Construction and characterization of a novel fusion protein MG7-scFv/SEB against gastric cancer.

Antibody-targeted superantigen has been developed into a new strategy to treat many malignant tumors. In this study, for specific targeting to gastric cancer cell, superantigen SEB (Staphylococcal Enterotoxin B) was genetically fused to the single-chain variable fragment of gastric carcinoma-associated antibody MG7(MG7-scFv) that recognizes the MG7 antigen frequently expressed in gastric cancer cell. The recombinant MG7-scFv/SEB fusion proteins are expressed in E. coli as inclusion bodies, and the purified MG7-scFv/SEB retains high binding affinity with gastric cancer cell SGC-7901 (positive MG7 antigen expression). When incubated with effector cell-peripheral blood mononuclear cells (PBMCs), MG7-scFv/SEB could effectively inhibit the proliferation and induce apoptosis of SGC-7901. After being treated with MG7-scFv/SEB, PBMCs remarkably increased the production of Th1 cytokines (IFN-gamma, IL-2), and slightly increased the production of Th2 cytokines (IL-4, IL-10) in vitro. It was observed that gastric-tumor-bearing rats administrated with MG7-scFv/SEB showed more inflammatory cell infiltration, more significant tumor inhibition, and longer survival time than those of rats treated with SEB or NS (Normal Saline). The data indicated that MG7-scFv/SEB fusion protein could specifically target gastric cancer cell, enhance the activity of T cells and induce tumor cell apoptosis to exert the antitumor effect on gastric cancer.

[Effects of magnetic gemcitabine stealth nano-liposomes on the characteristics of breast cancer cell line MCF-7].

The magnetic responsibility and antitumor effect of magnetic gemcitabine stealth nano-liposomes (MGSL) on breast cancer cell line MCF-7 in vitro and in vivo was evaluated. The magnetic response and targeting effect of MGSL in vivo were investigated. Morphological feature and ultrastructure changes of apoptosis of MCF-7 cells were observed. The effect of MGSL on proliferation inhibitory rate of MCF-7 cells was measured with MTT method. The FCM analysis was carried out to examine the cell cycle distribution and cell apoptotic rate. The antitumor effect on human breast cancer xenografts in nude mice was also studied. MGSL was able to converge at the targeting tissue under tridimensional magnetic field and the gemcitabine concentration around it increased, while the amount of gemcitabine in other organs decreased, such as in kidneys and heart. MCF-7 cell line was sensitive to MGSL and the cytotoxity was correlated with the loaded drug dose. The effect of MGSL on apoptosis of MCF-7 was obvious and the rate of apoptosis was 51.62%. The growth speed of tumor in the group of MGSL (+) significantly slowed down than that of other groups. MGSL prepared by reverse-phase evaporation method met with the demand of targeted delivery system, and it might be an effective antitumor agent.

Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper-binding site of [3-His] or [2-His-1-Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non-heme proteins.

Prognostic and clinical value of Sirt1 expression in gastric cancer: A systematic meta-analysis.

Many studies have reported that the expression of silent information regulator 1 (Sirt1) is associated with the clinical features and prognosis of patients with gastric cancer, but the exact function remains controversial. We conducted this study to illustrate the clinical and prognostic value of Sirt1 in gastric cancer. The related publications before December 2015 were searched in the databases including Pubmed, Cochrane Library, Embase and China National Knowledge Infrastructure (CNKI). The studies were included and excluded according to the inclusion criteria and exclusion criteria. The 3- and 5-year overall survival (OS) and clinical features such as age, T stage, N stage and differentiation were analyzed by software RevMan 5.3. A total of 1650 patients in 7 studies were included according to the inclusion criteria and exclusion criteria. The high expression of Sirt1 was found in 58.4% cases by immunohistochemistry. High expression of Sirt1 was closely linked with the 3-year OS (OR=0.25, 95% CI: 0.16-0.39, P<0.00001, fixed), patient's age (≥60 years old vs. <60 years old; OR=1.43, 95% CI: 1.06-1.93, P=0.02, fixed), T stage (T3+T4 vs. T1+T2; OR=1.45, 95% CI: 1.08-1.94, P=0.01, fixed), N stage (N1+N2+N3 vs. N0; OR=3.47, 95% CI: 2.39-5.05, P<0.00001, fixed) and tumor differentiation (G1+G2 vs. G3; OR=0.50, 95% CI: 0.35-0.69, P<0.0001, fixed). Nevertheless, it seemed that high expression of Sirt1 was not associated with 5-year OS (OR=0.44, 95% CI: 0.15-1.28, P=0.13, random). It was suggested that the high expression of Sirt1 implies a poor prognosis of gastric cancer patients in a relatively short period (3 years), but not in a long time (≥5 years). The expression of Sirt1 is also linked with patients' age, T stage, N stage and tumor differentiation.

Computational insight into nitration of human myoglobin.

Protein nitration is an important post-translational modification regulating protein structure and function, especially for heme proteins. Myoglobin (Mb) is an ideal protein model for investigating the structure and function relationship of heme proteins. With limited structural information available for nitrated heme proteins from experiments, we herein performed a molecular dynamics study of human Mb with successive nitration of Tyr103, Tyr146, Trp7 and Trp14. We made a detailed comparison of protein motions, intramolecular contacts and internal cavities of nitrated Mbs with that of native Mb. It showed that although nitration of both Tyr103 and Tyr146 slightly alters the local conformation of heme active site, further nitration of both Trp7 and Trp14 shifts helix A apart from the rest of protein, which results in altered internal cavities and forms a water channel, representing an initial stage of Mb unfolding. The computational study provides an insight into the nitration of heme proteins at an atomic level, which is valuable for understanding the structure and function relationship of heme proteins in non-native states by nitration.