Volume 14 - 2012

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Gene therapy in a mouse tumor model of breast cancer by siRNA-mediated down-regulation of STAT3

Author(s): Dr. Qingwei, Zhou,

Abstract: Breast carcinoma is one of the most common forms of cancer, with a high prevalence and mortality rate worldwide. Signal transducer and activator of transcription 3 (STAT3) plays a key role in tumor cell survival and proliferation, angiogenesis, apoptosis. It is aberrantly activated in several types of cancers, including breast cancer. We assessed the therapeutic effects using a DNA vector-based STAT3-specific small interfering RNA (pSi-STAT3) on a murine breast cancer model. We observed the tumor growth in evry groups and further discussed the mechanism underlying. STAT3 was significantly down- regulated at both the mRNA and protein levels in the pSi-STAT3 group. The growth of the tumors was significantly reduced in the pSi-STAT3-treated mice. Flow cytometry revealed that the number of early apoptotic cells was significantly elevated in the pSi-STAT3 group. Moreover, in the pSi-STAT3 group, the mRNA expression of the STAT3 downstream genes Bcl2 and c- Myc was also significantly inhibited, and immunohistochemistry revealed that the expression of STAT3, HIF1 and PCNA protein were reduced in the tumor tissues. Our results suggested that STAT3-specific siRNA significantly suppressed tumor growth in breast cancer-bearing mice. It might be a useful therapeutic strategy in malignancies.

Keywords: Breast cancer, STAT3, RNA interference, gene therapy


The Mycobacterium tuberculosis DNA binding protein Rv3488 binds to the intergenic region between Rv3488 and lipF

Author(s): Dr. Beatrice, Saviola,

Abstract: The Rv3488 gene encodes a protein that contains homology to transcriptional regulators. The immediate upstream gene, lipF, of Mycobacterium tuberculosis encodes an esterase and is upregulated by acidic stress. We over expressed recombinant Rv3488 and bound it to an intergenic promoter DNA region of M. tuberculosis between lipF and Rv3488 in the presence of specific and nonspecific competitor. Specific binding of recombinant Rv3488 to this intergenic region was observed at pH 7.5, pH 6.5, and pH 5.5.

Keywords: Acidic stress, lipF, Rv3488, transcriptional activator, lipase, Mycobacterium tuberculosis


The effects of antioxidative additives on electroporation efficacy

Author(s): Dr. Ľubomíra , Tóthova,

Abstract: Gene transfer into eukaryotic or prokaryotic cells is an emerging field in scientific research, however with transfection success being low. Many approaches to increase this efficiency rate are being tried, one of which is electroporation. The aim of this experiment was to analyze the possible enhancing effects of antioxidants on gene transfer mediated by in vitro and in vivo electroporation. E. coli DH5α cells were electroporated with plasmid pBR322. The antioxidants, melatonin and a combination of vitamin C and E were added directly to the electroporation buffer or the cells were pre-treated by growth in a medium containing these antioxidants. VEGF plasmid in the presence or absence of the antioxidants was administered. Vitamins C and E, and melatonin improved the electroporation efficacy in vitro, if they were applied directly into the electroporation buffer. A significant decrease in electroporation efficiency appeared if the cells were pre-treated with melatonin compared with the control group. The presence of antioxidant vitamins in electroporated muscles increased VEGF expression twofold. The results show that antioxidants are able to increase the efficiency of electroporation-mediated gene transfer, however further studies regarding dosing and timing should be performed.

Keywords: ascorbic acid; α-tocopherol; electropermeabilization; gene delivery; melatonin


Similar effects of chronic voluntary alcohol intake and high dose superoxide dismutase gene delivery on oxidative and carbonyl stress in rats

Author(s): Dr. Roman , Gardlik,

Abstract: Experimental and clinical studies demonstrate a crucial role of alcohol in the etiology of various diseases. Oxidative stress is among the major mechanisms of detrimental effects of alcohol. Our previous study showed a protective effect of low molecular weight antioxidants on the level of oxidative and carbonyl stress markers in a model of chronic compulsory alcohol intake. The aim of this study was to evaluate the effects of an enzymatic high molecular weight antioxidant (manganese superoxide dismutase - Mn-SOD) gene delivery on oxidative and carbonyl stress in a rat model of chronic voluntary alcohol intake. Male Wistar rats received either water (control, SOD) or alcohol solutions (alcohol, alcohol + SOD) instead of drinking water during a period of 28 days. The concentration of ethanol gradually increased every week (5, 10, 15, 20% v/v). Plasmid encoding mitochondrial Mn-SOD or saline was injected intramuscularly once a week. At the end of the study, the levels of advanced glycation end products (AGEs) and ascorbic free radicals in serum were decreased by both, chronic alcohol treatment and SOD gene application independently. These effects had an additive potential, as the combination of alcohol and SOD treatment resulted in the lowest levels of these markers (p<0.001). As expected, SOD gene therapy decreased the levels of serum advanced oxidation protein products (AOPP). Interestingly, chronic alcohol intake had no effect on the oxidative damage of proteins and the combination of treatments significantly increased AOPP (p<0.001). Further, alcohol and SOD independently decreased lipoperoxidation in cerebellum, as estimated by malondialdehyde measurement (p<0.05). Malondialdehydelevels in alcohol + SOD group were similar to those observed in the control group. In summary, carbonyl and oxidative stress markers were decreased by a high dose SOD gene therapy as well as by chronic voluntary alcohol intake. The combination treatment resulted in contradictory effects on the monitored parameters – additive, if considering antioxidative status and AGEs production; antagonistic, if considering malondialdehydeand AOPP levels. Further studies are needed to explain and prove these results.

Keywords: alcohol; oxidative stress; carbonyl stress; antioxidants; gene delivery


Bax and APPL1 are involved in DCC-ADD induced colorectal carcinoma cells apoptosis

Author(s): Dr. Zhongyi, Cong,

Abstract: DCC (deleted in colorectal carcinoma) is a tumor suppressor whose expression is significantly reduced or absent in most advanced colorectal carcinoma. DCC is a transmembrane receptor for netrin-1 functioning as a dependence receptor that triggers apoptosis in the absence of netrin-1. The addiction dependence domain (ADD) that located in the intercellular region of DCC is prerequisite for the pro-apoptotic activity, which is released when DCC is cleaved by caspase3 and interacts with caspase9 and activates downstream caspases. However, the concrete molecular mechanism is not very clear. In the present study, we constructed the recombinant plasmid pIRES2-EGFP-ADD, and transfected it into human colorectal carcinoma cells SW1116. We investigated the cell proliferation and apoptosis through CCK-8 cell proliferation assay, AO/EB fluorescence staining assay and TUNEL assay. And the expression of Bax and APPL1 was detected through immunocytochemistry method and flow cytometry. The results revealed that DCC-ADD gene transfection significantly inhibited SW1116 cells proliferation (P<0.05). The number of apoptotic cells of pIRES2-EGFP-ADD transfection group was obviously more than that of pIRES2-EGFP transfection group and medium. And the expression of Bax and APPL1 was increased post DCC-ADD gene transfection. The results supported the viewpoint that DCC is a tumor suppressor, which can inhibit the propagation of tumor cells by inducing apoptosis. Moreover, our data show that in addition to caspases, Bax and APPL1 are closely related to the DCC-induced apoptosis, which may give new clues for the DCC-induced apoptosis.

Keywords: DCC (deleted in colorectal carcinoma); colorectal carcinoma; apoptosis; Bax; APPL1


Engineered Smart Biomaterials for Gene Delivery

Author(s): Dr. F. Azizi , Jalilian,

Abstract: The delivery technology of gene into cells has been increasingly paid attention for gene therapy and the generation of genetically engineered cells. If it is possible to artificially introduce exogenous genetic materials into cells at a high transfection efficiency by a delivery technology, the technology will give academically, clinically, and practically great impacts to gene therapy, cell and molecular biology or pharmaceutical and food industries for bio-productions. The major aim of gene therapy is to effectively deliver the genetic materials into cells, genetically modifying and repairing cell functions, which may induce therapeutic healing of disease conditions. The genetic material involves DNA, RNA, antisense, DNA decoy, and ribozyme, and it is expected that their appropriate transfection allows disease cells to turn to a good direction of recovery. The genetic manipulation often manifests the mechanisms of intracellular machineries of gene and protein, while it may play an important role in making clear the appropriate genes associated with various diseases. Based on the basic and scientific knowledge, the delivery technology of gene is applicable to produce various proteins pharmaceutically valuable, e.g. cytokines, growth factors, and antibodies as well as seeds strong against harmful insects and cold weather damage. In other words, the cells genetically innovated work as the microfactory to produce valuable pharmaceutical and food products. This review provides a critical view of different approaches of gene therapy with a major focus on smart biomaterials transfection agent technologies to control the in vitro and in vivo localization and function of administered genes.

Keywords: non-viral delivery, gene therapy, gene vectors, gene uptake, cationic polymers, cationic lipids


In silico analysis of chimeric polytope of cancer/testis antigens for dendritic cell-based immune-gene therapy applications

Author(s): Dr. Mohammad Mahdi , Forghanifard,

Abstract: Background: Cancer cells benefit from different biological processes that allow them to grow successfully even in the presence of native defense mechanisms. The development of effective cancer vaccines could benefit from in silico techniques that evaluate tumor cell biology. Cancer/testis antigens (CTAs) exhibit limited expression patterns in normal human germ line and placenta cells, while overexpression of CTAs has been reported frequently in a variety of cancer cell types. Among CTAs, MAGEA4, LAGE1, and NY-ESO1, which are highly overexpressed in esophageal squamous cell carcinomas, may have crucial roles in tumor cell progression and metastasis. Results: In this study we designed a synthetic chimeric gene encoding the MHC class-I HLA-restricted epitopes of MAGEA4, LAGE1, and NY-ESO1. This polytope molecule was designed based on human codon optimization. The epitopes were linked using five hydrophobic amino acid repeats. The structure of the synthetic gene, related mRNA, deduced protein, and their stabilities were analyzed by bioinformatic software. Furthermore, the cleavage sites, T- cell epitopes, and MHC-binding affinity of peptides in the construct were predicted using web-based servers. The solvent accessibility and post-translational modifications of the construct’s protein were also predicted. Conclusion: A new combination of antigenic epitopes of CTAs, which may define accessibility, solubility, and immunogenicity, is presented to develop a chimeric gene as an effective structural model for cancer immune-gene therapy.

Keywords: Cancer/testis antigens, DC-immune-gene therapy, Chimeric construct, In silico analysis, Esophageal squamous cell carcinoma

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