Cancer is characterized by uncontrolled cell growth and proliferation. Cell maintains homeostasis by regulating many attributes of cellular behavior such as growth, proliferation and apoptosis. Any mutations in the normal cell results in increase or decrease in the expression of genes that maintain homeostasis. This aberrant gene expression interferes with normal apoptotic pathway of the cell, leading to cancer. As per the CRUK report (July 2010), 1 in 4 deaths caused in UK are due to cancer and it is estimated that in UK alone, nearly 1 in 3 people are likely to develop cancer in their life time. So it is necessary to devise a robust technology to control the incidence of cancer.
PROBLEMS IN CONVENTIONAL METHOD
Conventional method of cancer treatments relies mainly on monotherapy which involves the use of therapeutic drugs to mutilate different stages of solid tumors. Solid tumors are heterogeneous at molecular level and are observed within the cells of the same tumor and at different developmental stages like primary or the metastatic phase. Moreover the cell undergoes selective phenotypic resistance in response to a particular treatment. This molecular heterogeneity observed within the tumors of same cancer types along with selective phenotypic resistance is a crucial task for treatment (). The above problem was overcome by combinational treatment were the cancer cells are subjected to different treatment conditions with the aim to counteract the resistance developed due to monotherapy. In combinational therapy chemotherapeutic drugs are given with other treatments like radiation therapy, surgery, hormonal therapy etc. But the use of these treatment methods also had several disadvantages. Chemotherapeutic drugs are highly hydrophobic in nature that has very low water solubility. Such insoluble drugs aggregates and causes embolization of blood vessels. A well-known example is the drug paclitaxel, that causes local toxicity when the drugs accumulates in high concentration. In addition, the bio-distribution of chemotherapeutic drugs remains a substantial problem. The non-specific uptake of therapeutic drugs by normal tissues along with the malignant tissue leads to low tumor selectivity. Also, in radiation therapy normal cells are destroyed along with tumor cells. Cancerous cells acquire resistance to chemotherapeutic drugs by altering the binding site of the target protein or having an efflux mechanism by which the drugs are expelled out of the cell. Considering all the disadvantages there is a need for following a strategy that would restrict the drug action only to tumor, sustain in the system for a long time and also allow targeted release of therapeutic drugs.
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Chemo sensitization involves sensitization of cancer cells with targeted therapies like gene therapy followed by treatment with chemotherapeutic drugs. Chemo sensitization offers accessibility of chemotherapeutic drugs to cancer cells that are chemo resistance. It also enhances tumor selectivity and thereby reduces side effects. Choice of targeted therapies that specifically affect the signaling molecules involved in survival of cancer cells is crucial for chemo sensitization (Dickerson, 2010).
REGULATION OF APOPTOTIC PROTEIN IN CANCER CELLS
The cancerous cells evade apoptotic pathway by switching off the gene responsible for apoptosis. There are two major classes of cancer genes involved in this complex process; (a) proto-oncogenes that become cancerous as a result of mutation and (b) tumor suppressor genes which when inhibited allow progression of tumors. P73 is a member of P53 family of genes that encodes for pro-apoptotic and anti-apoptotic protein. P73 proteins are expressed as TAp73 and âˆ†Np73 forms. TAp73 is a full length protein that activates the target gene involved in cell cycle regulation and induction of apoptosis. âˆ†Np73 proteins cannot induce target gene involved in apoptosis as they lack N-terminal trans-activation domain found in TAp73 (M S Irwin,2004). Also âˆ†Np73 has anti-apoptotic activity by blocking the action of p53 and TAp73. During tumorogenesis and development, the relative expression of TAp73 and âˆ†Np73 form is substantially regulated that determines the fate of the cell (M Rossi et al, 2005). In cancerous cells, expression level of p73 is highly regulated by ITCH E3 ligase. ITCH E3 ligase catalyses the transfer of ubiquitin to p73 protein. This ubiquitinated protein is thus presented for degradation by proteasome. In response to DNA damage, ITCH is downregulated which in turn affects the p73 turn over by ubiquitination reaction. Therefore the level of TAp73 increases and promotes cell cycle arrest and apoptosis. Whereas under non stressed condition the ITCH controls the p73 level and induce cell proliferation.
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Recent research work in cancer molecular medicine has taken advantage of gene therapy for treating cancer. Gene therapy involves the use of nucleic acid vectors that increase the activity of the target by producing therapeutic protein or delivery of nucleotide sequence that would counteract the action of target gene (RNA interference) involved in tumor formation. shRNA and siRNA are widely used for gene silencing. ShRNA are short hairpin RNA transcribed from plasmid DNA. Dicer enzyme then processes the shRNA in to siRNA (short interfering RNA) which is in turn activated by RISC (RNA-induced silencing complex) complex to cleave the target mRNA sequence. RNA interference with siRNA involves direct administration of double stranded sequence of complimentary to the target mRNA sequence which is then converted to single stranded nucleotide by RISC complex and cleaves the corresponding target mRNA sequence involved in tumor formation.
CARRIERS FOR GENE DELIVERY
Nucleic acids, such as shRNA and siRNA used for RNA interference, are degraded after systemic administration. So delivering this nucleic acid efficiently to the site of action requires a carrier that would protect the therapeutic nucleic acid from the point of administration till it reaches the target site. Different types of vectors have been used with this porpoise (REF review about gene therapy). Vectors in gene delivery can be separated in two main groups, viral and non-viral vectors. Moreover, the use of viral and non-viral delivery system in gene therapy has added advantage over the conventional therapy in achieving selectivity towards the target tissues. 70% of the gene therapy clinical trials are based on viral gene therapy due to its high efficacy, however, due to safety issues like immunogenicity and oncogenicity, as well as the difficult large scale production hamper the use of viral vectors (Dutta 10). For these reasons, even though have a lower efficacy, non-viral gene delivery systems have become a promising alternative in gene therapy.
Strategies in non-viral gene delivery comprises of physical method and synthetic chemical vector. Physical method includes gene gun, electroporation and hydrodynamic delivery which in turn had disadvantages of poor uptake and bio-stability. These problems were overcome by the use of cationic biomaterials used as synthetic non-viral vectors by the condensation of negative nucleic acids as nanoparticles (REF) to deliver therapeutic gene sequences.
NON VIRAL SYNTHETIC VECTOR FOR GENE DELIVERY
Cationic biomaterials, such as polymers, Liposomes and dendrimers have been succesfully used to condense nucleic acids (REF). Condensation of anionic DNA by electrostatic interaction with cationic vectors protects the nucleic acids from enzymatic degradation; improve the cellular uptake as well as the cellular trafficking (Dufes, 2005). In this report I will show the…dendrimers (TO COMPLETE).
DENDRIMERS- STRUCTURE AND CHARACTERISTICS FEATURES
Dendrimers have unique structure and physiochemical properties over the other cationic polymers that had driven the interest of many scientistto use it as gene delivery system. Its molecular architecture has a symmetrically branched globular structure that is synthesized in a stepwise manner to ensure monodispersity (Grayson, 2008). Commercially available polyamidoamine (PAMAM) and polypropylenimine (PPI) dendrimer are the more used dendrimers in gene therapy (REF). This project is focused in the use of PPI dendrimers as non-viral vector for gene delivery, for a detailed review about the use of PAMAM dendrimers please see (REF). PPI dendrimers are synthesized by divergent mehthod with diamino butane as the core molecule. The surface of the PPI dendrimer present a high concentration of terminal amino groups that can interacts with the corresponding phosphate backbone of DNA. The degree of surface charge increases with increase in dendrimer generation. For this reason high generations PPI dendrimers (generation 5, 64 amino group) have shown a low haemocompatibility (Ravina, 2010). However, in 2002, the research group leaded by Prof. Uchegbu and Dr. Schatzlein reported that low generation PPI dendrimers (generation 2, 8 terminal amino group and generation 3, 16 amino group) favored reduced toxicity for use in vivo gene delivery studies. Dendrimers achieve selectivity to the tumor site by passive and active tumor targeting. Size of the dendriplexes is crucial for passive tumor targeting. Tumor blood vessels are characterized by poor vasculature with pore size varying between 100 to 1200 nm diameters. Dendriplexes of size around 100nm permeates the tumor vessels but retain in the tumor as the size of the dendriplex is large enough to drain through the impaired tumor lymph system. Whereas, macromolecular prodrug varies between size 2nm to 10nm which will permeate the blood vessel but quickly pass through the lymph system due to smaller size. This enhanced permeation and retention effect (EPR) of nanoparticle is an important contributing factor for achieving tumor selectivity (Kratz, 2008).
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DAB-16 is a third generation PPI dendrimer having 16 terminal amino groups. It has low cytotoxicity with IC50 of 39 µg.mL-1and good transfection efficiency compared DAB-8. The transfection efficiency also depends on the N/P ratio. N/P ratio is the ratio of amino group of DAB-16 to the phosphate group of DNA (Zinselmeyer, 2002). DAB-16 at N/P ratio 30 had shown optimum transfection efficiency and for gene delivery studies DAB-16 at N/P 30 and N/P 8 was used.