Smart Aptamer -Based Therapy
The role of bio-functional Aptamer in delivery system for cancer cells
1.1 causes of cancer:
1.2 The treatment of cancer
1.2.3 Gene Therapy and Chemotherapy
2 Targeted therapy
2.1 Targeted Therapy types:
3.1. Aptamer properties
3.2 Aptamer isolation
3 .4 Factors
3.5 Aptamer targets
3.6 Aptamer improvement
3.7 Aptamer modification
3.7.1 Chimera aptamer
4 Aptamer application
4.3 Drug delivery
5 Thrombin and cancer
5.1 Thrombin aptamer
6 Nucleolin and cancer
6.1 Nucleolin aptamer
7.1 Aptameric Enzymatic Subunits(AESs):
7.2 Structure switching
In spite of the huge volume of research that has been invested in the development of cancer treatment, the disease is still a major problem. The global incidence in 2008 was 2.7 million cases. Several statistics has been taken of cancer worldwide and in UK particularly. In 2008, there is about 7.56 million death worldwide caused by cancer, while in UK the death counted for about 156.000 cases. Whereas about 12.66 million cases are diagnosed in 2008, about 309.500 cases are diagnosed in the UK. In terms of the most common cancer which causes death in 2009 in UK, Lung has the majority in about 22 %, and breast for about 8 %, and they are summarized in the Pie chart below:
(CancerResearch UK, 2008)
1.1 causes of cancer:
Many factors can cause cancer disease. Oncogenes are often present in the genetic profile of cancer victims. When these genes are activated, they interrupt the process of programmed cell death and so cause normal cells to proliferate indefinitely as cancer cells. However, the activation of such oncogenes are often requires some additional external trigger, like a virus or a carcinogenic chemical. Thus, the etiology of cancer is a complex mix of genetic and environmental factor (Belpomme et al., 2007, Anand et al., 2008).
1.2 The treatment of cancer
Various treatments have been discovered in the management of cancer disease. They are chemotherapy and gene therapy and targeted therapy. However, due to the limitation of one type than the other, in several cases a combination of more than one type has overcome some of this limitation(Cross and Burmester, 2006)
I n term of immunotherapy, many research have been done to assess the influence of immunotherapy on metastatic cancer patients. Among these, vaccines, which are active immunization that can cause tumor growth treatment. The significant factors that lead to the use of vaccines are that viral diseases have been shown to be prevented when the treatment of vaccine is applied. In addition, clinic, side affects were not detected in patients receiving vaccine treatment. The immunologic rejection of vascularized tissue can be affected by the responses of immune cellular(Rosenberg et al., 2004).
For common tumor to be immunologically destroyed, three elements are essential. First, the generation of immune cells, which identify antigene for tumor. Second, the movement of these cells and their penetration for the staruma tumor is required. Third, in order to destroy the tumor at its site, the immune cells have to be triggered. Importantly, to facilitate antitumor influence, T cell has to be activated and differentiated correctly(Rosenberg et al., 2004). In addition, to patient with cancer, efficient immunotherapy can be developed, because of the recognizing of cancer antigenes(Rosenberg et al., 2004). Indeed, nano particle Heparin-polyethyleneimine (HPE1) has been used in the treatment of C26- colon carcinoma as non viral vector(Gutierrez et al., 1992).
1.2.2 Gene Therapy and Chemotherapy:
Since there is a genetic component in many cancers, it may be that in longer term gene therapy, which aims to replace or repair defective genes, could be developed as both a preventive and therapeutic strategy (Cross and Burmester, 2006). Some work has already been done in this direction. However, at present the main treatments offered to patients are surgery and chemotherapy(Kelsen et al., 1998). The former can control the disease in some cases, especially when tumours are localised and slow-growing. However, often the best that it can offer is a delay in the progress of the disease. What is really needed is a therapy which can kill cancer cells wherever they are located in the body. Chemotherapy aims to do precisely this. However, the cytotoxin drugs currently used are lack selectivity. This means that, although they kill more cancer cells than normal cells, they still cause many undesirable side-effects and often cannot achieve a total clearance of the malignancy(Kelsen et al., 1998).
Recent developments in cell biology have introduced a raft of new therapeutic approaches that promise to be able to target cancer cells in a much more selective way. There are two main types of such therapy: targeted immunotherapy and therapy based on molecular targets(Folkman, 1971).
2.1 Targeted Therapy types:
There are different kinds of targeted therapies.
– Signal transduction and angiogenisis:
They include signal-transduction inhibition, in which the growth factors receptors or enzymes that stimulate the growth of cancer cells, are inhibited by small molecules. Another kind is the angiogenesis which occur in both healthy as in the healing of wound ,and in disease, and it can encourage the growth in tumor by transfer nutrients and oxygen, therefore, the inhibition of these agents are significant(Folkman, 1971)
– Cell-cycle kinase and monoclonal antibodies:
Over expression of Cell-cycle kinase on the other hand, can lead to increase the proliferation of cells which results in cancer, therefore, the inhibition of this kinase is targeted(Folkman, 1971). An example of these agents are, Glivec, Tarceva, Iressa (Goldman, 2003). Another crucial agent of targeted therapy is the monoclonal antibody. Antibodies and other immune materials called biological response modifiers (BRMs), which work as a defender for the body when having disease. Antibodies can target the antigen which is expressed in tumor cells (Folkman, 1971). The first approval antibody by the US food and the drugs administration (FDA) for the cure of cancer is MabThera, Which targets CD20 which is the antigen of B lumphocytes whose markers are also targeted by Campath, Epratuzumab. For those who are not affected by MabThera, efficient therapy obtained when co-treatment of radiotherapy and antibody are applied (Lau and Chan, 2011)
It was discovered that several types of antibodies are capable of the inhibition of cancers, such as hematological and sarcoma malignancy, over expressed Hypatocyte Growth Factor (HGF) and MET, whose inhibition has shown regression in the growth of several cancer cells. Due to this expression, HGF can be activated by the participation of the paracrine circuit. The signalling of HGF has been shown in cancer cells in different studies. In addition, in human sarcoma, it was detected that MET expression has an increase rate. As the results of these findings, Head and Neck Squamous Carcinoma cells(HNSCC)have been examined in vitro with HGF, which involved in the metastasize of HNSCC. Moreover, with HNSCC patients, considerable level of interleukin-8 and VEGF has been shown. Thus, HGF has been a target for drug generation to find an efficient treatment for cancer (Lau and Chan, 2011). HGF activity is inhibited when MET receptor binds to MetMab(OA5D5), which is an anti-Met antibody monovalent. Moreover, the tumor cell invasion, growth, angiogenesis, has been inhibited by XL820, which has an increase affinity for VEGFR and MET, and it is TKI multi target (Lau and Chan, 2011).
The use of aptamers in targeted therapy:
There are several hurdles that must be overcome for targeted therapy to prove successfully. If a cytotoxic agents have to be delivered in a highly selective way into tumour cells, it must be small and innocuous enough to penetrate the cellular membranes. Yet it must be complex enough to be able to bind specifically to the target tumour cells and no others. Interest is being shown in the potential of aptamers to meet these requirements.
Aptamers are small sections of nucleotides or chemical antibody or protein which, because of the fragment of genetic information they contain, can be designed to bind selectively with a chosen target molecule. This target could be a protein on the surface of a cancer cell, or some key intermediary in the biochemical process of cell replication (Dua et al., 2011, Pestourie et al., 2005, Mallikaratchy et al., 2011).
This review surveys recent work in the development of aptamer-based targeted cancer therapy, as a preliminary study for a research project.
3.1 Aptamers properities:
Aptamers have been used instead of antibodies as they confer several benefits(Musheev and Krylov, 2006). In antibodies, the cytotoxic molecules which need to be delivered to specific targets cannot pass through the membrane via antibodies because of their huge size, aptamers, however, are ideal for passing through the membrane due to their small size. In addition, while the synthesis of antibodies is expensive and takes a long time and needs hard work, the synthesis of aptamers is simpler and cheaper. In addition, antibodies are immunogenic and sensitive to temperature. Furthermore, it is not possible to alter and change the kinetics of their binding; the structure of aptamers, however, can be changed once they bind to their target; They lack immunogenicities and have low toxicity (Torres-Chavolla and Alocilja, 2009, Luzi et al., 2003a); even when they are fluorescent marking, their affinity remains(Musheev and Krylov, 2006, Dua et al., 2011). In addition, in vitro and in cells, aptamers are capable of inhibiting the role of certain proteins(Pestourie et al., 2005). Many positive effects result from the use of aptamers in living cells. First, targets, which are connected with a particular phenotype, can be identified. Second, there is no need to purify targets that bind to membranes before being selected. Finally, protein in the membrane surface can be targeted, when these proteins are in their original form(Dua et al., 2011). In addition , at the time of using molecules in biosensors as a bio-recognition element,(Liss et al., 2002)has reported a comparison between aptamer and antibody for this molecules.
3.2 Aptamer isolation:
In terms of aptamers identification, the exponential enrichment of systematic of ligands has been used (SELEX). SELEX is protocol, by which aptamers can be identified in vitro(Musheev and Krylov, 2006). It was reported initially in 1990((Ellington and Szostak, 1990, Tuerk and Gold, 1990). This procedure contains several steps. First, chemical synthesis is used to create various DNA molecules in a huge library. The following step is the use of PCR to amplify them. Then, transicription in vitro is done to create an RNA library in order to select an RNA aptamers, which may be modified chemically. After that, the aptamer which does not bind to the target is separated from the one that binds. This then is followed by the amplification of molecules that composed of the binded target activity by RT-PCR. The target then is affected by the enriched library. The process of amplification and selection are repeated several time to control the sequences that react with the targets (Proske et al., 2005, Tombelli et al., 2005, Luzi et al., 2003a).
Figure 1.A, the basic stages of SELEX. 1- The target is incubated with a pool of random RNAs. 2- then , sequences that are unbound are removed. 3- the enriched pool is obtained by the reamplification and transcription of the binding sequences using RT-PCR 4- after repeated cycles , the selection of aptamers occur(Proske et al., 2005).
Proteins, which are purified are utilized to make the action of SELEX. However, the folding of cell surface receptors makes this process of purification difficult. Thus the number of aptamers produced, which attached to the surface is reduced (Musheev and Krylov, 2006, Keefe et al., 2010). Therefore a novel approach has been invented to overcome this limitation called Cll-SELEX.
Due to the need of the purification and solubility of targets when typical SELEX is used, target a complex mixture is made successfully by new SELEX method (Cell SELEX) (Torres-Chavolla and Alocilja, 2009, Tan et al., 2011)Figure1.B. Indeed, several kinds of aptamers such as agonists and antagonists are generated. In addition, aptamer selected in this procedure has several advantages (Pestourie et al., 2005). Furthermore, targets can be within cell surface or bloodstream, or inside cells, to which aptamers can transfer independently or with drugs into the targets. In addition, considerable therapeutic effects are evident as a result of the use of high affinity aptamers for antagonists. Accordingly, increasing affinity and selectivity occur (Keefe et al., 2010).
Figure1.B. The sequence of DNA that recognise target cells is enriched. This if followed by the coloning of selected pool. Then, in order to identify individual aptamers, the clones which are positive are sequenced.
The incubation of the pool of single stranded DNA (ssDNA) occur firstly. Then the DNA that is bound is removed by thermal denaturation. After the removal, PCR is used to amplify them. Then, the division of the product of double-stranded PCR to ssDNAs is facilitated. Following this, by the use of flow cytometry, the strand DNAs are examined or they are kept for the selection in the following cycle, in order to evaluate the progression of SELEX. Then, after the enrichment of the selected pool, the PCR product of this pool is sequenced and cloned for the identification of aptamer (Tan et al., 2011).
3.3 Aptamer types:
Several aptamers have been reported but they are currently in different phases in the clinic. , All of them have various affinities to the targets and different definition as well as various beneficial for therapeutic application.
|Pegaptanib ,(cugen aptamer)||Inhibits the reaction between receptors VEGFRL and VEGF.|
|. AS1411 aptamer||Its linkage reaction with nucleolin stimulates the therapeutic reaction|
|REG1 aptamer(Povsic et al., 2011)||in anticoagulation|
|ARC1779 aptamer(Firbas et al., 2010)||acts as inhibitors for domain within willebrand factor, called A1, resulting in removing an antithrombotic influence, when Ib receptors of platelet membrane glycoprotein is bound.|
|N4172 aptamer||inhibitory affect on thrombine at exosite 1 and consists of non-modified 26-mer oligodeoxynucleotide.|
|ARC1905 aptamer||since this aptamer is pro-inflamatory and include within AMD patients, it has an affect on AMD.|
|NOX-E36||affected by chemokine, in which its ligand in turn binds to NOX-A12 aptamer|
3.4 Factors affect aptamers:
The several factors that affect aptamers within the target are as follow. The resistant to nuclease, it was discovered that modification has an effect on the half live of nucleutides in blood. Chemical modification can be introduced to increase the half life within the linkage of internucleotide phosphodiester or within the nucleutide sugar. Several modifications illustrate the stability of serum for andogenous serum nuclease, which has considerable rate of separation, and which can be stabilized by terminal capping. In addition, to reduce nucleas -mediated degradation and increase the stability of aptamers, medicinal chemistry is utilized. Another factor is the filtration within the renal. PEG conjuagation and cholesterol has been shown to decline the renal filtiration rate as well as increase the half life, cholesterol however, has less affect on renal filtration than that of PEG. When remarkable volume of the molecule PEG is conjugated with aptamers, these aptamers are declined significantly in the filtration renal, which occur due to their large size which is 5-15 kDa. The toxicology is another factor which can affect aptamers (Keefe et al., 2010).
3.5 Aptamers targets:
Aptamer has two target types, extracellular and intracellular target. Although different procedures have been utilized in intracellular targets, the extracellular binding aptamer are preferably used. This is due to its beneficial properties. The stability of the latter, however, is being stimulated. In therapy, aptamer can target extracellular either for therapy or for diagnosis application. In terms of therapy, the extracellular can be targeted directly by their activation and inhibition, or indirectly by drug delivery using aptamer as a vehicle. In diagnosis, however, tumor cells can be distinguished from normal cells by an aptamer(Meyer et al., 2011).
3.6 Aptamer Improvement:
For the improvement of aptamer, several expectations have been suggested. If high-throughput selection and the clearance and generation resistant of engineered aptamers are used, this can lead to increase the number of therapeutic aptamers. Therefore, to obtain such results, several strategies have been followed; encourage the creation of aptamers, when the selection is frequently repeated by apatmer themselves. In addition, by the reduction of affinity that was catched to beads. Although the obvious role of oligonucleotide is activity inhibition, the assembly was more the purpose of this oligonucletides. The use of PEG or cholesterol in conjugation with aptamers, which results in that renal filtration, is declined in animal. OX40-specific aptamer has been shown to act as receptor agonist after the dimerization to oligonucleotide organiser using hybridization. Although the aptamers selected to aim at purified targets has limited use, the one which selected against whole cell has many utilities. For example, as real time imaging props, which are attributed and neutralized ligands. Recent discovery has shown that some therapeutics agents that are delivered by aptamers, such as siRNA and toxins and drugs, can be remained in particular tissue. The most common targeted therapy for aptamer is Prostate Membrane Antigen PSMA(Keefe et al., 2010).
3.7 Aptamer Modificatin:
For therapeutic effect, the chemical modification of oligonucletides is utilized most by SELEX. This is because of the sensitivity of DNA molecule, wild type RNA to nuclease mediated separation, which results in less therapeutic effect. In addition, these oligonucleotides can be stabilized by this modification. Aptamers that are currently known are chemically modified (Keefe et al., 2010).
The stability of aptamer against nucleases can be enhanced by the use of phosphor-rothionate in the place of DNA phosphate backbone. Another example is that RNA aptamer can be stabilized when 2´-riobose is derivatized (Kanwar et al., 2011).
3.7.1 Chimera aptamer:
– Chimera property:
Particular study showed that chimera has more stability in therapy than the use of single stranded DNA. An crucial example of chimera, small interference(RNA) , which is Anti-human immunodeficiency virus (anti-HIV) was examined in terms of its delivery into infected cells using anti-gp120 RNA aptamer, to benefit from its bindind to gp120glycoprotein. In addition, it was shown that the potency and efficacy of RNAi can be stimulated by 27-mer siRNA chimera (Zhou et al., 2008).
– Chimera and cancer:
The proliferation of cancer cells has been shown to be regressed when chimera aptamer is utilized. In previous research, the connection between prostate-specific membrane antigen (PSMA) and small interfering RNA (siRNAs) was generated. This chimera then was tested on the proliferation of cells, which shows regression of PSTMA prostate cancer cells, but the non-defected cells were not influenced(Kanwar et al., 2011)
Example of Chimera:
4 Aptamer Application:
Since age-related macular degeneration was treated utilizing pegap-tanibs sodium, an anti-VEGF165(VEGF165)aptamer, which the food and drug administration has approved, the utility for using aptamer in clinical therapy has since been developed(Khati, 2010). However, there are other several applications, such as utilizing aptamer in sensors and as inhibitors and in diagnostic and therapy as well as delivery of drugs. In protein purification, aptamer can be used as a significant tool, especially when they have affinity with cells, thereby this may lead to the identification of targets molecule such as proteins using mass spectrometry. In addition, particular cells can be isolated using cell-specific aptamer, which Cowrek and Tan used. An example of this is the isolation of small cell lung cancer(S CLC) with other cells in the media by the conjugation of nanoparticle with aptamers for these cancer cells.
For aptamer to be used in diagnosis and biosensing, the sequence of aptamer has to be joined with mechanism of signal transduction(Khati, 2010).The use of a signal in sensor generation, whose contents are the recognition domain, is crucial to utilize a ligand-caused structural alteration. The discovery of Ramos cells by the conjugation of gold nanoparticle aptamers on a lateral flow device used the sensor based cell specific aptamers (Meyer et al., 2011).
The use of aptamer as in therapy has increased in recent years. In 1990 the oligonucleotides have been used as therapeutic agents as the replication of HIV(human immunodeficiency virus,which contains the transactivating responsive (TAR) RNA, was prevented by RNA-decoys. Aptamer are considered as a vehicle in therapy. Aptamer which are nucleic acid are used nowdays as a vehicle for targeted delivery more than that of either antibodies or small molecules.Targeted drug delivery can be more efficacy when aptamer used as a vehicle to deliver drug or toxin, this is due to its high affinity and specificity which is particularly can deliver the cytotoxic drug specifically into the target(Kanwar et al., 2011). Thus, inhibitory or stimulatory affect occur on specific cells. Indeed, aptamer has been used as inhibitors or stimulators for cells. In mice, it was found that the function of CTLA-4 can be inhibited in vivo by specific aptamer, which in turn enjoin the anti tumor response. In addition, the immunity of tumor was discovered to be affected by CD8*T-cells, which contains and encouraged by a receptor called 4-1BB of tumor necrosis factor(TNF), this family include 4-1BB receptor ,whose enhancement was thought to cause tumor suppression. Therefore, aptamers bind to this receptor was detected, which corroborate these receptors. Thus, the response of anti-tumor occur(Kanwar et al., 2011).
Aptamer can be used for small molecules to be high through-put screened, during which the small molecule leads compound can be identified when it is replaced with aptamer that binds target. In addition, it can be used indirectly either for structure-based design as template, and for small molecule drugs to be screened biochemically. Either hydrogen bond or ionic interaction make protein interacts with targets(Khati, 2010).
4.3 Drug and toxins Delivery:
Cargo molecules can be delivered to specific tissue or cells using aptamers. It was shown that RNA aptamer that altered 2-F, binds with the extracellular portion of prostate PSMA. Importantly, this aptamer A10 can be used as vehicle to deliver drugs this is since the use of clatherin-coated pits, which shows that within cells, particular endocytosis of PSMA presents. This means that after the binding of this PSMA with aptamer, they are endocytosed. Thus drug is delivered into targeted cells, and the creation of aptamer drug chimera is raised (Meyer et al., 2011). Aptamers such as A10 and PTK7-particular sgc8c, have delivered therapeutics of small molecules known as Doxorubicin. Gelonin, which is a toxin and a biopolymer therapeutics and its affect tested on tumor cells after its conjuation with proteins. These drugs and toxin can cause decreasing of side effects after the conjugation. siRNA was successfully delivered into targets using aptamers , in distinct steps. PSMA atamer is connected to siRNA when the streptavidine is used as conjugation for biotin labelled siRNAs and biotin labelled aptamers. Second, hybridization of aptamers to siRNA, or by connecting between tat/rev-particular siRNA and HIV gp120-specific aptamer. Another significant delivery role of aptamer into tumor cells is the delivery of nanoparticle which are supra molecular structure. In addition, docetoxel drug that capsulated was delivered to xenograft LNCap in nude mice(Keefe et al., 2010).
Having discussed about the chimera of PSMA-aptamer and si RNA , the possibility of utilizing these chimera in cancer cells, is worth addressing. When PSMA –tumor cancer cells are exposed for gelonin variant, which was created by the covalent binding of gelonin with PSMA-aptamer. Thus the cancer cells were damaged by the internalizing with gelonin variant(Kanwar et al., 2011).
Another toxin was delivered by aptamer is Doxorubicin which is a chemotherapeutic agents that can be delivered without modification neither for this toxin nor for aptamer. Dox is anthroacycline-derived drug molecule used in chemotherapy. However, the cytotoxicity resulted in this therapy has enhanced its delivery into the desired cells by targeted therapy to reduce its toxicity. Indeed when this has been applied for PSTMA cancer cells, both the proliferation and the cytotoxicity were decreased(Kanwar et al., 2011).
5.1 Thrombin and cancer:
Several protein substrates can be attached to thrombin, which consists of a domain of serine protease. α- thrombin of the trypsin like serine protease can be released by any vascular injury. In addition, thrombin is found in three forms, its original enzyme form, inactive form and coupled with Na²(Lin et al., 2011).
Thrombin is found to play a significant role in the development of cancer cells and it can cause apoptosis in tumor cells. Furthermore, it influence several conditions such as thrombosis, hemostasis and wound healing. This leads various human cells either normal or malignant to be proliferated and reinforce their metastatic such as endothelial cells and fibroblast and monocytes and other tumor cells. Moreover, since thrombin possesses many biological activities such as inflammation and curing tissue, the thrombin receptors are the interceder of these activities. G-protein coupled receptors, which are the components of the seventh transmembrane group within the receptors, have an extracellular N terminal region, which may accept the inclusion of a tethered peptide ligand. Moreover, protease, which can liberate the tethered ligand peptide by penetrating the extracellular region of the receptors and converting the signal, results in the activation of the receptors.
The 1- Alb3 integrine expression rises in tumor cells by the connection of these cells to endothelial cells, which is caused by thrombin. Moreover, the activation of NF-KB stimulates the expression of ICAM-1(CD54) and VCAM-1(CD106). Thrombin has been found to cause the] regulation of programmed cell death in human tumor cells. However, in their research, hirudin was used to inhibit thrombin, while in this research thrombin aptamer will be used for the same purpose(Ahmad et al., 2000).
1.5.2 Thrombin aptamer:
As thrombin play a significant role in the regulation of cell process, aptamers for inhibiting thrombin have been generated. Thrombin is a serine protease which is multifunctional and it has a role in anticoagulant and in pro-coagulant. The activity of the thrombin enzyme is inhibited by thrombin aptamers, and blood clots occur at the end of these processes. Additional properties make use of these aptamers. For example, chemical groups can be used to alter aptamers, which cause them being bound at different sites. Thus make biosensing atomics force microscopy benefits from these aptamers. Consequently, the generation of aptamers that bind to thrombin binding site acts as an anticoagulant to inhibit thrombus creation(Paborsky et al., 1993). In addition, thrombin aptamer first targets those proteins, which are incapable of binding to nucleic acids, and accordingly participate in therapeutic process(Lin et al., 2011).
This sequence of aptamers that inhibits thrombin is d(GGTTGGTGTGGTTGG); these aptamers were generated using SELEX technology(Tuerk and Gold, 1990). Within the process of the generation of the thrombin aptamer, the oligonucletide pool, is enriched and binds to thrombin with increased affinity. It is also cloned and sequenced. Preceding that, immobilized thrombin with affinity columns are used, through which the enriched oligonucletides that bind thrombin passed multiple times. After every stage, the pool of sequences that are bound and isolated, is applied or treated by a polymerase chain reaction(Lin et al., 2011)
The structure of thrombin aptamer has been explained in several studies. X-ray crystallography has been used to alter the structure of thrombin, which is three -dimensional, and NMR spectroscopy has been used to illustrate the tertiary organiztion of thrombin aptamer. The reaction of the ssDNA protein can be determined when the binding area of the aptamer on thrombin is recognized, and can increase the therapeutic properties of thrombin(Paborsky et al., 1993). There are two exosites, which are surrounded by positive charge residues, and another site is an active site of thrombin; the two exosites are the heparine-binding site, which has a central of hydrophobic region, and the other is the fibrinogen-recognition site, which has a hydrophobic notch(Lin et al., 2011).
These regions are from the catalytic pore, this creates binding areas of thrombin, which in turn make receptors and macromolecular substrates react in a particular way with thrombin. At the time of the availability of heparin, when the antithrombin III inhibits the activity of thrombin, this process is promoted by the former site. In the latter site, however, in the tiny space between this particular complex and fibrinogen, the platelet thrombin receptor as well as thrombomodulin and hiruden, are promoted. These sites on thrombin are those capable of binding with aptamers, and this is because the phosphodiester nucleic acid component of the backbone is polyanionic(Paborsky et al., 1993).
1.6.1 Nucleolin and cancer:
The identification of the nucleolin protein and its structure and roles explains its therapeutic benefit. Nucleolin belongs to nucleolar protein, In terms of its location, it is detected in the contents of febrile surrounding the fibril centre, and this is illustrated using either electro-microscopy analysis or immune-fluorescence ultra-structural or both. Nucleolin has remarkable properties, it is highly methylated and phosphorlated as well as ADP-ribosylated. A number of studies have shown that the cell surface contains this protein and it is also found within the cytoplasm of nucleus.
Previous research has shown the participation of nucleolin in various significant processes. These include RNP gathering, the mRNA function, snRNA modification as well as the transport from RNA, of these the most well known is ribosome biogenesis. Other activities among the cells have been affected by nucleolin. When nucleolin comes together with ribosomal proteins, it creates pre-ribosomal particles, which then link to the many RNA and DNA components of nucleolin. In addition, these appeared to contribute to ribosomal biogenesis. In this process of the ribosomal biogenesis of RNA transcription, maturation, modification ribosomes are present. This is followed by the distribution of pre-ribosomal particles into the cytoplasma making up mature ribosome.
This protein was first identified by Orrick et al (1973), then this protein is known as C23. In addition, it was later purified from different eukarytic cells and in particular, Chinese hamster ovary cells. Although it was mostly known that this protein has mass of 100-110 Kda, when the cDNA of the hamester nucleolin is cloned, it appeared to have a molecular mass of 77 Kda, this is because it consists of 713 amino acids; nucleolin composed of amino acids whose sequence analysis shows three distinct domains. The first one is the central domain, which contains four RNA binding domain. The second one is N-terminal domain which composed of various sites of phosphorlaytion in acidic areas. The third one is the c-terminal domain(GAR) or (RGG), which includes arginine and phenylalnine residues and glycine as well as considerable level of Ng, Ng dimethylarginines (Ginisty et al., 1999).
1.6.2 – Nucleolin aptamer and cancer:
A huge research has been conducted to discover a drug to inhibit the proliferative of cell cancer, among these the discovery of AS1411 aptmer. There is a detectable cellular protein which is found to be bound to Guanin-Rich Oligonucleutides (GROs) whose biological activities matched to this protein, and is known as nucleolin. The AS1411 nucleolin aptamer is an unmodified 26-mer guanosine rich aptamer, and is developed by Aptamera (Louisville, KY) as AGRO100. AS1411 is GRO29A-OH with the 3 × 5′ thymidines removed(Srivastava and Pollard, 1999) and its sequence is 5 ̀- GGT-GGT-GGT-GGT-TGT-GGT-GGT-GGT-GG-3 ̀. In addition, the stable structure which consists of G-quarters, is formed by this aptamer, and this gives specific GROs such as 29-mer GRO29A, its anti proliferative power. In particular, the typical SELEX was not used for the detection of anti-cancer properties. Instead, It was developed in vitro, by a mechanism, which is nonantisense, due to the detectable of anti-proliferative properties, which guanosine-rich oligonucletides (GRO) have against cancer. During the cell cycle, in the S phase, cells are stored stepwise, and this is the GROs affect on cancer cell lines(Xu et al., 2001). In Addition, the activity alteration of an enzyme, arginine methyltransferase 5(PRMT5), by which the symetrica dimethyl arginine formation(SDMA) is catalyzed , was reported to be influenced by AS1411(Soundararajan et al., 2009, Bates et al., 2009), Several cells such as the HeLa cancer cell line and the prostate (DU145) cell line, are inhibited using GRO29A (Ireson and Kelland, 2006). Moreover, Gastric cancer cell lines are inhibited by AS1411 aptamer as well.
– HeLa Cell:
Was isolated from women called Henrietia Lacks who had grandular cervical cancer,50 years ago. The significant of this cells starts from the continuous growth of these cell line. 50 years ago these cells were grown by George Gey, and since then, it was the model of cancer cell which is widely used in cancer research (John, 2002).
–Nucleolin aptamer as a vehicle:
Aptamer that binds to the receptor of the cell surface protein can deliver cargoes to several cells. This is because when the ligands bind to cell surface protein, the receptors are internalized. In addition, it is due to the intracellular cycle of proteins in the surface In this research I will use nucleolin protein as surface protein in HeLa cell. The utility of using nucleolin is in the delivery of cargo into cancer cells(Ray and White, 2010)This is due to its shuttling among nucleus and cytoplasm and cell surface in cells which are highly proliferated(Ray and White, 2010).
7.1 Aptameric Enzymatic Subunits(AESs):
Aptamers have been combined by the sequence in specific procedure. The substrate can lead to the improvement of biosensing system, which is in turn can solve the problem of screening and finding a stable enzyme to the target molecule because within the reaction of the enzyme, multi amplification for sensing signal can occur. In addition because of the structural changes of aptamers, which caused by the binding between them and the target, and which results in signal generation, have been established(Yoshida et al., 2006).
In addition, aptamer has been used as subunits which facilitate the discovery of target molecules by the affect of these subunits on the activity of the enzyme when it binds to the target molecules. Therefore, recently, reseachers have designed the aptameric enzymatic subunits (AESs), for the detection of target molecule, by measuring the activity of the enzyme in homogenous solution. The researchers have used the AESs subunits to design sensor system to avoid the need of aptamer modification in the homogenous sensing system, which depends on structural changes of the enzyme when measuring its activity. Such modification made the loss of enzyme activity(Yoshida et al., 2006).
Two AES s have been designed by the team. First, they linked target binding aptamer, which is a sensing probe that once binds to the target, its inhibitory activity rises(AES 1), to the thrombin enzyme aptamer, which is a sensing probe that by binding to the target molecule, its activity of inhibition decreases(AES2). with AESs, the aptamer that inhibits thrombin is destabilized when it is connected to the aptamer that binds adenosine. However, when the adenosine was added into the complex, thrombin aptamer has been stabilized. In AESs, on the other hand, thrombin inhibiting aptamer has been separated into two oligonucleotides. While the adenosine complementary strand is connected to one of them, the other oligo is linked to adenosine binding aptamer. Consequently, the researchers found that within the complex, the activity of thrombin is inhibited by the hybridization of the two oligonucleotides. However, after the binding of adenosine –binding aptamer with adenosine, at the time of adding the adenosine, the complex lost its inhibitory activity and destabilized. Thus, the researchers have concluded that addition of adenosine can affect thrombin inhibitory activity, and it can be detected using the design of AESs. They suggest that the AES2 can be used widely to any aptamer of target molecule(Yoshida et al., 2006).
‘Figure 2 Pre-summed structures of AES 1 (left) and AES 2 (right). Main structure of each AES 1 series and AES 2 series is the same, and only the changed sequences are boxed(Yoshida et al., 2006).
This is the principle of which we use similar idea of the structure for two aptamers, Pb+2 and Nucleolin, but different purpose, instead of detection purpose, the delivery of toxin to cancer cells is our purpose.
7.2 Structure Switching:
Structural changes occur once aptamer binds to the target. Although there are various methods for the detection of the change in the structure, the popular one is using fluorescence. This is due to the wide availability of quenchers and fluorophores. Aptamere can be converted into florescence probes by strategies of rational design. In this strategy, complementary DNA strands which labeled with quencher and DNA aptamer labeled with fluorescence.
At the present of thigand, this and aptamer form a complex, in which the oligonucleotide labeled with quencher is dissociated from aptamer labeled with fluorophore. Thus fluoresecence signal rises. At the absence of ligand, however, the duplex of DNA is created between oligonucleotides which are labeled with quencher and aptamer which are labeled with fluorophor, which thereby quenched the fluorophore (Lin, Lawrence,2011).
Switch of signal aptamer. Structure-switching of aptamer is stimulated by target (star) from a duplex of DNA-DNA to a complex of DNA-target( in right). When the signal of fluorescence is increased, aptamer dissociation from DNA-strand that is labelled with quencher (QDNA) is detected ( Lin, Lawrence,2011).
8- Pb(II) Ion detection:
Several metal ions have been quatified and detected such as Ca(ll) and Zn(II). In addition, these metal ion can be analysed using atomic absorbtion spectrometry and microprobes. Sensitive and selective sensors is required for metal ions which is toxic in order to prevent its risk. For metal ions, the method of fluorescence is significantly used. However, the fluorosens method is limited in the possibility of binding the specific and the non-specific metal ions(Lu et al., 2003).
Various positive effects can result from using metal ion sensors such as in vitro selection of DNA/RNA , through which ahug e pool of sequence can be utilized. In addition, the polymerase chain reaction(PCR) can be used to amplify the target sequences. With this procedure, the specificity of the metal ion ca be altered when catalytic DNA/RNA is in vitro selected. Researchers have introduced various methods for the in vitro DNA catalytic selection, which was then proceed by fluorescent sensor. This is used in Lead(Pb ) detection in Lake water. They found that DNA Pb(II) sensor can detect Pb(II) in water sample, even in the presense of other metal ions(Lu et al., 2003).
(GROs) – Guanin-Rich Oligonucleutides
(AESs)- aptameric enzymatic subunits
(BRMs) biological response modifiers
(HNSCC)- Head and Neck Squamous Carcinoma cells
HGF – Hypatocyte Growth Factor
(SELEX)- systematic evolution of ligands by exponental enrichment
(RNA)- small interference
(anti-HIV)- Anti-human immunodeficiency virus
(PSMA)- prostate-specific membrane antigen
(S CLC)- small cell lung cancer
(TNF)- tumor necrosis factor
(TAR)- transactivating responsive
(SDMA)- symetrica dimethyl arginine formation
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