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Volume 12 - 2008


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1-6

Advantages of intracerebral versus systemic administration of a DNA-based vaccine in treatment of an intracerebral tumor

Author(s): Dr. Terry, Lichtor,

Abstract: Structural differences between malignant and nonmalignant cells of the same individual form the basis of clinical immunotherapeutic strategies. Previously, we reported the therapeutic properties of a vaccine prepared by transfer of a cDNA-expression library from breast cancer cells into a highly immunogenic allogeneic fibroblast cell line where genes specifying an array of breast cancer antigens were expressed. In addition we have demonstrated the application of this cell-based vaccination strategy for breast cancer metastatic to the brain. In this study we explored the efficacy of the vaccine upon intracerebral versus subcutaneous injection in the treatment of an intracerebral tumor. Although the vaccine was efficacious in prolonging survival upon subcutaneous or intracerebral injection, there did not seem to be any synergy in either the development of systemic antitumor immunity or in prolonging survival when the vaccine was administered both intracerebrally and subcutaneously. However the intracerebral lymphocytic response was more intense following injection of the vaccine into the brain in the region of the tumor cells. Furthermore regulatory T cells (CD4+CD25+Foxp3+-positive) which inhibit antitumor immunity were not increased in the spleen cells from tumor-bearing mice injected intracerebrally but were increased in those injected subcutaneously with the enriched vaccine. This data suggests that local delivery of the cytokine-secreting DNA vaccine in treatment of an intracerebral tumor has certain advantages.

Keywords: Brain Tumors, cDNA, Gene Therapy, Interleukin-2, Local Delivery, Tumor Vaccine



7-14

Pro-apoptotic gene enhances the immunogenicity of glycoprotein B gene of herpes simplex virus-1

Author(s): Dr. Zuhair Muhammad, Hassan,

Abstract: Increasing apoptosis in transfected host cells has caused a significant enhance in the immunogenicity of DNA vaccine. It has been known that pro-apoptotic protein Bax induces apoptosis and adjuvant effect of Bax is achieved when suitable dose of the Bax gene is used as a molecular adjuvant. We compared three doses of Bax encoding plasmid (pbax) including 10, 25 and 50

Keywords: Apoptosis; Bax; DNA vaccine; HSV-1



15-24

Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection

Author(s): Dr. Richard, McCurdy,

Abstract: Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection

Keywords: John J. McGrath, Alan Mackay-Sim



25-30

Health economics of nutrigenomics in weight management

Author(s): Dr. Kenneth Blum,

Abstract: The emerging field of nutritional genomics presents various clinical questions that require further investigations. One of those questions is whether nutrigenomic interventions reduce the cost of illness (COI). Prior to this study, there has been an absence of data evaluating the health economic implications of this new field of research. In this theoretical modeling study, we sought to evaluate the health economics implications of a nutrigenomic product for weight loss. We constructed a nutrigenomic economic model by linking 1) published study data related to the efficacy of a product and/or ingredients, 2) validated clinical assessments that have already been tied to health economics data, and 3) data involving condition prevalence and overall cost of illness. In this theoretical model, we demonstrate that LG839 variant positively reduces the cost of illness at the macroeconomic and microeconomic level based upon a cost-effectiveness and cost-benefit analysis. From this proposed model, we have forecasted the prognostic health economic implications of a nutrigenomic intervention to demonstrate a theoretical model of nutrigenomic economics. This study is hypothesis-generating and should be used in the definition of protocols to prospectively test the health economic benefits of nutrigenomics.

Keywords: Bioeconomics, LG839 variant, nutrigenetics, obesity, Reward Deficiency Syndrome (RDS)



31-38

Antigenic epitopes of viral polyprotein: an approach for fragment based peptide vaccines from Papaya Ringspot virus

Author(s): Dr. Karbhari V Kale,

Abstract: Papaya ringspot is a destructive disease characterized by a yellowing and stunting of the crown of papaya trees and assay was designed to help assign putative genome polyprotein analysis of Papaya ringspot virus strain W. We used different methods for the prediction of linear epitopes using a combination of a hidden Markov model and a propensity scale method. Data set was collected from the literature, and data sets of epitopes in the genome polyprotein having twenty four antigenic determinants in 675 residues long sequence. The structural homology modeling method is allows potential drug targets to identify active sites i.e. linear epitopes, which form antibodies in host cells. The method integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency. The challenges for the future are to establish the function of all of protein structures. In this assay we use of multiple methods towards the accurate identification of antigenic epitopes. The proposed approach is useful not only for plant and viral biology but it covers the wide area of vaccines and antibodies for therapeutic purposes in humans.

Keywords: HMM, MHC, linear epitopes, cleavage sites



39-44

Activity and Integrating Expression of Human Endostatin Produced by Pichia pastoris

Author(s): Dr. Chongbi Li,

Abstract: Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of expressed proteins for purification. Endostatin is a potent and specific antiangiogenic protein capable of inhibiting the growth of murine and xenotransplanted human tumors. Thus far, however, recombinant endostatin prepared from Escherichia coli has been insoluble after purification and therefore inappropriate for clinical settings. In this study, human endostatin gene was integrated into the chromosome of host pichia pastoris by using the yeast inserted plasmid pPICZaA-endo including human endostatin gene, native Saccharomyces cerevisiae a-factor secretion signal, zeocin resistant gene and the AOX1 promoter and transcription terminator (TT) of pichia pastoris electroporation. The recombinant clones were then selected by the plates containing antibiocin-zeocin (100ug/ml), and finally one highly expressed clone was selected by PCR, SDS-PAGE and Western-blot. The yield of expressed endostatin from P. pastoris depended critically on growth conditions, and attainment of high cell densities by fermentation had been shown to improve protein yields up to 15mg/L estimated. Moreover, the protein was easily purified by using a heparin-agarose column. A strengthened antiangiogenic activity in vivo has been identified by a novel and simple CAM (chorioallantoic membrane) technique.

Keywords: rhEndostatin, Pichia pastoris, integrating expression, CAM



45-68

The impact of biomics technology and DNA directed anti-obesity targeting of the brain reward circuitry

Author(s): Dr. Kenneth Blum,

Abstract: Obesity-related medical conditions are the second leading cause of death in the U.S. Classified as a chronic disease in 1985, the understanding of obesity and its causes and effects has been further elucidated through additional research into the evolution of genetic and biologic influencing factors of this deadly disease. What used to be understood as primarily a behavioral problem of overeating and under-exercising, has only contributed to continued increases in the rates of obesity despite increases in dieting and exercise. Successful strategies to effectively induce sustainable fat loss and manage obesity have been elusive. For the most part, the tactics employed 45 I. Introduction An Evolutionary Genomic Trieste Obesity used to be understood in fairly elementary behavioral terms: eating too much and exercising too little results in excess body weight, due in large part to a lack of willpower or self-restraint. But as people have increased their dieting and exercise, the rates of obesity continue to rise as the combined prevalence of overweight and obese persons in the US have increased from 46% of the adult population (NHANES II, 1976 to 1980) to over 60% of the adult population in NHANES III (1988-1994). In 1985, obesity was recognized as a chronic medical disease with serious health implications caused by a complex set of factors. Obesity-related medical conditions contribute to 300,000 deaths each year, second only to smoking as a cause of preventable death (Hill, 2006). Obesity has been established as a major risk factor for hypertension, cardiovascular disease, Type 2 diabetes, and some cancers in both men and women. Obesity affects 58 million people across the nation and its prevalence is increasing (source: U.S. Census Bureau). Approximately one-third of American adults are estimated to be obese, and 60% are overweight. In response to this rising epidemic, the medical, food and fitness communities have consistently told Americans to just make behavioral modifications, such as diet and exercise. As scientific advancements have demonstrated in other neurological healthcare conditions such as alcoholism, there are important biological and genetic components that limit the efficacy of behavioral adjustments alone. Eighteen years ago, Blum and colleagues published in 1990 landmark research suggesting that another prevalent healthcare condition which had been traditionally characterized in behavioral terms like obesity, namely alcoholism, also had a hereditary or genetic component and that genetic information could explain why such a condition could be found to “run in the family”. Blum’s research (Blum et al, 2007) continued to explain how knowing this important genetic information could then caution certain genotypes to adjust their dietary intake and environments to overcome this genetic predisposition. In a recent study of 11,000 Americans, results suggested that more than 75% of obese Americans (n=3,100) say they have healthy eating habits. According to this survey, 40 percent of obese people also said they do “vigorous” exercise at least three times per week. In this survey by Thomson Medstat, a Michigan-based healthcare research firm, obese people reported similar behaviors in snacking, reading nutritional labels, and eating out when compared to normal weight people. Weight loss alone is difficult, but sustainable weight loss is exceedingly difficult. Most people regain as much as two-thirds of Blum et al: Obesity and reward circuitry have not been multi-faceted, multi-system approaches, but have been characterized by one-dimensional metabolic approaches targeted at achieving weight loss as measured by linear criteria (i.e. scale weight, Body Mass Index (BMI), percent body fat, etc). Recent evidence indicates a much more complex and multidimensional syndrome, characterized by the simultaneous breakdown of many facets of metabolism exacerbated or limited by the predispositions of inherited genetic traits. There is significant evidence to substantiate the existence of Reward Deficiency Syndrome (RDS) as a new paradigm shift in the understanding of Obesity. Specifically, there are genetic links to the role of catecholaminergic pathways in aberrant substance seeking behavior, in particular cravings for carbohydrates. These neurological factors, regulated by genetic predisposition, are a subtype of RDS that we classify as Neurobesigenics. In the treatment of this neurogenetic mechanism, there are leading prescription pharmaceuticals as well as nutritional therapies. There is growing evidence to support the augmentation of precursor amino acid therapy and enkephalinase and Catechol-o-methyl-transferase (COMT) inhibition leading to enhanced levels of neurotransmitters: serotonin, enkephalins, GABA and dopamine/norepinephrine. Utilization of combining substances/nutrients directed at replenishing the nutrigenomic needs of multiple pathways includes brain reward targets mechanistically mimicking the brain reward cascade. The effects of a novel nutraceutical supplement contained in a DNA-customized nutraceutical denoted as LG839, including Synaptamine, Super CitriMax and Passion flower, have been evaluated in an open label trial. Based on a number of case reports, it appears that this unique combination of ingredients may have a generalized anti-stress and anti-craving effect resulting in an inhibition of carbohydrate appetite, inducing energy increase, positive focus, improved performance, a sense of well-being, fat loss and enhanced body image/composition. Published research from our laboratory on an earlier complex denoted as SG8839 provides a classical example of a Structural Equation Modeling Methodology (SEM), a statistical regression model that is used to evaluate the effectual relationships between various systems in a multi-system matrix to measure the contributory roles of those PATHs, enabling targeted and effective nutraceutical interventions. We review the potential mechanisms and rationale for the utilization of this multifaceted approach to attenuate the pleiotropic defaults in obesity stemming from the evolution of the thrifty gene concept to modern times. This is the first time the components of this formula have been combined at the clinically tested dosage levels indicated, to promote successful and sustainable results in improved body recomposition. In summary, the impact of biomics technology and the DNA directed nutraceutical (LG839TM) targeting of the brain reward circuitry may provide a customized approach to prevent and treat high risk individuals who are carriers of a genetic predisposition to obesity. We are cognizant that the importance of this research is not focused on any particular product but to encourage the scientific community to incorporate pharmacogenetic/nutrigenetic testing prior to administration of any anti-obesity agent. Thus, in this review we present the necessity of exploiting systems biology and “omics” and have coined the term “Neurobesigenics”.

Keywords: Reward Deficiency Syndrome (RDS), nutrigenomics, polymorphisms, “Neurobesigenics,” reward cascade, dopamine, pleiotropic disease, Genotrim



69-76

Hepatitis Virus Protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

Author(s): Dr. Jennifer E. Embury,

Abstract: Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these vectors to Pahenu2 mice, we observed the unintentional development of neoplastic disease (44%) and hepatic pathology (70%) in WPRE-treated mice. Our vector contained a portion of the oncogenic hepadnoviral “X-protein” in the WPRE segment that had been intentionally modified in an attempt to prevent its expression. The hepadnoviral X- protein encoding sequence is known to function as a mediator in oncogenic activity (Murakami, 1999). We have evidence that the X-protein fragment unexpectedly formed a fusion protein with a phenylalanine hydroxylase transgene in our vector and suspect this fusion protein may have been responsible for the high rate of unusual types of cancer and hepatic pathology. These results are not to imply that the use of the WPRE element will always result in the development of cancer. But in this particular instance, an unanticipated event may have ensued when the X-protein formed a fusion protein with the transgene. This is a cautionary illustration to be considered when developing genetic therapies to treat diseases.

Keywords: Hepatitis Virus Protein X, fusion proteins, AAV-WPRE vectors, Serum phenylalanine assay, DNA extraction, immunohistochemistry, Histopathology, sequencing and quantitative PCR, Plasmid, vector construction, Western blot analysis



77-82

Transgenomics

Author(s): Dr. Virendra S Gomase,

Abstract: Papaya ringspot is a destructive disease characterized by a yellowing and stunting of the crown of papaya trees and assay was designed to help assign putative genome polyprotein analysis of Papaya ringspot virus strain W. We used different methods for the prediction of linear epitopes using a combination of a hidden Markov model and a propensity scale method. Data set was collected from the literature, and data sets of epitopes in the genome polyprotein having twenty four antigenic determinants in 675 residues long sequence. The structural homology modeling method is allows potential drug targets to identify active sites i.e. linear epitopes, which form antibodies in host cells. The method integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency. The challenges for the future are to establish the function of all of protein structures. In this assay we use of multiple methods towards the accurate identification of antigenic epitopes. The proposed approach is useful not only for plant and viral biology but it covers the wide area of vaccines and antibodies for therapeutic purposes in humans.

Keywords: Blastocyte, genetically modified organisms, germ line, oocyte, transgene



83-86

Prediction of MHC binder for fragment based viral peptide vaccines from cabbage leaf curl virus

Author(s): Dr. Virendra S Gomase,

Abstract: Cabbage leaf curl viral peptides are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. The MHC peptide binding of pathogenicity proteins is predicted using neural networks trained on C terminals of known epitopes. In analysis predicted MHC/peptide binding of pathogenicity proteins is a log-transformed value related to the IC50 values in nM units. We describe an improved method for predicting linear epitopes (Table 1). The region of maximal hydrophilicity is likely to be an antigenic site, having hydrophobic characteristics, because terminal regions of pathogenicity protein is solvent accessible and unstructured, antibodies against those regions are also likely to recognize the native protein. It was shown that a pathogenicity protein is hydrophobic in nature and contains segments of low complexity and high- predicted flexibility. Predicted antigenic fragments can bind to MHC molecule is the first bottlenecks in vaccine design.

Keywords: Antigen, Epitope, PSSM, SVM, MHC, Peptide vaccine



87-94

Development of MHC class nonamers from Cowpea mosaic viral protein

Author(s): Dr. Virendra S Gomase,

Abstract: Cowpea mosaic virus causes one of the most commonly reported virus diseases of cowpea (Vigna unguiculata), in which it produces chlorotic spots with diffuse borders in inoculated primary leaves. Cowpea mosaic viral peptides are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. Peptide binders identified through this approach tend to high-efficiency binders, which is lagers percentage of their atoms are directly involved in binding as compared to larger molecules. For development of MHC binder prediction method, an elegant machine learning technique support vector machine (SVM) has been used. SVM has been trained on the binary input of single amino acid sequence. We also found the SVM based MHCII-IAb peptide regions 51-PTINHPTFV, 113-PLPKFDSTV, 187-VYSKDDALE, 181- RKYAVLVYS, (optimal score is 1.034); MHCII-IAd peptide regions 138-AISAMFADG, 170-LSAMRADIG, 25- PSSADANFR, 191-DDALETDEL, (optimal score is 0.541); MHCII-IAg7 peptide regions 27-SADANFRVL, 151- LVYQYAASG, 159-GVQANNKLL, 158-SGVQANNKL, (optimal score is 1.692); and MHCII- RT1.B peptide regions 57-TFVGSERCR, 188-YSKDDALET, 68-YTFTSITLK, 44-KTLAAGRPT, (optimal score is 0.787) which represented predicted binders from genome polyprotein m. These antigenic epitope are sufficient for eliciting the desired immune response against viral infection. Study focused on computational approach to deciphering the peptide fragments, which are antigenic in nature for synthetic peptides viral vaccines and their function of genome polyprotein m. In analysis predicted antigenic epitopes of genome polyprotein m are predicted a successful immunization strategy against various diseases.

Keywords: Genome polyprotein, Epitopes, MHC, SVM, Crystal Structure, Hydrophilicity, Hydrophobicity



95-100

The Homeless youth and their exposure to Hepatitis B and Hepatitis C among in Tehran, Iran

Author(s): Dr. Fatemeh Fallah,

Abstract: Nowadays the issue of street children is one of the most important issues of societies from industrial city to developing cities. There are approximately one hundred million children spending their life in streets. With regard to the last description of the International Organizations for street children, many of statistical result and numbers are reported less than the actual facts. many countries are suffering from an individual relation the issue of street children despite possessing common properties, and by controlling this phenomenon each country will get in specific picture. 203 street children picked up from different places of Tehran and settled at welfare center, where provides shelter for street children, were chosen for this study. These children were clinically examined by pediatrician and requested to answer the questionnaire ( asking about their gender; age; birth place; educational status; the origin of the family; sleeping place; occupation, income and social security of parents; number of siblings; reasons for being in streets; period of living in the streets; street friends; means of earning money; substance use. Smoking level was classified as heavy (10 and more per day), medium (1-9 per day) and rare (1-2 per week).) In order to determine the existence of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infections, ELISA, PCR and RT-PCR methods were performed on serum samples. Among 203 street children studied in this research, 196 children were boys and 7 children were girls. 6 cases (3%) were HBsAg positive, 54 cases were HBs- Ab positive (26.6%) and 16 cases were HBc-Ab positive (8%). 7 cases (3.5%) were HCV Ab positive. All of the positive cases were boys. There were 3 Iranian and 3 Afghanian kids among HBsAg positive cases. They did not smoke,they did not have tattoo,all of them had family that one of them lived alone and other 5 cases lived with their family and their average age was <14. In HCV Ab positive cases there were 5 Iranian and 1 Afghanian kids. 3 children did not have family, 6 children did not smoke and one of them was addicted to crack and had tattoo on his body. The average age of this group in three cases was 14> and in four cases 14 < years. 4 cases were HBV PCR positive and 6 cases were HCVRT-PCR positive. According to this results, additional laboratory examination for screening of acquired infectious disease such as Hepatitis seem to be necessary. Although in this type of infection clinical symptom may appear a few months after exposure to the virus, it can be transmissible in this latent period.

Keywords: Hepatitis B, Hepatitis C, street children



101-110

Immunoresistant human glioma cell clones selected with alloreactive cytotoxic T lymphocytes: downregulation of multiple proapoptotic factors

Author(s): Dr. German G. Gomez#,

Abstract: We previously reported the cellular, functional and cytogenetic characterization of immunoresistant (IR) 13-06- IR29 and 13-06-IR30 human glioma cell clones isolated after immunoselection with alloreactive cytotoxic T lymphocytes (aCTL). Relative to the 13-06-MG parental cells, both clones resisted aCTL lysis at multiple effector to target ratios; the resistant phenotype was maintained for 13-41 cell doublings after cloning and when selective pressure was removed; cross-resistance to other inducers of apoptosis/cell death was also observed (Gomez et al, 2006; Gomez and Kruse, 2007). In this study we further characterize the IR clones for factors that may contribute to the resistance. Data obtained by in-vitro quantitative morphologic and 7-amino actinomycin D flow cytometric assays revealed reduced apoptotic cell death when IR clones were coincubated with aCTL, relative to the parental cells. Since changes in apoptosis were observed, we examined the expression patterns of apoptosis-related genes in several extracts of parental cells and IR clones using pathway-specific cDNA microarray analysis. In general, the apoptotic factors were downregulated in the IR clones. From three separate extracts analyzed separately on microarrays, three factors, ATM, caspases 3 and 8, were statistically downregulated in both IR clones. Immunoblotting of the proteins confirmed the findings. Therefore, a possible mechanism for immunoresistance in gliomas may be achieved by the downregulation of one or more genes in the apoptotic pathway.

Keywords: Apoptosis, Astrocytoma, Cellular Therapy, Adoptive Immunotherapy, CTL



111-128

Aptamers in oncology: a diagnostic perspective

Author(s): Dr. Huma Khan,

Abstract: Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called “aptamers” based on the Greek word aptus, which means “to fit”. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate binding characteristics. This technology has spawned the development of a new class of oligonucleotide therapeutic products. However, while interest among pharmaceutical companies continues to grow with some candidates already in clinical trials and one in the market, there appears to be some reluctance to fully explore the diagnostic potential of this technology. This article will review aptamer developments in diagnostics, compare them with other oligonucleotide therapeutics and highlight both potentials and pitfalls of technological development in this area.

Keywords: Aptamers, Diagnostics, Oncology, Aptazymes, Aptasensors



129-140

Dopamine D2 Receptor Taq A1 allele predicts treatment compliance of LG839 in a subset analysis of pilot study in the Netherlands

Author(s): Dr. Kenneth Blum,

Abstract: Various types of individuals having “Reward Deficiency Syndrome (RDS)“ related behaviors including sugar craving (e.g. obesity) have been described and heredity has been shown to be involved in some of these types. An important role of the mesolimbic dopamine system has been suggested in the reinforcing effects of a number of addictive substances (i.e. alcohol, nicotine, sugar etc) and recent molecular genetic studies are implicating the gene for the dopamine receptor (DRD2) as well as other genes in RDS and in particular obesity. We genotyped 1,058 Dutch subjects for polymorphisms of four candidate genes (PPAR gamma 2, MTHFR, 5-HT2a, and DRD2)

Keywords: Reward Deficiency Syndrome (RDS), Dopamine genetics, drd2gene, pharmacogenomics, pharmcogenetics, LG839, amino- acids



141-146

Vaccinomics

Author(s): Dr. Virendra S Gomase,

Abstract: Vaccinomics is the branch of omics, which deals with vaccine analysis; vaccine itself is used for boosting the immunity to diseases. Most of the vaccine reactions appear to be more common than vaccine-preventable diseases. Vaccinomics encompasses the fields of immunogenetics and immunogenomics as applied to understanding the mechanisms of heterogeneity in immune responses to vaccines and new drug targets. Recent advances in the fields of immunology, vaccines, genomics, proteomics and Human Genome Project allowed to discover and developed new vaccines.

Keywords: Attenuation, Influenzae, Genetic Engineering, Toxoid, Vaccine



147-166

Prediction of antigenic binders from c-terminal domain Human papillomavirus oncoprotein e7

Author(s): Dr. Virendra S. Gomase,

Abstract: Human papillomavirus (HPV) is one of the most common causes of sexually transmitted disease (STD). Human papillomavirus viral peptides are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. TAP is a transporter associated with MHC class I restricted antigen processing. The TAP is heterodimeric transporter belong to the family of ABC transporter, that uses the energy provided by ATP to translocate the peptides across the membrane. The subset of this transported peptide will bind MHC class II molecules and stabilize them. These MHC-peptide complexes will be translocated on the surface of antigen presenting cells (APCs). In this assay we predicted the binding affinity of Human papillomavirus oncoprotein e7 having 56 amino acids, which shows 49 nonamers. Small peptide regions found as 9-RHKILCVCC (score 6.186), 34-LRTLQQLFL (Score- 6.091), 31-AEDLRTLQQ (Score- 5.979), 8-QRHKILCVC (Score- 5.960), 45- LSFVCPWCA (Score-5.604), known as oncoprotein e7 TAP transporter. Adducts of MHC and peptide complexes are the ligands for T cell receptors (TCR). These complexes elicit the immune response for clearing various intracellular infections. Prediction methods based on the specificity of TAP transporter will complement the wet lab experiments and speed up the knowledge discoveries on the basis of these two computational algorithms.

Keywords: oncoprotein e7, TAP transporter, MHC, APCs, TCR



167-174

Retrovirally-mediated genetic correction of mesenchymal stem cells from patients affected by mucopolysaccharidosis type II (Hunter Syndrome)

Author(s): Dr. Carla Corradi-Perini,

Abstract: Mucopolysaccharidosis type II (MPSII) is an X-linked metabolic storage disorder due to the deficiency of iduronate-2-sulphatase (IDS) and accumulation of glycosaminoglycans (GAGs). Clinically it presents as a multi- system disorder with developmental delay, bone and joint disease and in the severe forms progressive mental retardation. At present little therapeutic options are available. Bone marrow transplantation is no longer recommended due to the severe side effects and lack of proven efficacy in correcting central nervous system and bone disease. Enzyme replacement therapy is under assessment and it requires weekly, expensive administration for the lifespan of the individual. Mesenchymal stem cells (MSC) are bone marrow-derived cells capable of differentiation into tissue such as bone and have been shown to contribute to bone repair. They are amenable to gene manipulation and therefore provide an excellent target for the correction of MPSII disease, especially with regard to bone disease. In this study we tested whether MSC from MPSII patients (hMSCMPSII) could be corrected with a retroviral vector expressing the IDS gene. Following transduction hMSCMPSII maintained the capacity to differentiate into osteoblasts and adipocytes and showed levels of IDS enzyme over 10 fold higher than those detected in MSC from healthy donors. This led to normalization of GAGs storage in hMSCMPSII. Such transduced cells were able to cross-correct MPSII fibroblasts by uptake of the IDS enzyme via the mannose-6-phosphate receptor. This study suggests that correction of autologous hMSCMPSII by retroviral gene transfer is effective and may be amenable for the improvement of the skeletal features of the disease.

Keywords: Marrow stromal cells, Iduronate-2-Sulphatase, MPSII



175-180

A characterization of genetic haplotypes in BRCA1 identifies linkage disequilibrium with a novel polymorphism in intron 7

Author(s): Dr. Josefa Salgado,

Abstract: Inherited mutations in the BRCA1 gene are known to confer a predisposition to breast and ovarian cancer. Mutations, and low-frequency variants, have been invariably detected on any of the two major haplotypes H1 and H2, albeit more frequently on the dominant haplotype H1. Deleterious mutations, detected in our patients, were studied in relation with different haplotypes. A group of 77 patients with hereditary and familiar breast and/or ovarian cancer have been studied. BRCA1 gene analysis was done by direct sequencing. A seven polymorphic site cassette is used to define the BRCA1-haplotypes in our population. The frequency of a novel polymorphism found was studied in a control population of 100 unrelated healthy volunteers. Two new haplotypes (H2+H4 and H2+H5) not defined before have been found. We have first characterized a novel polymorphism (IVS7+16(TTC)nTTTTC) at intron 7 of BRCA1 gene. The IVS7+16(TTC)nTTTTC polymorphism shows a significant linkage disequilibrium with a cassette of seven polymorphisms that define H2 haplotype in this population. Seven out of eleven patients with deleterious mutations show the polymorphic site cassette. The (TTC)7/7 and (TTC)6/7 genotypes in the intron 7 could be markers for H1 and H2 haplotypes respectively. Larger patient population studies would be needed to study the association between BRCA1 mutations and the presence of the cassette.

Keywords: BRCA1-novel polymorphism, BRCA1-haplotypes, breast cancer, linkage disequilibrium



181-188

Molecular targets in medullary thyroid carcinoma

Author(s): Dr. Sam W Moore,

Abstract: Summary A high percentage of thyroid cancer has been associated with a number of oncogenic genetic variations. The RET proto-oncogene (REarranged during Transfection; RET) is thought to play an important role in the etiology of thyroid tumours and is clearly implicated in Medullary thyroid carcinoma. RET variations which confer oncogenic gain of function, appear to result in the development of cancer due to uncontrolled cellular proliferation as well as failure to undergo normal differentiation and the loss of apoptotic functions. Understanding the biological role of the RET proto-oncogene in thyroid carcinoma is an important area of biomedical research which leads not only to better understanding of the pathophysiological changes involved in oncogenesis but also determines the optimal management and identifies molecular targets allowing the development of novel therapeutic approaches. This review looks at the structure and function of the RET proto-oncogene, its understood method of activation and important molecular targets identified to date. It also studies agents used for novel molecular targeting in treatment of thyroid carcinomas.

Keywords: medullary thyroid carcinoma, RET structure, RET activation, Molecular targets, prophylactic thyroidectomy



189-206

Let-7, miR-125, miR-205, and miR-296 are prospective therapeutic agents in breast cancer molecular medicine

Author(s): Dr. Debmalya Barh,

Abstract: Increasing evidences in recent years demonstrate that several biological processes and disease pathogenesis are regulated by micro RNAs (miRs) and restoration of normal miR activity can be new way of treating cancers. Several genetic alterations and deregulation of miRs have been reported in breast cancer. Similarly, side effects of conventional chemotherapeutic drugs are well known. In this research, using a broad bioinformatics approach we have identified critical disease pathways and drug targets in female breast cancer. Replacement therapy with let-7 is already under clinical trails for lung cancer. Here we have shown that restoration of let-7 along with miR-125 or miR-205 or miR-296 can potentially inhibit all critical disease pathways involved in breast cancer irrespective of patient specific molecular profile. Results also suggest that these miRs might be the future therapeutic agents in breast cancer molecular medicine with out side effects.

Keywords: breast cancer, critical disease pathway, cancer, drug targets, gene therapy, key nodes, let-7, microRNA



207-218

Suppression of Primary and Disseminated Murine Tumor Growth with eIF5A1 Gene Therapy

Author(s): Dr. John E.Thompson,

Abstract: Eukaryotic translation initiation factor (eIF5A) is the only known protein that is post-translationally modified to contain hypusine. The purpose of this study was to establish whether eIF5A1 gene delivery might be an effective therapy against primary and disseminated tumors. The effects of adenoviral-mediated eIF5A1 gene transfer on tumor growth and animal survival were examined using a syngeneic murine melanoma (B16-F0) model and a human lung xenograft (A549) model. Significant suppression was observed in both primary melanoma (p < 0.001; B16-F0) and lung tumor (p < 0.001; A549) growth following intra-tumoral injections of Ad-eIF5A1. Increased incidence of apoptosis was evident in melanoma tumors following Ad-eIF5A1 treatment. Gene transfer of the second member of the eIF5A family, eIF5A2, also gave rise to significant delays in growth of primary melanoma tumors. Animal survival experiments revealed prolonged survival [median survival time: 25 days (treated), 7 days (control) for B16-F0; and 54 days (treated), 24 days (control) for A549]. Systemic administration of DOTAP:pCpG- eIF5A1 complexes into C57BL/6 mice suppressed tumor growth (p < 0.05) in a B16-F10 model of experimental disseminated metastases. Our findings suggest that eIF5A1 may be an important target in the development of treatments for primary and disseminated cancers.

Keywords: eukaryotic translation initiation factor 5A (eIF5A), gene therapy, Adenovirus, DOTAP, melanoma, lung cancer, apoptosis



219-238

Hexavalent chromium exposure, genomic instability and lung cancer

Author(s): Dr. Ana M. Urbano,

Abstract: Worldwide, several million workers experience occupational exposure to different hexavalent chromium [Cr(VI)] compounds (chromates) which have long been recognized as human respiratory tract carcinogens through chronic exposure. Although the majority of lung cancers were found among Cr(VI)-exposed workers who smoked, smoking does not affect chromium accumulation in the lung and chromate exposure was clearly established as an independent risk factor for lung cancer. Compatible with the smoking-unrelated origin of the majority of chromate malignancies are the findings that the molecular features of chromate- and smoking-associated cancers are very different and that the location of chromate lung tumors, i.e. the bronchial bifurcations, corresponds to the sites of chromium accumulation in ex-chromate workers. More recent studies also revealed that environmental exposure to particulate Cr(VI) compounds is increasing due to chromium-containing dusts generated from industrial waste disposal, portland cement, concrete pavement, milling, demolition, cigarette smoke and fuel combustion. Experimentally, it has been demonstrated that Cr(VI) compounds can neoplastically transform cells in culture. They are also genotoxic and can induce a wide spectrum of DNA damage, gene mutations, sister chromatid exchanges and chromosomal aberrations. Albeit extensive information and studies on Cr(VI)-induced effects, the mechanisms mediating Cr(VI)-induced toxicity and carcinogenicity are still poorly understood. This happens mostly due to the use, in a vast majority of the studies, of inadequate model systems and exposure regimens, as well as to the restricted access to lung tumor tissues from Cr(VI)-exposed workers, critical for the identification of consistent cellular and molecular changes. Consequently, additional studies using adequate model systems and exposure regimens mimicking occupational exposure conditions will have to be performed in order to understand the signalling mechanisms involved in the cellular response to Cr(VI), as well as the role of genomic instability and of specific DNA repair pathways in the development of this pathology. Knowledge of these pertinent issues will be a step forward for the discovery of biomarkers of malignant transformation that might lead to new treatment approaches or new ways to prevent this particular subtype of lung cancer. This review will discuss several aspects underlying Cr(VI)-induced carcinogenicity. However, particular emphasis will be given to the recently identified roles of Cr(VI)-induced DNA lesions, particularly single- and double-strand breaks, on genomic instability, one of the hallmarks of lung cancer.

Keywords: Hexavalent chromium, lung cancer, genomic instability, microsatellite instability



239-246

Screening of coding region of metastasis suppressor genes KISS1 and KAI-1 for germ line mutations in breast cancer patients

Author(s): Dr. Fraz Arshad Malik,

Abstract: Breast cancer is one of the most common female cancers worldwide. Abnormalities of genetic or epigenetic factors are mainly responsible for the development and progression of mammary tumours. In patients with breast cancer, metastasis is the leading cause of death. In recent years, a group of genes has been identified as metastasis suppressor genes (MSGs), which are involved in the suppression of the growth of secondary tumours. Down regulations of MSG expression have been frequently observed in advanced tumours. The present study was designed to screen two of the most frequently down-regulated MSGs (KAI1 and KISS1) for germ line mutations in sporadic breast cancer cases of the Pakistani population. 170 cases of unilateral breast cancer patients, who had no prior history of breast cancer and no other disease in general in their families with age ranging from 35-75yrs, were included in this study. Mutational analysis for the entire coding region of KAI1 and KISS1 (including 10 exons and 3 exons, respectively) was carried out by using the Single Strand Conformational Polymorphism (SSCP) technique. No germ line mutation was observed on the entire coding region in the samples from patients with breast cancer in the Pakistani population. Splice site variants on these genes were also absent in breast cancer patients. Involvement of germ line mutations for these MSGs is thus considered to be an event that occurs less frequently in breast cancer patients of Pakistani population. Conserved coding regions of both MSGs indirectly enlighten the involvement of transacting factor on DNA sequence as major contributor in the progression and aggression of tumours rather than any high risk associated mutation itself. A detailed analysis of regulatory mechanism is required to explore the genetic basis of down regulation of these MSGs for a better understanding of breast cancer progression.

Keywords: sporadic breast cancer, KISS1, KAI1, Single strand Conformational polymorphism, SSCP



247-252

A new potential radiosensitizer- multi-walled carbon nanotubes modified by ammonium persulfate

Author(s): Dr. Jian-She Yang,

Abstract: Here we prepare carbon nanotubes modified with ammonium persulfate, very short carbon nanotubes with 50-100 nanometer length was obtained, and the higher Ζ potential of 52 mV was detected, these supporting the successful modification. HeLa cells were irradiated with γ rays via adding or absent above functionalized carbon nanotubes (f- WCNTs) into cell culture medium with different concentration and radiation dosage. Confocal microscopy images and fluorescence-labeled DNA detection verified the successfully pure multi-walled carbon nanotubes (p-WCNTs) and f-WCNTs penetrated into cells. Compared with pure radiation, by MTT test, f-WCNTs induced cell death markedly with about 8.7 times higher than former one under little dose of radiation; meanwhile, no obvious toxicity was observed both in p-WCNTs and f-WCNTs without of radiation exposure. We hypothesized that large amount of hydroxyl and carbonyl organs on the surface of very short f-WCNTs changed into free radicals result from radiations led cell damage. These implied that f-WCNTs could be regarded as a new radiosensitizer.

Keywords: ammonium persulfate, multi-walled carbon nanotubes



253-258

Decreased risk of bladder cancer in men treated with quinazoline-based !1-adrenoceptor antagonists

Author(s): Dr. Dr. Natasha Kyprianou,

Abstract: Previous studies documented that human bladder cancer cells are sensitive to the apoptotic effects of quinazoline- derived !1-adrenoreceptor antagonists and bladder tumors exhibit reduced tissue vascularity in response to terazosin. More recent evidence suggests that exposure to quinazoline !1-adrenorecptor antagonists leads to a significant reduction in prostate cancer incidence. This retrospective observational cohort study was conducted to determine whether male patients treated with quinazoline !1-adrenoceptor antagonists for either benign prostate hyperplasia (BPH) or hypertension have a decreased risk of developing bladder cancer. Review of the medical records of all male patients enrolled at the Lexington Veterans Administration (VA) Medical Center identified men exposed to quinazoline-based !1-adrenoceptor antagonists (Jan 1, 1998-Dec 31, 2002) for either hypertension and/or benign prostate obstructive symptoms. The whole group of 27,138 male patients was linked to the Markey Cancer Center’s Kentucky Cancer Registry (KCR), part of the NCI’s Surveillance, Epidemiology, and End Results (SEER) Program, to identify all incident bladder cancer cases diagnosed in this population. Measures of disease incidence, relative risk, and attributable risk were calculated to compare the risk of developing bladder cancer for !1-blocker-exposed versus unexposed men. A two-by-two contingency table of !1-antagonist exposure versus bladder cancer diagnoses was constructed and the relative risk was calculated. Our analysis revealed a cumulative bladder cancer incidence of 0.24% among the !1-blocker-exposed men compared to 0.42% in the unexposed group. Thus, there was a risk difference of -0.0018, which indicates that 1.8 fewer bladder cancer cases developed per 1000 exposed men. Alternatively stated, 556 men would need to be treated with quinazoline !1-blockers to prevent one case of bladder cancer. Exposure to quinazoline !1-blockers thus may have prevented 7 to 8 bladder cancer cases among the 4173 treated men during the study period .The data yield an unadjusted risk ratio of 0.57 (95% CI: 0.30, 1.08) and therefore, men treated with !1-adrenoreceptor antagonists have a 43% lower relative risk of developing bladder cancer than unexposed men (p=0.083). Our inability to determine person-years at risk of developing bladder cancer for each unexposed control patient, was a limitation for calculating an incidence ratio and rate difference. These results offer an initial indication that exposure to doxazosin and terazosin decreases the incidence of bladder cancer. This is the first epidemiological evidence that the anti-tumor action of quinazoline-based !1- antagonists may potentially translate into a protective effect from bladder cancer development.

Keywords: Bladder Cancer, Prevention, !1-adrenoceptor Antagonists, Apoptosis



259-266

Hematopoietic growth factors in the elderly

Author(s): Dr. Wassim Mchayleh,

Abstract: The Hematopoietic System is subject to the aging process. This translates in a blunted response to Hematopoietic stress in the elderly population. The clinical use of Hematopoietic growth factors (HGFs) has helped transform the care of the elderly cancer patient. The indications for the use of hematopoietic growth factors in the elderly population are no different from the general population. In fact, given the increased susceptibility of the elderly cancer patient to treatment related morbidity and mortality, there may be even more compelling reason for the use of growth factors, to obviate complications of myelosuppression. We review the biology of aging and hematopoiesis, and the indications for the use of HGFs in the elderly.

Keywords: Elderly, Growth Factors, G-CSF, Erythropoeitin



267-276

HSP70 variations in the acute treatment with mood stabilizers in patients with bipolar disorder: results of a preliminary work

Author(s): Dr. Chi-Un Pae,

Abstract: A pharmacogenetic approach was used to investigate the role of heat shock protein (HSP) 70 on the effect of mood stabilizers since a line of evidence has proposed a possible involvement of its chaperone activity in the pathophysiology of bipolar disorders. Forty five patients with bipolar I disorder were treated for an average of 36.5 (±19.9) days with mood stabilizers (lithium, valproate, or carbamazepine), were evaluated with using the Clinical Global Impression (CGI) scale and the Young Mania Rating Scale (YMRS), and were genotyped for their HSP 70 variants (rs2227956 C/T, rs2075799 A/G, rs1043618 C/G, rs562047 C/G, rs539689 C/G). Results: No association was found between the investigated variations and response to mood stabilizer treatments even considering possible stratification factors. The small number of subjects is an important limitation to the present study, nonetheless HSP 70 gene variants seem not to be involved in acute antimanic effect. Adequately-powered study would properly address the potential role of HSP 70 gene variants for the effect of mood stabilizers.

Keywords: Pharmacogenetics, lithium, heat shock proteins, bipolar disorder, manic



277-292

Recombinant adeno-associated virus as vaccine delivery vehicles

Author(s): Dr. Shree R. Singh,

Abstract: Adeno-associated viruses (AAV) are non-enveloped, replication defective, single-stranded DNA virus and require co-infection with a helper virus, such as adenovirus or herpes virus, to undergo a productive infection. The AAV genome is 4.7 kb in size and is framed by two inverted terminal repeats (ITRs) at both ends of the DNA strand, and contains two open reading frames (ORFs): Rep and Cap. In total, 11 strains of AAV have been isolated and characterized from humans and primates, and new serotypes are continuously discovered. All serotypes share similar structure, genome size and organization. Serotype 2 (AAV2) has been the extensively studied and presents natural tropism towards skeletal muscles, neurons, retinal cells, vascular smooth muscle cells and hepatocytes. The most divergent serotype is AAV5 with notable differences at the level of the ITR size. AAV`s natural defectiveness, its lack of pathogenicity, and the ability to infect cells in vivo have led to the study of its potential use as a gene therapy vector. Recent studies have begun to test AAV vectors as vaccine carriers against human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis, severe acute respiratory syndrome (SARS), and many other viruses. rAAV vectors can evade the immune response and mediate a durable expression of transgene in vivo. However, evidence has been gathering that in some circumstances, the rAAV vector may initiate a cellular and humoral response to the expressed gene product in vivo. It is therefore important to understand the factors, which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV- based gene therapies or vaccine delivery. Various factors seem to influence the immune response of AAV vaccine vectors. These include the AAV serotype, the transgene, route of administration, dose of vector, transgene expression levels, immune responses to the viral capsid, and others. A more thorough understanding of the interplay between rAAV and their encoded transgenes and the host immune system is necessary for the optimal development of rAAV vaccine system.

Keywords: Adeno-associated virus, Vector, Vaccine, Transgene, Cellular response, Humoral response



293-300

Inhibition of focal adhesion kinase with her-2 targeted antibody pertuzumab (Omnitarg®, 2C4) in breast cancer cells

Author(s): Dr. Emel Canbay,

Abstract: Pertuzumab (Omnitarg!, 2C4) is a recombinant humanized monoclonal antibody targeted to extracellular region of HER-2. Previous results proved the inhibitory effect of Pertuzumab on the survival of breast cancer cells via MAPK and Akt pathway. Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. Here, we aimed to investigate the effects of Pertuzumab on ligand activated total FAK expression and phosphorylation in the HER-2 overexpressing BT-474 breast cancer cell line. Heregulin was used for ligand activation. We have found that FAK expression and phosphorylation were inhibited in with Pertuzumab in breast cancer cells.

Keywords: Pertuzumab, Omnitarg®, 2C4, focal adhesion kinase, breast cancer



301-311

Enhanced splenic protection and reduction of parasitaemia following transplantation of splenocytes overexpressing Bcl-2 in a murine malaria model

Author(s): Dr. Martha Legorreta-Herrera,

Abstract: Malaria is one of the most important infectious diseases worldwide. During acute infection, the activation of the immune response and parasite clearance by the spleen are key host defence mechanisms. Apoptosis of lymphocytes is believed to compromise the immune response in malaria cases. As limited progress has been made using gene therapy against parasitic infections, we explored the potential beneficial effect of reconstituting Plasmodium chabaudi infected mice with transduced splenocytes overexpressing Bcl-2. An ecotropic retrovirus encoding GFP and Bcl-2 was packaged in 293T cells. A second retrovirus expressing only GFP was used as control. Initially, CBA/Ca mice were transplanted with 2 x 107 total splenocytes from syngenic donor mice transduced with retroviral particles. Two hours later recipient mice were infected with 5 x 104 P. chabaudi parasitised erythrocytes. Parasitaemia was evaluated every 24 h. Seven days post-infection, we evaluated splenic histology and Bcl-2 expression via RT-PCR and western blot. The protection against H2O2- induced apoptosis was tested on isolated total splenocytes. The efficiency of retroviral infection, expression of Bcl-2 and reporter gene was confirmed by Fluorescence microscopy, RT-PCR and western blotting of splenocytes from transplanted with cells transduced with GFP- or Bcl-2-expressing retroviruses. Parasitaemia in mice transplanted with GFP-transduced splenocytes transiently decreased 10-fold at day five after Plasmodium infection and was further decreased when mice were transplanted with Bcl-2-transduced splenocytes. The splenic architecture of mice that received transduced cells was preserved compared to control mice. Cytoprotection in vitro was enhanced with the Bcl-2 construct. The protective effect correlated with increased the cellularity in the spleen and overexpression Bcl-2. We tested the use of standard gene therapy strategies based on the reconstitution of irradiated mice with genetically modified heterologous cells in 301 Martínez-Flores et al: Enhanced splenic protection and reduction in a murine malaria model an experimental murine model of malaria. We observed transient reduction of parasitaemia, preservation of splenic architecture and increased cell survival in vitro against oxidative stress. Although the experimental design aimed to test the effect of Bcl-2 overexpression, transplantation of cells transduced with the empty retroviral vector resulted in a significant protective effect accompanied by the increased expression of endogenous Bcl-2.

Keywords: Apoptosis, retroviral vectors, promoter PolII, oxidative stress



313-357

Microtubule-targeted antitumor drugs: chemistry, mechanisms and nanoparticle formulations

Author(s): Dr. Teni Boulikas,

Abstract: The ingenious application of vinca alkaloids (vinblastine, vinorelbine, vincristine, vindesine, vinflunine) destabilizing microtubules and of taxanes (paclitaxel, docetaxel) stabilizing microtubules has been a milestone achievement in oncology. Recent investigations into their molecular mechanism revealed that all compounds possess additional pleiotropic effects that converge on induction of apoptosis in cancer cells via activation of signaling pathways. Their success has prompted vigorous investigations into microtubule-targeting activity from natural products as well as synthetic molecules arising from molecular modeling that led to the identification of Epothilones A, B, D, Ixabepilone, Sagopilone, Discodermolides, Dictyostatin, Peloruside A, and ABT-751; an additional purpose for epothilone drug discovery has been to bypass paclitaxel resistance mainly arising from the efflux function of P- glycoprotein. Nanoparticles provide a new mode of cancer drug delivery functioning as a carrier for entry through fenestrations in tumor vasculature. The 130-nm nanoparticle formulation albumin-bound paclitaxel (Nab- paclitaxel, AbraxaneTM) utilizes the natural properties of albumin to reversibly bind paclitaxel, transport it across the endothelial cell and concentrate in tumors; AbraxaneTM received regulatory approval in the USA from a higher response rate and longer time to progression than Taxol in patients with metastatic breast cancer. The success of AbraxaneTM led to an explosion in research on polymer nanoparticle formulations for taxanes using micellar PEGylated hyperbranched polyesters, polyglycerol-polyethylene glycol copolymers, cyclodextrin nanoparticles, polylactide-co-glycolide PEG and many others. Several such formulations are expected to enter the market. Other tubulin polymerization inhibitors reviewed here include tubulysin A, a highly cytotoxic peptide from myxobacteria, CC-5079, bisbenzylisoquinoline alkaloids, aplidine, nocodazole, GMC-5-193, cyclostreptin, colchicine, TLK-286 and vinflunine, a novel third generation vinca alkaloid. Structural similarities have been used to further modify the successful microtubule-targeted drugs in order to seek molecules of improved efficacy, of lower toxicity or able to overcome tumor resistance. We review the molecular mechanism of these drugs, whenever feasible, we suggest correlations between their chemical structure and mechanism and point to the importance of drug delivery for success.

Keywords: Microtubules, tubulin, vinca alkaloids, vinblastine, vinorelbine, vincristine, vindesine, vinflunine, taxanes, paclitaxel, docetaxel, epothilones A, B, D, ixabepilone, sagopilone, discodermolide, P-glycoprotein, dictyostatin, abraxane, colchicine, aplidin



359-370

Gene expression signature-based chemical genomics and activity pattern in a panel of tumour cell lines propose linalyl acetate as a protein kinase/NF-κB inhibitor

Author(s): Dr. Saadia Bashir Hassan,

Abstract: The essential oil of Lebanese sage, Salvia libanotica, was reported to have anti-tumour activity; however, the mechanism of action has not been identified yet. In this study, 14- cancer cell lines including drug-sensitive and resistant lung, leukaemia, and colon, as well as primary human tumours of chronic lymphocytic leukaemia (CLL) and primary normal mononuclear cells (PBMCs) were used to characterize the anti-tumour activity and mechanism of action of linalyl acetate, a component of the Lebanese sage essential oil. Drug activity and gene expression data sets were utilized to identify drugs with similar activity patterns and genes involved in drug sensitivity/resistance. In addition, the Connectivity Map, a gene expression signature-based screening approach, assisted in predicting further the molecular action of linalyl acetate. Small cell lung carcinoma and colorectal cancer cell lines were the most sensitive to the drug and greater tumour selectivity was observed against chronic lymphocytic leukaemia cells compared to normal mononuclear cells. Only limited effect of some of the classical mechanisms of multi-drug resistance on the activity of Linalyl acetate was noted which makes it potentially interesting for drug-resistant patients. There was high similarity between the activity-pattern/gene expression profile of linalyl acetate and that of protein kinase/NF-κB inhibitors. Validating this, linalyl acetate was found to strongly inhibit Janus kinase, JAK3, and p38α kinases in a cell-free assay as well as the NF-κB translocation in a dose-dependent manner. Taken together, our results show that the NF-kB inhibitor, linalyl acetate, may represent a new therapeutic compound in the management of inflammation and cancer.

Keywords: Linalyl acetate, NF-κB, Connectivity Map



371-382

A short term pilot open label study to evaluate efficacy and safety of LG839, a customized DNA directed nutraceutical in obesity: Exploring Nutrigenomics

Author(s): Dr. Kenneth Blum,

Abstract: We hypothesized that genotyping certain known candidate genes would provide DNA-individualized customized nutraceuticals that may have significant influence on body re-composition by countering various genetic traits. It is well known that obesity and related symptoms significantly aggravates type 2 diabetes. Both obesity and diabetes are influenced by the interaction of both genes and environmental factors. Exploration of the current literature has identified a number of candidate genes to be associated with both of these two disorders and include amongst others the dopamine D2 receptor (DRD2), methylenetetrahydrofolate reductase (MTHFR), serotonin receptor (5-HT2a)Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ), and Leptin (OB) genes. In the present study, we systematically evaluated the impact of polymorphisms of these five candidate genes as important targets for the development of a DNA-customized nutraceutical LG839 [dl-phenylalanine, chromium, l-tyrosine other select amino-acids and adaptogens]) to combat obesity with special emphasis on body recomposition as measured by Body Mass Index (BMI). A total of 21 individuals were evaluated in a preliminary investigational study of LG839. Based on the results of buccal swab genotyping of each subject, an individualized customized nutraceutical formula was provided as a function of measured gene polymorphisms of the five gene candidates assessed. At the inception of the study and every two weeks subsequently, each subject completed a modified Blum-Downs OPAQuE ScaleTM [Overweight Patient Assessment Questionnaire]. The alleles included the DRD2 A1; MTHFR C 677T; 5HT2a 1438G/A; PPAR-γPro12A1a and Leptin Ob1875<208bp. Pre- and post ad hoc analysis revealed a significant difference between the starting BMI and the BMI following an average of 41 days (28-70d) of LG839 intake in the 21 individuals. The pre- BMI was 31.2 (weight/Ht2) compared to the post BMI of 30.4 (weight/Ht2) with a significance value of P < 0.034 (one tailed). Similarly the pre -weight in pounds (lb) was 183.52 compared to the post weight of 179 lb with a significance value of P<(0.047). We also found trends for reduction of late night snacking, carbohydrate craving reduction, reduction of stress, reduction of waist circumference. Moreover, in the 41 day period we found a trend in weight loss whereby 71.4% of subjects lost weight. Thus 15 out of 21 subjects lost weight with a z score of 2.4 and significance value of P <(0.02). In this group 53% lost on average over 2.5% of their starting weight. Further confirmation of these preliminary results (ongoing) warrants investigation and should ultimately provide novel DNA directed “omic” therapeutic targets of novel anti-obesity agents especially in diabetes and other related diseases.

Keywords: LG839; SynaptamineTM, Reward Deficiency Syndrome (RDS), Obesity, Diabetes, nutrigenomics, polymorphisms, genes



383-386

Severe phenotype of Morquio A disease in a child with S287L N-acetylgalactosamine-6-sulfate sulfatase mutation

Author(s): Dr. Zoran S Gucev,

Abstract: Mucopolysaccharidosis IVA is caused by a deficiency of lysosomal N-acetyl-galactosamine-6-sulfate sulfatase (GALNS; E.C.3.1.6.4). One hundred fourty-eight GALNS mutations were described in patients with different phenotype severity. Our patient was a 6.5 year old boy with severe kyphoscoliosis and growth delay. He developed coarsening of the facial features and megalencephaly. Both corneas were cloudy. His gait was difficult with limited and painful movements in the hips, the spine and the knees. X-ray studies showed platispondily with ovoid vertebrae, bulging sternum and flaring of the rib cage. The long bones were short with irregular trabeculation. Metaphyses were widened, femoral head was flattened. The metacarpals had conical bases. Total excretion of glycosaminoglycans (GAG) in urine was increased. Thin layer chromatography of urinary GAG showed massive excretion of keratan sulphate. N-acetyl Galactosamine-6-sulphate sulfatase activity in leukocytes was low (0.7 nmol/MU17h/mg protein). DNA sequencing detected a S287L mutation (c.860C>Tc.860 C>T). This is the first GALNS mutation described in our population. The same mutation conferred a severe MPS IVA phenotype in a American, Austrian and Polish patients.

Keywords: Morquio A disease; N-acetyl galactosamine-6-sulphate sulfatase; S287L mutation; severe phenotype



387-394

Attenuation of experimental liver fibrosis by hepatocyte growth factor gene delivery mediated by adenovirus

Author(s): Dr. Bin Wu,

Abstract: The hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, has an essential part in the development and regeneration of the liver. In this study, HGF expression in vitro and in vivo was determined using ELISA. Rats were injected subcutaneously with CCl4 for eight weeks to induce liver fibrosis, and then divided randomly into groups for administration of various doses of adenovirus HGF (Ad-HGF) or vehicle. All rats were sacrificed 8 weeks after obtaining samples of serum and hepatic tissue. The results showed that glutamic oxaloacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), total protein (TP), albumin (ALB), and total bilirubin (TBil) in serum were significantly recovered after gene therapy (P<0.05). Ad- HGF also attenuated the expression of TGF-!1 and the deposition of collagen. We conclude that intravenous administration of Ad-HGF may be useful for the treatment of fibrotic liver by promoting liver function recovery and collagenolytic capacities.

Keywords: Liver fibrosis; Hepatocyte growth factor; Transforming growth factor; Gene therapy; Adenovirus



395-404

Regulation of hormonal therapy resistance by cell cycle machinery

Author(s): Dr. Ratna K. Vadlamudi,

Abstract: Estrogen Receptor (ER) plays a central role in the development and progression of breast cancer. Hormonal therapy substantially improves disease-free survival of ER+ve breast tumors, however acquired resistance to endocrine therapies frequently occur. Emerging data implicate growth factor signaling pathways and their cross talk with ER as major cause of resistance. Both these pathways have been recently shown to use cell cycle machinery as downstream effectors in mediating therapy resistance. Several studies have demonstrated deregulation of cell cycle regulators and their cross talk with ER in therapy resistant tumors. The objective of this article is to review the underlying mechanisms by which tumor cells use cell cycle machinery to override hormonal therapy and to explore cell cycle machinery components as novel therapy targets for overcoming hormonal therapy resistance.

Keywords: Cell Cycle, CDKs, Estrogen, Estrogen Receptor, Co-regulators, Breast cancer, Therapy resistance, Antiestrogens



405-414

Reversal of MDR1/P-glycoprotein associated drug resistance in human Hepatoblastoma

Author(s): Dr. Steven W. Warmann,

Abstract: Despite impressive improvements of treatment results in children suffering from hepatoblastoma (HB), advanced tumor stages still provide unsolved problems for treating physicians. A major factor for this phenomenon is the phenotype of drug resistance. The mechanism related to the Multidrug Resistance Gene 1 (MDR1) and its product P-glycoprotein (P-gp) has been identified in experimental studies as major factor contributing to drug resistance in HB. P-gp is an ATP dependant membrane channel, which pumps cytotoxic agents out of the tumor cells. Modulation of P-gp using atoxic chemosensitizers improved treatment results in several tumor types. There is a correlation between MDR1 gene expression levels and the amount of applied chemotherapy courses in resected HB. Relapses and metastases show the highest expression followed by primary tumors after neoadjuvant chemotherapy. Lowest expression levels are found in tumors that were not pre-treated before surgery. We also observed significant treatment improvements of chemosensitizers compared to respective mono-therapies both, in cell lines and in xenotransplanted HB. In cell cultures, cell viabilities decreased significantly without an increase of MDR1 gene expression levels. In xenotransplanted HB, tumor growth and serum alpha-fetoprotein levels were decreased significantly. Again, there were no effects of chemosensitizers on MDR1/P-gp expression levels. Our data underline the hypothesis that chemosensitizers may represent a promising tool for the treatment of advanced types of HB.

Keywords: MDR1/P-glycoprotein, Hepatoblastoma, Standard Therapy (IPA), CARBO/VP-16, High dose CARBO/VP-16




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