Generating Aptamers for Cancer Diagnosis and Therapy

Aptamers are single-stranded DNA or RNA oligonucleotides or peptides that are capable of binding to specific target proteins. Aptamers are generated in a process named SELEX or cell-SELEX. They resemble antigens in respect of their highly specific binding in the lower nanomolar and/or picomolar range. In contrast to antibodies, they can be generated in vitro, are more stable molecules and have less immunogenicity, better tissue penetration and faster clearance. Aptamers can be applied in many conditions, one of them being cancer medicine, because of their ability to detect cell-specific markers within complex systems that have no specified target molecules. Moreover, aptamers can be used as therapeutic agents, which are indirectly or directly conjugated with drugs, due to their toxic, inhibitory or activating effect on target cells. *Corresponding author: Avrelija Cencic, Faculty of Medicine, Department of Biochemistry and Nutrition, University of Maribor, Eu-slovenia, Tel: 00386 2 330 5871; E-mail: avrelija.cencic@uni-mb.si Received May 21, 2012; Accepted June 21, 2012; Published June 22, 2012 Citation: Marolt U, Cencic A, Gorenjak M, Potrc S (2012) Generating Aptamers for Cancer Diagnosis and Therapy. Clin Exp Pharmacol 2:111. doi:10.4172/21611459.1000111 Copyright: © 2012 Marolt U. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The term aptamer is derived from the Latin word "aptus", meaning "to fit", since aptamers are specific three-dimensional in vivo structures that are capable of binding to specific target proteins. They are generated in a process named SELEX. End products of this method are singlestranded DNA or RNA oligonucleotides (12-30 bases) or peptides with high specificity and affinity to their target molecules, which are produced from libraries that contain 10 13 to 10 16 single stranded DNA (ssDNA) or RNA molecules [1,2].
The concept was first described in 1990 by three different research groups. Robertson et al. reported discovering an RNA enzyme (mutated tetrahymena ribozyme) that cleaves single-stranded DNA in a specific sequence [3]. Tuerk et al. described a procedure that depends on alternated cycles of ligand selection from pools with different nucleotide sequences and species-bound amplification. This is when the term "Systematic Evolution of Ligand by Exponential Enrichment"-SELEX was introduced [4]. The Ellington study group named the end products of the in vitro selection process "aptamer" [2]. Since 1990, numerous researches were carried out. To illustrate, a simple pubmed search for the term aptamer shows 2781 hits. Small molecules (dyes, metal ions, amino acids, small peptides) [5][6][7][8][9][10], proteins [11], viruses [12], biological cells [13,14] and bacteria [15] were studied as potential aptamer targets. Moreover, cancer related proteins were investigated, such as PDGF, VEGF, HER3, NFƙB, tenascin-C and the prostatespecific membrane antigen [16][17][18]. In general, researches have proved that aptamers can be used in cancer medicine for detecting cancer cells, cancer biomarkers and enriching rare cancer cells with nanoparticle technology. Aptamers can be further implemented as target therapy for specific cancer. In this manner they can be toxic per se or increase the efficiency and sensitivity of specific chemotherapy [19].

Aptamers against Antibodies
Monoclonal antibodies that bind to tumor antigens are commonly used as therapeutic agents in different neoplasms. One such agent is a tyrosine kinase inhibitor (imatinib mesylate; industrial name Gleevec) that is used for treating chronic myeloid leukemia. Therapy with imatinib mesylate can lead to a regression of advanced tumors by inhibiting thyrosine kinase activity in overexpressed Brc-Abl oncoprotein [20,21].
Aptamers resemble antibodies in respect of their highly specific binding in the picomolar and/or nanomolar range. When comparing these two agents, we see that aptamers possess some advantages over antibodies. First of all, they are generated in an in vitro setting, which allows them screening against molecules with weak immunogenicity and toxicity [30,31]. Antibodies lack this property, because they are produced in an in vivo session. Aptamers are also stable molecules; they can be heated to 80 º C, exposed to harsh environments and stored in different solvents without any irreversible conformation changes. Furthermore, compared to antibodies, they have little immunogenicity, as it was proven in a study on monkeys, which received doses that were 1000-fold larger than the therapeutic one, but little or no immunogenic effect was observed [32]. In addition, they can be chemically modified to improve stability in blood, biodistribution, tissue penetration and clearance [33]. The most often used chemical modifications of aptamers include 2´-amino pyrimidines, 2´-fluoro pyrimidines, 2´-O-methyl nucleotides, 2´-ribo nucleotides, 5-modofied pyrimidines and 4´-thio pyramidines [34]. Finally, the synthesis of aptamers is quite easy to perform.
It has also been determined that the vascular endothelian growth factor (VEGF) is a potential target. It was tested in a mice model of renal cancer and was associated with tumor regression [37][38][39].
Different aptamer substances are at this time in various phases of clinical and preclinical trials. Studies especially focus on therapeutic use in conditions, such as age-related macula degeneration, acute myloid leukemia, autologous stem cell transplantation, type 2 diabetes mellitus, heart disease, coronary artery disease etc. In 2004, the first aptamer therapeutic was approved by the FDA. The agent (Pegaptanib, Macugen) has a binding domain on VEGF 165 and is used against agerelated macula degeneration [40]. Some of the other aptamers that are in clinical trials are listed in Table 1.

SELEX Tecnology (Systematic Evolution of Ligand by Exponential Enrichment)
SELEX technology ( Figure 1) is an evolutionary in vitro iterative panning procedure that is used for selecting aptamers, which specifically identify targets that range from small molecules to proteins [2,4]. However, cancer cells are characterized by cell surface molecules that overexpress or alter due to multiple oncogenic mutations. Different approaches have been discovered to target such surface molecules. The initial step of the SELEX procedure is the synthesis of a single-stranded nucleic acid (RNA/DNA) library of large sequence complexity (typical length is 20 to 100 nucleotides, flanked by two constant regions with primer sites for PCR amplification). The second is selecting oligonucleotides, which are capable of binding to target molecules with high affinity and specificity. DNA aptamers can be selected directly from a DNA library, whereas in case of RNA aptamers, an in vitro transcription of a DNA library into an RNA library has to be performed. A typical aptamer library with a variable region of 24 to 25 nucleotides has a predictive complexity of 4 24 to 4 25 different molecules.
The next steps of the SELEX procedures are: (a) nucleic acid pool folding in a specific buffer, in order to obtain specific conformational changes; (b) incubating the aptamer library with a target molecule, such as a recombinant protein within a binding buffer (smart aptamers with predefined equilibrium constant Kd, generated by the Equilibrium Capillary Electrophoresis of Equilbrium Mixtures (ECEEM), can be used [41]); (c) partitioning unbound nucleic acids from specifically bound ones to the target molecule; (d) dissociating the nucleic acidprotein complexes and (e) amplifying the nucleic acid pool that is enriched with sequences that allow binding to selected target molecules with PCR (DNA SELEX) or reverse transcriptase (RT)-PCR for RNA SELEX. The new generated nucleic acid pool serves as a starting library for a new SELEX cycle that consists of the same steps as the first round. The number of SELEX repetitions depends on the used library type and on specific enrichment achieved per selection cycle. After the last round of aptamer selection, PCR products are cloned and sequenced. Typically, about 50 aptamer clones are analyzed through sequencing [2,4,42]. After that, sequence alignments are performed, in order to assess the complexity of the selected aptamer pool and to group together aptamer clones with homologous sequences. Alignments are mostly performed with internet programs, such as CLUSTAL W (http://www.ebi.ac.uk/clustalw) [43]. Aptamer sequences are analyzed, in order to obtain relevant structures for binding. This step is conducted by the mfold program that is available online at http://www.bioinfo. rpi.edu/applications/mfold/DNAforml.cgi. The main purpose of this step is to identify possible configurations of single-stranded nucleic acids, such as stems, loops or bulges [44]. Binding studies follow, in order to determine specificity and affinity of selected aptamers. Besides, aptamer-target interactions are defined with footprinting [45][46][47]. The three-dimensional binding structure of the aptamer-target complex is visualized by using nuclear magnetic resonance (NMR) spectroscopy, X-ray structure analysis and crystallization [48,49].
Partitioning unbound nucleic acids from specifically bound ones represents one of the key steps in SELEX technology. That is why some new tools for isolation were implied, such as flow cytometry, surface plasmon resonance [50], capillary gel electrophoresis [41,51,52] and microfluid devices [53]. All this methods have simplified the procedure, making it more effective and faster.

Cell-SELEX Technology and Biopanning and Rapid Analysis of Selective Interactive Ligands
Cell-SELEX technology ( Figure 2) allows targeting molecules on the surface of living cells, which has some advantages over classic SELEX technology. Cell-SELEX seems to be appropriate for identifying cancer cells and generating aptamers that bind specifically to altered surface molecules on cancer cells without the exact knowledge of changed sequences. The steps in cell-SELEX technology resemble the SELEX procedure, but have some differences. The DNA library, which consists of 30-40 bases that are flanked by primer sequences, is being incubated with target cells in specific conditions. The unbound DNA probes are washed out and the bound ones are collected with heat denaturing or phenol-chloroform and EtOH precipitation. The eluant is then amplified by PCR or used for counter selection. Counter selection represents a step in cell-SELEX, where aptamer probes are incubated with negative (control) cells. This time, the unbound probes are collected and amplified. This step is crucial for removing nucleic acids that bind to normally expressed membrane proteins. The final step consists of a double-stranded DNA (dsDNA) amplification that is treated with streptavidin beads, which results in antisens strands with biotin retaining on the beads and sens strand elution with sodium hydroxide solution. At the end, eluted ssDNA represents the enriched library for the next round of cell-SELEX. To prove that high affinity ligands are being enriched, flow cytometry or fluorescence microscopy is used to detect fluorescin isothiocyanate (FITC) labelled as a 5´ forward primer. The pool is usually composed of high affinity ligands after 15-25 cycles of the cell-SELEX procedure. In the last step, the pool is cloned and sequenced, in order to determine the sequence of enriched DNA pools. The identified sequences are then chemically synthesized and labelled with fluorophore to evaluate the binding affinity [40,54].
Another method, which allows targeting of cell-surface proteins in a cell population, is called biopanning and rapid analysis of selective interactive ligands (BRASIL). The method allows separation of phagecell complexes from the remaining unbound phage. The separation is possible due to differential centrifugation, which leads cells from a hydrophilic environment to the hydrophobic-organic phase. This allows separation of unbound phage, which remains soluble in the upper hydrophilic phase, in just one centrifugation step. The above mentioned characteristics make BRASIL a simple and efficient cell panning method. Moreover, it can be used for targeting and isolating ligand-receptor pairs in cell populations, which are isolated from clinical samples as fine-needle aspirates [55]. Nevertheless, in SELEX technology, aptamers, which act against complex targets, are selected in 8-10 repetitions of the cycle, while cell-SELEX requires more than 14 repetitions. BRASIL, on the other hand, requires a single centrifugation step. Despite of this, cell SELEX has one clear advantage over SELEX. The process can produce multiple ligands that have high affinity to target-positive cells, but not the control cells. This is the end product of counter-selection, which can be further used to discriminate two closely related cell populations as observed in malignant glioma cells, T-cell limphobastic leukemia against B-cell lymphoma, as well as small-cell and large-cell cancer [13,56,57]. Cell-SELEX technology makes it feasible to discriminate amongst closely related cell types or even within the same cell under different conditions [42].
When comparing RNA aptamers against DNA aptamers, some differences can be observed. The selection procedure for DNA aptamers is simpler and cheaper. Furthermore, reverse transcription is essential. DNA aptamers are generally more stable than RNA aptamers. On the other hand, RNA aptamers possess a higher level of structural diversity due to the frequent occurrence of modified nucleotides within their structure, their base paring properties and tendency to form threedimensional structures [58].

Aptamers in Cancer Cell Diagnostics and Treatment
Cancer represents a group of diseases with a genetic compound, which can affect any part of the body. It is a leading cause of death worldwide, affecting around 13% of the population. Cancer is a major public health problem and an economic burden. The goal is to detect and treat the disease in its early stage, in order to achieve a better outcome. However, the main problem is the lack of sensitive and specific methods for discovering cancer in its early stages, due to the small amount of circulating cancer cells and low expression of biomarkers or specifically molecular markers. On the other hand, chemotherapy, as a main or supplementary therapy, is toxic for healthy cells. However, this is where aptamers can be applied, due to their ability to detect cell-specific markers within complex systems that are devoid of specified target molecules. Moreover, aptamers, which are highly specific for cancer cells, can be conjugated with therapeutic agents or small interfering RNA (siRNA). Aptamers can also be used directly, as medicinal substances, because of their toxic effect on certain target cells. They can be applied as regulators of the intrinsic intracellular pathway, due to their inhibitory effect on oncogens, and thus inhibit tumor growth and invasion.
As previously mentioned, aptamers represent one alternative   on how to capture and visualize cancer cells. Usually advanced biotechnology, nanotechnology or analytical methods are used in addition. So far, five promising approaches for cell detection seem to be: endogenous nucleic acid analysis [59]; flow cytometry analisis [60]; nanoparticle-based biosensing [61]; microfluid cell separation [62] and histological examination [63,64]. Also, non-invasive in vivo imaging techniques have been developed, in order to provide information on tumor progression and examination of cellular and biomolecular events.
Besides, aptamer-functionalized nanoparticles can be modified, so that they feature diagnostic and therapeutic properties, which were observed in a research by Bagalkot et al. that uses the anti-PSMA aptamer [65,66].

Aptamers Used in Alimentary Neoplasm
By reviewing recent research, we found some applications for aptamers in HCC (hepatocellular carcinoma), pancreatic adenocarcinoma and colorectal cancer. RNA and DNA aptamers were developed to target different cell surface proteins associated with tumor growth and biomarkers for certain types of cancer.
In hepatic neoplasm, osteopontin and the alfa-feto protein were observed as specific targets for aptamers. Osteopontin (OPN) is associated with HCC (hepatocellular carcinoma) growth and metastasis, and could therefore represent a unique therapeutic target. The study determined adhesion, migration/invasion and epithelialmesenchymal transition (EMT) markers by using an aptamer or a mutant control aptamer (Mu-aptamer). The study found out that aptamer that targets OPN significantly decreases EMT and tumor growth of HCC [77]. An RNA aptamer that specifically binds alphafetoprotein was also developed by using the SELEX technology. The identified aptamer specifically and efficiently inhibited the AFPmediated proliferation of hepatocarcinoma cells and down-regulated AFP-induced expression of oncogenes in the cells in a dose dependent manner [78]. Cell-SELEX was used to generate aptamers for liver cancer recognition. Two liver cell lines from Balb/cJ mice were used: a BNL 1ME A.7R.1 liver cancer cell line (MEAR) and a BNL CL.2 non-cancer cell line (BNL). In multiple rounds of selection and by using the BNL as  a control, we identified aptamers that specifically recognize the MEAR cancer cell line in vivo and in vitro [63]. In a research by Zhang et al., a mouse's MEAR-specific ssDNA aptamer TLS9a was used to fabricate a quantum dot-labelled aptamer bioprobe (QD-Apt). A bioprobe with optimal bioactivity was obtained by conjugating streptavidin-modified quantum dots (SA-QDs) with a biotin-derived aptamer in a ratio of 1:16. QD-Apt was able to specifically recognize MEAR cells, but not BNL cells and Hela cells, indicating that the probe was biocompatible and suitable for live cell imaging [79].
The P12FR2 RNA aptamer, which is specific for the pancreatic adenocarcinoma up-regulated factor (PAUF), was generated with the potential to be effective in treating human pancreatic cancer. The research group proved that the P12FR2 aptamer inhibits PAUFinduced migration of PANC-1 human pancreatic cancer cells and decreases tumor growth by about 60%, when injected intraperitoneally, without causing loss of weight in treated mice [80]. Along with this, the A1 high mobility group (HMGA1) proteins and their overexpression were investigated. These proteins are related to anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. That is why bio-stable l-RNA oligonucleotides, the so-called Spiegelmers (NOX-A50 and NOX-f33), were generated, which specifically bind to HMGA1b. In a xenograft mouse study that used the PSN-1 pancreatic cancer cell line, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes, showing an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume [81]. Watanabe et al. proved in their research that aptamers can enhance chemotherapy efficiency in pancreatic cancer treatment. They used HMGA-targeted AT-rich phosphorothioate DNA (AT-sDNA) aptamers to suppress HMGA (high mobility group A), which is, when elevated, associated with resistance to the chemotherapy agent gemcitabine [82].
In colon cancer diagnostics and therapy, aptamers were used to identify specific biomarkers and to inhibit certain receptors involved in carcinogenesis. By using cell-SELEX technology and the DLD-1 and HCT 116 colorectal cancer cultured cell lines, a panel of targetspecific aptamers was selected with high affinity and selectivity to colon cancer cells, but not to normal colon cells or other cancer cells [83]. In a study by Kwak et al., RNA aptamers, which are specific for peroxisome proliferator-activated receptor delta (PPAR-delta), were developed, which efficiently inhibited transcription and reduced mRNA levels of vascular endothelial cell growth factor-A and cyclooxygenase-2 resulting in a striking loss of tumorigenic potential, especially in HCT116 colon cancer cells with high-level expression of PPAR-delta-specific aptamers [84]. Moreover, the role of beta-catenin in transcription and splicing of oncogenic target genes was studied by using high-affinity RNA aptamers that connect to beta-catenin in vivo. The selected aptamer prevented alternative transcription and splicing induced by stabilized beta-catenin [85]. Furthermore, a DNA aptamer was created to target the CEA (human carcinoembryonic) antigen that is characteristic for gastrointestinal adenocarcinomas. The binding of the DNA aptamer was monitored with flow cytometry in the MC-38 (CEA-) and MC (CEA+) mouse-colon adenocarcinoma lines. Results indicated high specific binding of the DNA aptamer in the CEApositive MC-38 cell line, but not in the CEA-negative line. This way, aptamers could be used as intracellular delivery vehicles for aptamerdirected drug conjugates [86].

Conclusion
Cancer remains one of the leading causes of death worldwide. The main cancer types are lung (1 370 0000 deaths), stomach (736 000 deaths), liver (695 000 deaths), colorectal (608 000 deaths), breast (458 000 deaths) and cervical cancer (275,000 deaths) (76). The main challenge in cancer medicine is associated with early detection and specific treatment of cancer. The critical point is especially low expression of biomarkers in early stages and toxicity of chemotherapy. However, recent researches are investigating the therapeutic and diagnostic role of oligonucleotides. Oligonucleotides which are capable of binding to molecules other than nucleic acids are named aptamers. These are single stranded DNA or RNA oligonucleotides (12-30 bases), which represent a new way of targeting cancer cells and cancer biomarkers, as well enrich rare cancer cells with biotechnological, nanotechnological and analytical methods. Furthermore, they can be used as therapeutic agents. Aptamers are selected by using either the SELEX, the cell-SELEX or the brasil technology. Cell-SELEX seems to be especially appropriate for identifying cancer cells and generating aptamers that bind specifically to altered surface molecules on cancer cells without the exact knowledge on changed sequences. In the field of abdominal surgery, researches with aptamers were performed on pancreatic, colorectal and liver cancer. The selected aptamers could target certain biomolecules or inhibit receptors that are involved in carcinogenesis. This characteristic is of particular value in pancreatic cancer, due to late diagnosis and insufficient treatment opportunities resulting in poor prognosis. Further researches are required, in order to determine ,whether aptamers are capable of detecting biomarkers and inhibiting receptors or intrinsic pathways that are involved in carcinogenesis.