was successfully developed with novel features that natural antibodies do not possess.

Hydrogel functionalization with DNA aptamers for sustained PDGF-BB release.

We demonstrate that hydrogel functionalization with DNA aptamers can be applied for developing a novel sustained-release system.

Development of a novel pretargeting system with bifunctional nucleic acid molecules.

This study was aimed at exploring a novel pretargeting system based upon bifunctional nucleic acid molecules that are comprised of a nucleic acid aptamer and a nucleic acid tail. The properties of bifunctional molecules were investigated by both theoretical prediction and experimental determination. Different from the algorithm-based structure prediction, the experimental data showed that some nucleic acid tails could significantly decrease the binding capability of the aptamer. It was also found that the effectiveness of bifunctional molecules in labeling cells was dependent on the hybridization length. Based on these understandings, one bifunctional molecule was selected to study pretargeting. The results demonstrated that the bifunctional molecule could not only bind to target cells, but also hybridize with its complementary oligonucleotide on the cell surface. Thus, bifunctional nucleic acid molecules hold great potential for pretargeting applications.

A hybrid DNA aptamer-dendrimer nanomaterial for targeted cell labeling.

Antibodies are natural nanomaterials and have been widely used for targeted cell labeling. However, the applications of antibodies are often limited by their large size and instability. The purpose of this study is to develop a new type of multifunctional nanomaterial that is comprised of a nucleic acid aptamer and a dendrimer, both of which are stable. This nanomaterial is approximately 8 nm in size. Moreover, it could not only carry multiple signal molecules, but also bind to target cancer cells with high affinity and specificity. This sub-10 nm multifunctional nanomaterial is expected to be useful in basic biomedical research and clinical medicine.