[Research advances on hypolipidemic effect of polysaccharides].

Hyperlipidemia is a systemic chronic metabolic disease caused by dyslipidemia in the body. It is an important risk factor of accelerating atherosclerosis, which will cause coronary heart disease, thrombus and other cardiovascular diseases, so it is a "invisible killer" for human health. Controlling and lowering blood lipids can reduce the risk of cardiovascular and cerebrovascular diseases. The current therapies for hyperlipidemia mainly include chemical synthetic medicines. However, long-term use of hypolipidemic drugs would cause various side effects, and the demand of effective and nontoxic drugs for hyperlipidemia patients is eager. Polysaccharide has attracted worldwide concerns due to its characteristics of good biocompatibility and less side effects. Polysaccharide is a kind of biological macromolecule which is widely found in plant cell walls, animal cell membranes and microorganism cell walls. A number of studies have shown that polysaccharides from natural materials have broad biological activities, such as anti-tumor, immunomodulatory, antioxidant, hypoglycemic, and hypolipidemic effects, with broad application prospect. This paper has reviewed and summarized the polysaccharides with hypolipidemic effect and their mechanisms which have been reported at home and abroad, hoping to provide certain reference for their development and application in lowering blood lipids.

[Relationship between hepatitis B virus X protein and hepatic cellular cancer].

Hepatic cellular cancer (HCC) is one of the most common cancers in the world, which is a serious threat to human health and life quality. More than 700,000 people die of HCC each year on average, and its incidence increases in many countries. Chronic hepatitis B virus (HBV) infection has been identified as a dominant risk factor for HCC. The pathogenesis of HBV-induced hepatocarcinogenesis is, however, incompletely understood. Evidence currently available supports a key role of the HBV X protein (HBx) in the cancer transformation and malignant tumor metastasis. HBx is a multifunctional regulator that may cooperate with the host factors to exert its effects on transcription, signal transduction, cell cycle progression, apoptosis, protein degradation, expression of oncogene and anti-oncogene. This review presents the current knowledge in the molecular pathogenesis of HBx in the induction of HCC.

[Expression, purification, and characterization of fusion protein TAT-cytoglobin].

he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.

[Adeno-associated vector mediated intracellular biological activity of human Kallistatin].

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.

Cytotoxic activity of the alkaloids from Broussonetia papyrifera fruits.

CONTEXT: Broussonetia papyrifera (L.) Vent. (Moraceae), a traditional Chinese medicinal herb, has been extensively applied for many years to treat various diseases. Recently, a number of compounds with biological and pharmacological activities have been extracted from the plant and used as chemotherapeutic candidates to treat a range of diseases such as cancer. OBJECTIVE: The current study was designed to isolate the alkaloid compounds from ethyl acetate extraction of Broussonetia papyrifera fruits, and to evaluate the cytotoxic activity of total alkaloids as well as individual isoquinoline alkaloids from B. papyrifera fruits. METHODS: Alkaloid compounds were isolated from the ethyl acetate extraction by silica gel column chromatography methods using CHCl3/MeOH as eluents. The compounds' structures were determined by detailed analysis of NMR, MS spectral data, and chemical methodology. Cytotoxic activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods against human A375, Hela, BEL-7402 cancer cells, and non-cancer cells. RESULTS: Two isoquinonline alkaloids were isolated and characterized as N-norchelerythrine and dihydrosanguinarine. The total alkaloids and seven individual alkaloids had higher activities on BEL-7402 and Hela cell lines with low IC50 values 6.61-47.41 and 5.97-40.17 µg/mL (<50 µg/mL). Nitidine, broussonpapyrine, and chelerythrine had strong toxic on non-cancer cells with IC50 value 18.01, 19.91, and 22.31 µg/mL, respectively. DISCUSSION: N-Norchelerythrine and dihydrosanguinarine were isolated from this plant for the first time. Our data implicated that seven isoquinoline alkaloids had cytotoxity with structure-activity relationships, which provided fundamental information for further modification of their anticancer effect.

Luciferase assay to screen tumour-specific promoters in lung cancer.

OBJECTIVE: Specific promoters could improve efficiency and ensure the safety of gene therapy. The aim of our study was to screen examples for lung cancer. METHODS: The firefly luciferase gene was used as a reporter, and promoters based on serum markers of lung cancer were cloned. The activity and specificity of seven promoters, comprising CEACAM5 (carcinoembryonic antigen, CEA), GRP (Gastrin-Releasing Peptide), KRT19 (cytokeratin 19, KRT), SFTPB (surfactant protein B, SP-B), SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA), SELP (Selectin P, Granule Membrane Protein 140 kDa, Antigen CD62, GMP) and DKK1 (Dickkopf-1) promoters were compared in lung cancer cells to obtain cancer-specific examples with strong activity. RESULTS: The CEACAM5, DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB promoters were cloned. Furthermore, we successfully constructed recombinant vector pGL-CEACAM5 (DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB) contained the target gene. After cells were transfected with recombinant plasmids, we found that the order of promoter activity from high to low was SERPINB3, DKK1, SFTPB, KRT19, CEACAM5, SELP and GRP and the order for promoters regarding specificity and high potential were SERPINB3, DKK1, SELP, SFTPB, CEACAM5, KRT19 and GRP. CONCLUSION: The approach adopted is feasible to screen for new tumour specific promoters with biomarkers. In addition, the screened lung-specific promoters might have potential for use in lung cancer targeted gene therapy research.

[Interaction of E3 ligase HUWE1 and eukaryotic translation initiation factor eIF4E].

To explore the regulation of eIF4E, we screened the protein interacting with eIF4E from human cDNA library by using yeast two-hybrid system. Several clones interacting with eIF4E were identified. One of them was homologous with HUWE1 (HECT, UBA and WWE domain containing 1, also named as ARF-BP1, HECTH9 or HUWE1). Cell co-immunoprecipitation showed that eIF4E could bind to HUWE1 in mammalian cells. We also found that HUWE1 bearing the HECT domain is necessary for its association with eIF4E.

[Strategy for keeping efficient expression and pharmacodynamics in reducing rAAV gene medicine immune response based on the decrease of vector dosage].

How to reduce immune response is an unprecedented challenge for rAAV gene medicine. Recent studies suggested that lowering dosage of the vector used could reduce immune response caused by rAAV gene medicine. Nevertheless, it would also decrease the transgene expression, leading to failure of gene treatment. It is therefore important to take appropriate steps to maintain high gene expression level and pharmacodynamic, while the dosage of rAAV used is reduced. Here, steps to enhancing gene therapy, such as optimization of the administration, reconstruction of the viral vector and selection of the promoter, are discussed in order to achieve maximum outcome.

[Ubiquitination of recombinant adeno-associated viral vector and its application].

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

[Research progresses of plant polysaccharides on cell adhesion].

Cell adhesion mediated by cell adhesion molecules (CAMs) constitutes essential life phenomenon. In inflammation, immunity, infection, thrombosis, tumor metastasis and wound healing, cell adhesion comes into being the basic physiological and pathological process. Intervening with cell adhesion has been the important therapeutic and prophylactic strategies for diseases. Accumulated evidence has indicated that plant polysaccharides especially those exacted from Chinese traditional and herbal drugs displayed various pharmacological effects such as anti-inflammation, anti-cancer, anti-infection, immunomodulation, cardiovascular protective effects and so on. In this paper, the research progress of plant polysaccharides on cell adhesion is reviewed.

[Combinatorial RNAi and its application in cancer gene therapy].

RNA interference (RNAi) has been proved as a novel approach for gene therapy. However, RNAi mono-therapy only aims at single gene, it therefore may ultimately fail to cure cancers caused by polygene variation. To overcome the deficiency of RNAi mono-therapy, "combinatorial RNA interference" (coRNAi) was put forward as a new strategy. By co-expressing the inducers of RNAi triggering single or multiple targets directly and other RNA- or protein-based silencers, coRNAi keeps target genes silent, prevents carcinogenic progression and induces apoptosis of tumor cells. This paper mainly reviews the major strategies of coRNAi and their applications in cancer gene therapy.

[Engineering and screening of artificial riboswitch as a novel gene control element].

Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.

[Role of cytoglobin in protecting hepatic stellate cells against oxidation induced damage].

The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.

[Recombinant adeno-associated virus vector related impurities].

Recombinant adeno-associated virus (rAAV)-based vectors that can stably express therapeutic genes in vivo without detectable side-effect have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is how to establish clinically compatible purification methods in separating rAAV from potentially pathogenic impurities, especially rAAV vector-related impurities, a class of impurities corresponding to AAV particles that closely resemble bona fide vectors and are difficult to remove. In this review we summarize the assembly process of rAAV vector-related impurities and their characteristics differed with rAAV vectors, and evaluate several current technologies to prevent their formation or separate them from rAAV stocks.

[Capsid assembly and DNA encapsidation of adeno-associated virus].

Recombinant adeno-associated viral vectors (rAAV) have been widely used as gene therapy vectors in clinical trials. Here, we reviewed the genomic structures and replication mechanisms of wt-AAV. Then, the assembly of capsid and the encapsidation of genomic DNA, two major events during AAV pakaging, was discussed in detail. Although the overall pattern of virus assembly and encapsidation is known, the molecular mechanisms and the structure-function relationship involved in these processes are not well understood. Further elucidatation of these processes may improve the production technology of rAAV and develop gene drug based on rAAV.

Protection of the liver against CCl4-induced injury by intramuscular electrotransfer of a kallistatin-encoding plasmid.

AIM: To investigate the effect of transgenic expression of kallistatin (Kal) on carbon tetrachloride (CCl(4))-induced liver injury by intramuscular (im) electrotransfer of a Kal-encoding plasmid formulated with poly-L-glutamate (PLG). METHODS: The pKal plasmid encoding Kal gene was formulated with PLG and electrotransferred into mice skeletal muscle before the administration of CCl4. The expression level of Kal was measured. The serum biomarker levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malonyldialdehyde (MDA), and tumor necrosis factor (TNF)-α were monitored. The extent of CCl4-induced liver injury was analyzed histopathologically. RESULTS: The transgene of Kal was sufficiently expressed after an im injection of plasmid formulated with PLG followed by electroporation. In the Kal gene-transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST, MDA and TNF-α levels compared to those in control mice (P<0.01 to 0.05 in a dose-dependent manner). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in mice after CCl4 administration. In contrast, the damage was markedly attenuated in the Kal gene-transferred mice. The expression of hepatic fibrogenesis marker transforming growth factor-ß1 was also reduced in the pKal transferred mice. CONCLUSION: Intramuscular electrotransfer of plasmid pKal which was formulated with PLG significantly alleviated the CCl4-induced oxidative stress and inflammatory response, and reduced the liver damage in a mouse model.

[Selection and anti-cancer effects of siRNAs targeting HMGA2 gene].

High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.

The asymmetric division and tumorigenesis of stem cells.

Stem cells use asymmetric and symmetric cell division to generate progeny. Symmetric cell division is defined as the generation of daughter cells that are destined to acquire the same fate. Stem cells divide asymmetrically to generate one daughter with a stem-cell fate and one daughter with different fate. Disruption of the machinery that regulates asymmetric division may be a reason for the generation of cancer. The asymmetric mechanism is maintained by cell polarity factors, cell fate determinants, and the spindle apparatus. The mutation or dysregulation of these factors may change stem cells from asymmetric to symmetric cell division, then leading to tumorigenesis. Therefore, further study is needed on the mechanisms of stem cell control between asymmetric and symmetric cell division, as well as the relationships among stem cells, cancer stem cells, and tumor cells. It may bring us a new approach for the resistance, recurrence, and metastasis of tumors.

[Cellular immunotoxicity of rAAV gene medicine and possible solutions].

Gene medicine based on recombinant adeno-associated virus (rAAV) vector has rapidly become the prior-choose reagent for gene therapy, since it had been shown that the rAAV was able to stably express many genes in vivo without detectable side-effect. However, recent findings of CTL immune responses to AAV capsid in a clinical trial highlighted a new issue regarding safety that previously was not identified in animal studies. Obviously it is so important to understand the interaction of rAAV with the immune system in details for the safety and success of rAAV gene medicine. In this review we evaluate several current hypotheses aiming to explain the cellular immunotoxicity, also analysis the current findings including the presentation kinetics of the capsid antigen and the activation of CTL. Focusing on the key steps of the immune response several solutions are proposed, including immunosuppression, optimization of vector and improvement of purity, in order to insure clinical safety and efficacy of rAAV.

[Oral recombinant adeno-associated virus gene medicine].

The efficacy of recombinant adeno-associated virus (rAAV) vector-mediated gene delivery to the gastrointestinal tract has been paid a considerable attention over the last 10 years, since our first report on the oral gene pill strategy in Nature Medicine, even though there are still several potential obstacles for this route to overcome in order to obtain efficient gene delivery. The preclinical results of oral rAAV gene medicine are summarized in this review, and special attention is paid on its pharmacokinetic and pharmacodynamic aspects with an emphasis on drug delivery, absorption, distribution and transduction. The rAAV based vectors have been shown promising results in human clinical trials with fewer safety concerns over other gene medicines. However, the underlying mechanisms and biopharmaceutical features of oral rAAV gene medicine remain to be explored extensively and intensively to develop this novel technology as a treatment for a wider range of diseases.