Structure Elucidation of a Novel Polysaccharide Isolated from Euonymus fortunei and Establishing Its Antioxidant and Anticancer Properties.

Euonymusfortunei polysaccharides (EFPs) have not been extensively investigated yet in terms of their extraction and biological activity. The orthogonal experimental design was employed in this study to evaluate the optimum yield of EFPs. A maximum yield of 2.63 ± 0.23% was attained using material-liquid ratios of 60 mL/g, extraction temperature of 80°C, ultrasonic power of 144 W, and extraction time of 75 mins. The polysaccharide content reached 53.47 ± 0.31% when deproteinized thrice. An analysis of monosaccharide composition revealed that these polysaccharides consist of Gal, Glc, Man, Fuc, and Rha with a molar ratio of 7.14 ∶ 23.99 ∶ 6.29 ∶ 6.55 ∶ 1.00, respectively, in EFPs. Subsequently, the in vitro scavenging capacities of 2,2-diphenylpicrylhydrazyl (DPPH) and ·OH and superoxide anion radicals, along with the reducing power of EFPs, were studied. Results revealed that EFPs have higher antioxidant activity, particularly ·OH scavenging, as well as reducing power, as compared to Astragalus polysaccharides (ASPs) and Lycium barbarum polysaccharides (LBPs). The Cell Counting Kit-8 (CCK-8) method was used to evaluate the effects of different concentrations of polysaccharides on SKOV3 cell proliferation, and the results revealed their inhibition at concentrations in the range of 200-800 µg/mL. In addition, findings from flow cytometry further confirmed that EFPs blocked the cell cycle at G0/G1 and S phases and induced SKOV3 cell apoptosis. In a word, EFPs could be exploited and used further based on the experimental results from this study.

[Bioinformatics analysis and construction of eukaryotic expression vector of MAGE-D4].

Objective To analyze the physicochemical properties, structure and function of melanoma-associated antigen D4 (MAGE-D4) protein, and then construct the eukaryotic expression vector of MAGE-D4. Methods The physicochemical properties, structure and function of MAGE-D4 protein were analyzed by bioinformatics. Using MAGE-D4/pMAL-C2 prokaryotic recombinant plasmid as the template, PCR product digested by restriction enzyme was connected with pEGFP-C1 eukaryotic expression plasmid and transformed into E. coli. Ligation products were identified by antibiotic screening, enzyme digestion and sequencing. Then the recombinant plasmid was transfected into A549 lung cancer cells by liposome. Results MAGE-D4 protein was an unstable hydrophilic protein without transmembrane structure and signal peptide. Its secondary structure was mainly α-helix. MAGE-D4 contained multiple functional modification sites and was mainly located in the nucleus. SLLLVILGV might be a restricted T cell epitope of HLA-A*0201 derived from MAGE-D4. The first three proteins to potentially interact with MAGE-D4 were NSMCE4A, MLANA/MART-1 and BAGE5. DNA sequencing showed that the recombinant plasmid contained full-length coding sequence (CDS) of MAGE-D4 and it could be successfully transfected into A549 lung cancer cells. Conclusion MAGE-D4 protein is an unstable nuclear protein, which may play functions by interacting with a variety of melanoma-related proteins. The peptide derived from MAGE-D4 may have strong immunogenicity. The eukaryotic expression vector of MAGE-D4 has been successfully constructed.

Effect of IPP5, a novel inhibitor of PP1, on apoptosis and the underlying mechanisms involved.

Genes encoding apoptosis-inducing proteins are postulated to be candidate tumour suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumours. In the present study, we characterized the function of a novel human BMSC (bone marrow stromal cell)-derived protein {IPP5 [inhibitor-5 of PP1 (protein phosphatase 1)]} by large-scale random sequencing of a human BMSC cDNA library. hIPP5 (human IPP5) cDNA encodes a protein of 116 amino acid residues, which shares high homology with human PPI-1 (inhibitor-1 of PP1). The effect of IPP5 on apoptosis and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results showed that overexpression of active mutant IPP5 inhibited anchorage-dependent growth and induced apoptosis in HeLa cells, which may be attributed to the up-regulation of p21(waf/cip1) (a 21 kDa cell-cycle regulatory protein), p53 and Bcl-2-antagonist/killer, and down-regulation of Bcl-2 and Bcl-X(L). We also showed that the expression of active mutant IPP5 in HeLa cells was further enhanced on TNF (tumour necrosis factor) treatment and overexpression of active mutant IPP5 sensitized HeLa cells to TNF-induced JNK (c-Jun N-terminal kinase) and p38 activation as well as TNF-mediated apoptosis. Thus overexpression of active mutant IPP5 may increase cell susceptibility to TNF-induced apoptosis by the activation of p38 and JNK pathways. In addition, IPP5 active mutant could interact with PP1alpha as demonstrated by the co-precipitation assay.

[Preparation and application of melanoma-associated antigen D4 polyclonal antibody].

Objective To prepare the rabbit polyclonal antibody recognizing human melanoma-associated antigen D4 (MAGE-D4), and identify its immune characteristics for preliminary application. Methods MBP/MAGE-D4 fusion protein was expressed from pMAL-C2/MAGE-D4 recombinant plasmid constructed in the previous work. Then the purified MBP/MAGE-D4 protein was used to immunize the New Zealand white rabbits for generating polyclonal antibody. Subsequently, the anti-MAGE-D4 antibody was purified with protein A affinity chromatograph and identified by SDS-PAGE. The titer and specificity of the antibody were further detected by indirect ELISA and Western blotting, respectively. MAGE-D4 expression and localization of lung cancer tissues were analyzed by immunohistochemical staining. Results MAGE-D4 polyclonal antibody with high purity was obtained. Its titer was about 1:256 000. Western blot analysis demonstrated that the antibody could specifically react to the recombinant MAGE-D4 protein. Immunohistochemical staining showed that the antibody could recognize the endogenous MAGE-D4 protein in lung cancer tissues, and its positive rate was 69.6% (17/23). Conclusion The MAGE-D4 polyclonal antibody with high specificity and sensitivity has been successfully prepared.

High expression and frequently humoral immune response of melanoma-associated antigen D4 in glioma.

MAGE-D4 is a novel member of MAGE super-family. It has preliminarily been demonstrated that MAGE-D4 mRNA is not expressed in majority of normal tissues except for brain and ovary in which only trace amount of MAGE-D4 mRNA can be detected, but predominantly expressed in glioma. MAGE-D4 protein expression and its immunogenicity in glioma have not been elucidated well. This study was designed to analyze MAGE-D4 expression both at mRNA and protein level, characteristic of humoral immune response, and their relationships with glioma patients' clinicopathological parameters. Recombinant MAGE-D4 protein and antiserum were generated. Quantitative RT-PCR analysis revealed that MAGE-D4 mRNA expression was overall up-regulated in 41 glioma specimens compared with that in 14 normal brain tissues. Immunohistochemistry analysis showed that 78% (21/27) glioma tissues expressed MAGE-D4 protein, which was predominantly located in the cytoplasm of tumor cells, but absent in any neuroglia cell of normal brain tissues. ELISA analysis demonstrated that humoral response against MAGE-D4 was detected in 17% (7/41) of glioma patients' sera but not in 77 healthy donors. No apparent correlation was observed between the expression and immunogenicity of MAGE-D4 with clinicopathological parameters of glioma. In summary, these results indicate that MAGE-D4 is highly expressed in glioma and can develop specifically humoral response in glioma patients, which supports that it may be a promising biomarker for glioma diagnosis and immunotherapy.

Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target.

Melanoma-associated antigen D4 (MAGE-D4) is a novel member of MAGE family. This study aimed to examine the expression and immunogenicity of MAGE-D4 in colorectal cancer (CRC) to determine its potential as a prognosis and immunotherapeutic target. The expression of MAGE-D4 mRNA and protein was determined by RT-PCR and immunohistochemistry (IHC) in CRCs with paired adjacent non-tumor tissues, colorectal adenomas and normal colorectal tissues, respectively. Sera from 64 CRC patients were tested for MAGE-D4 antibody by ELISA. MAGE-D4 mRNA was more frequently expressed in CRCs (76.7%, 46/60) than in adjacent non-tumor tissues (15.0%, 9/60). MAGE-D4 protein was detected in all the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; P=0.050). Humoral immunity to MAGE-D4 was detected in 12 of 64 (18.8%) CRC patients' sera but not in 77 healthy donors. There was no correlation between MAGE-D4 expression, serum antibody and clinicopathological parameters. These findings suggest MAGE-D4 may serve as a potentially prognostic biomarker and an attractive target of immunotherapy in CRC.

Soluble expression of recombinant human SMP30 for detecting serum SMP30 antibody levels in hepatocellular carcinoma patients.

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti- SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

Cancer testis antigen OY-TES-1 expression and serum immunogenicity in colorectal cancer: its relationship to clinicopathological parameters.

Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because their expression is restricted in normal germ line tissues but frequently detected in variety of tumors. OY-TES-1 is identified as a member of CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer (CRC) is solely based on mRNA analysis. None of previous researches has studied OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24 colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC patients were also tested for OY-TES-1 antibody by ELISA. Our results showed that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC (73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was found in normal colon by either RT-PCR or immunohistochemistry. Furthermore, OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004) and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%, 7/73) of CRC patients' sera but not in 76 healthy donors. This finding demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce humoral immune response spontaneously in CRC patients, suggesting that it might be a promising immunotherapy target for CRC.

[Construction of eukaryotic expression vector encoding ACRBP and its expression in hepatocarcinoma cells].

AIM: To construct the eukaryotic expression vector pEGFP-N1/ACRBP and stably express ACRBP in human hepatocarcinoma cells, providing functional clues for ACRBP. METHODS: A recombinant plasmid pMAL-C2/ACRBP was used as a template to amplify ACRBP cDNA. The PCR product was ligated into an eukaryotic expression vector pEGFP-N1 to construct a recombinant plasmid pEGFP-N1/ACRBP. Then the pEGFP-N1/ACRBP was transfected by Fugene HD into ACRBP-negative HepG2 cells. The stably transfected clones were selected by G418. RT-PCR and immunohistochemistry were used to detect the expression of ACRBP in HepG2 cells. RESULTS: The eukaryotic expression vector pEGFP-N1/ACRBP was constructed and confirmed by sequencing. The stably transfected HepG2 cells expressed ACRBP. CONCLUSION: The eukaryotic expression vector pEGFP-N1/ACRBP has been successfully constructed and transfected into HepG2 cells, resulting in stable expression of ACRBP.