WM130 preferentially inhibits hepatic cancer stem-like cells by suppressing AKT/GSK3β/β-catenin signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE The eradication of cancer stem cells(CSCs)is signifcant for cancer therapy and prevention.METHODS In this study,we evaluated WM130,a novel derivative of matrine,for its effect on CSCs using human hepatocellular carcinoma(HCC)cell lines,their sphere cells,and sorted EpCAM+cells. RESULTS We revealed that WM130 could not only inhibit proliferation and colony formation of HCC cells, but also suppress the expression of some stemness-related genes and up-regulate some mature hepatocyte marker genes, indicating a promotion of differentiation from CSCs to hepatocytes. WM130 also suppressed the proliferation of doxorubicin-resistant hepatoma cells, and markedly reduced the cells with CSC biomarker EpCAM.Moreover,WM130 suppressed HCC spheres,not only primary spheres but also subsequent spheres,indicating an inhibitory effect on self-renewal capability of CSCs.Interestingly,WM130 exhibiteda remarkable inhibitory preference on HCC spheres and EpCAM+cells rather than their parental HCC cells and EpCAM- cells respectively. In vivo, WM130 inhibited HCC xenograft growth, decreased the number of sphere-forming cells, and remarkably decreased the levels of EpCAM mRNA and protein in tumor xenografts. Better inhibitory effect was achieved by WM130 in combination with doxorubicin.Further mechanism study revealed that WM130 inhibited AKT/GSK3β/β-catenin signaling pathway. CONCLUSION Collectively, our results suggest that WM130 remark-ably inhibits hepatic CSCs, and this effect may via the down-regulation of the AKT/GSK3β/β-catenin pathway.These findings provide a strong rationale for the use of WM130 as a novel drug candidate in HCC therapy.

Mass spectrometry based proteomics profiling of human monocytes

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases.

Preparation and characterization of a monoclonal antibody with high affinity for soluble Abeta oligomers.

Amyloid beta-protein (Abeta) has been causally implicated in the neurodegenerative processes that accompany Alzheimer's disease. Soluble oligomers of the Abeta(1-42) fragment are thought to be significantly more neurotoxic than higher molecular weight aggregates. We report the isolation and characterization of a mouse monoclonal antibody (MAb) directed against soluble Abeta(1-42) oligomers. Synthetic Abeta(1-42) oligomers were assembled in vitro; these were used to immunize mice, and hybridomas were isolated following myeloma fusion of splenocytes from immunized animals. Screening for reactivity against Abeta(1-42) resulted in the identification of MAb A8 with high affinity for soluble oligomers. The isotype of A8 was found to be IgG(2b). Experiments using sub-peptides of Abeta(1-42) revealed that the epitope identified by A8 lies within the 1-6 region of Abeta. The antibody displays high affinity for soluble Abeta(1-42) oligomers in the molecular weight range of 16.5-25 kDa, and detected target antigen in brain sections from senescence-accelerated SAMP 8 mice. The sensitivity and optimal titers for the detection of soluble Abeta(1-42) oligomers were determined to be 0.625 microg/mL in indirect ELISA, and 1:10(6), 1:4000, and 1:150 for ELISA, Western blotting, and immunohistochemistry, respectively. The A8 antibody specific for soluble Abeta(1-42) oligomers will provide a valuable tool for Alzheimer's disease research.

Enhanced interferon-gamma secretion and antitumor activity of T-lymphocytes activated by dendritic cells loaded with glycoengineered myeloma antigens / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Immunotherapy is emerging as a promising cure for cancer. However, a severe problem in this area is the immune tolerance to tumor cells and tumor-associated antigens, as evidenced by the ability of cancer to escape immune surveillance. To overcome this problem this work examined the potential of improving the antigenicity of myeloma by metabolic engineering of its cell surface carbohydrate antigens (i.e., glycoengineering) and presentation of the modified tumor antigens by dendritic cells (DCs) to generate cytotoxic T-lymphocytes (CTLs).</p><p><b>METHODS</b>CD138+ myeloma cells were isolated from 11 multipe myeloma (MM) patients by the immunomagnetic bead method. The MM cells were treated with N-propionyl-D-mannosamine (ManNPr), a synthetic analog of N-acetyl-D-mannosamine (ManNAc), the natural biosynthetic precursor of N-acetyl sialic acid (NeuNAc), to express unnatural N-propionylated sialoglycans. The glycoengineered cells were then induced to apoptosis, and the apoptotic products were added to cultured functional DCs that could present the unnatural carbohydrate antigens to autologous T-lymphocytes.</p><p><b>RESULTS</b>It was found that the resultant DCs could activate CD4+ and CD8+ T-lymphocytes, resulting in increased expression of T cell surface markers, including CD8CD28 and CD4CD29. Moreover, upon stimulation by glycoengineered MM cells, these DC-activated T-lymphocytes could release significantly higher levels of IFN-gamma (P < 0.05). Lactate dehydrogenase (LDH) assays further showed that the stimulated T-lymphocytes were cytotoxic to glycoengineered MM cells.</p><p><b>CONCLUSIONS</b>This work demonstrated that glycoengineered myeloma cells were highly antigenic and the CTLs induced by the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma. This may provide a new strategy for overcoming the problem of immune tolerance for the development of effective immunotherapies for MM.</p>

Prokaryotic Expression,Purification and DNA Binding Activity of DEK Protein's Carboxy-terminal DNA-binding Region / 中国生物工程杂志

DEK protein's carboxy-terminal DNA-binding region(CBD)is a newly found DNA-binding domain of DEK,which contains several phosphorylation sites and has a close correlation with DEK protein's function in vivo and in vitro.Using prokaryotic expression system,the peptide of DEK protein's carboxy-terminal DNA-binding region(CDB)was expressed and purified.In detail,the CDB DNA fragment was constructed into pET-30a(+)vector,and E.coli BL21(DE3)competent cells were used as host cells.The fusion protein His-CBD was expressed by induction of IPTG and purified by Ni-NTA agarose.The result of SDS-PAGE showed that the molecular weight of the purified protein was about 10.7kDa.Electrophoretic mobility shift assay(EMSA)indicated that DEK-CDB prefered to bind to supercoiled form of DNA in vitro,it had similar character to the binding of whole length DEK protein with DNA.This suggested that the carboxy-terminal DNA-binding region of DEK protein might function on the binding of DEK protein to DNA partly.

Application of IL-13R?2-directed Toxin Fusion Protein in Tumor Therapy / 中国生物工程杂志

The research of interleukin-13 receptor ?2(IL-13R?2) chain is a rising pop these years.Previous studies have shown that many human tumors overexpress IL-13R?2 chain,while normal cells do not express this receptor or express very low level.This difference of express level is significant to diagnose and cure tumors by the IL-13R?2-directed toxin fusion protein.During the past decade,the structure and the function of IL-13R?2 together with the relationship between this receptor and tumors has been further developed.Therefore new therapies and theories can be proposed as the clinical tumor treatments.In this case,the expression in various tumor cell lines was not only focused on but also on the IL-13 and toxin fusion protein killing effect in both the cell level and the in vivo level.Besides,an overview of the mechanism in the treatment of IL-13R?2-directed toxin fusion protein together with the improved methods for fusion protein purification and other relative tumor therapy was given.In conclusion,the current status and progress of IL-13R?2 as well as IL-13R?2-directed toxin fusion protein in tumor therapy were represented.