SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with colorectal cancer progression.

Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of ß-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

Galectin-14 promotes hepatocellular carcinoma tumor growth via enhancing heparan sulfate proteoglycan modification.

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy and lacks effective treatment. Bulk-sequencing of different gene transcripts by comparing HCC tissues and adjacent normal tissues provides some clues for investigating the mechanisms or identifying potential targets for tumor progression. However, genes that are exclusively expressed in a subpopulation of HCC may not be enriched or detected through such a screening. In the current study, we performed a single cell-clone-based screening and identified galectin-14 as an essential molecule in the regulation of tumor growth. The aberrant expression of galectin-14 was significantly associated with a poor overall survival of liver cancer patients with database analysis. Knocking down galectin-14 inhibited the proliferation of tumor growth, whereas overexpressing galectin-14 promoted tumor growth in vivo. Non-targeted metabolomics analysis indicated that knocking down galectin-14 decreased glycometabolism; specifically that glycoside synthesis was significantly changed. Further study found that galectin-14 promoted the expression of cell surface heparan sulfate proteoglycans (HSPGs) that functioned as co-receptors, thereby increasing the responsiveness of HCC cells to growth factors, such as epidermal growth factor and transforming growth factor-alpha. In conclusion, the current study identifies a novel HCC-specific molecule galectin-14, which increases the expression of cell surface HSPGs and the uptake of growth factors to promote HCC cell proliferation.

Improving the cytotoxicity of immunotoxins by reducing the affinity of the antibody in acidic pH.

BACKGROUND: Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated. METHODS: In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions. RESULTS: Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin. CONCLUSIONS: These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.

Improving antibody affinity through in vitro mutagenesis in complementarity determining regions.

High-affinity antibodies are widely used in diagnostics and for the treatment of human diseases. However, most antibodies are isolated from semi-synthetic libraries by phage display and do not possess in vivo affinity maturation, which is triggered by antigen immunization. It is therefore necessary to engineer the affinity of these antibodies by way of in vitro assaying. In this study, we optimized the affinity of two human monoclonal antibodies which were isolated by phage display in a previous related study. For the 42A1 antibody, which targets the liver cancer antigen glypican-3, the variant T57H in the second complementarity-determining region of the heavy chain (CDR-H2) exhibited a 2.6-fold improvement in affinity, as well as enhanced cell-binding activity. For the I4A3 antibody to severe acute respiratory syndrome coronavirus 2, beneficial single mutations in CDR-H2 and CDR-H3 were randomly combined to select the best synergistic mutations. Among these, the mutation S53P-S98T improved binding affinity (about 3.7 fold) and the neutralizing activity (about 12 fold) compared to the parent antibody. Taken together, single mutations of key residues in antibody CDRs were enough to increase binding affinity with improved antibody functions. The mutagenic combination of key residues in different CDRs creates additive enhancements. Therefore, this study provides a safe and effective in vitro strategy for optimizing antibody affinity.

Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma.

BACKGROUND: Glypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC. METHODS: In this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines. RESULTS: Both humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC. CONCLUSIONS: We provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.

Proteomics and molecular network analyses reveal that the interaction between the TAT-DCF1 peptide and TAF6 induces an antitumor effect in glioma cells.

Glioblastoma is the most lethal brain cancer in adults. Despite advances in surgical techniques, radiotherapy, and chemotherapy, their therapeutic effect is far from significant, since the detailed underlying pathological mechanism of this cancer is unclear. The establishment of molecular interaction networks has laid the foundation for the exploration of these mechanisms with a view to improving therapy for glioblastoma. In the present study, to further explore the cellular role of DCF1 (dendritic cell-derived factor 1), the proteins bound to TAT-DCF1 (transactivator of transcription-dendritic cell-derived factor 1) were identified, and biosystem analysis was employed. Functional enrichment analyses indicate that TAT-DCF1 induced important biological changes in U251 cells. Furthermore, the established molecular interaction networks indicated that TAT-DCF1 directly interacted with TAF6 in glioma cells and with UBC in HEK293T (human embryonic kidney 293T) cells. In addition, further biological experiments demonstrate that TAT-DCF1 induced the activation of the RPS27A/TOP2A/HMGB2/BCL-2 signaling pathway via interaction with TAF6 in U251 cells. Taken together, these findings suggest that the TAT-DCF1 peptide possesses great potential for the development of glioblastoma therapy through the interaction with TAF6-related pathways and provides further theoretic evidence for the mechanisms underlying the antitumor effects of TAT-DCF1.

The antitumor effect of TAT-DCF1 peptide in glioma cells.

BACKGROUND: Glioblastoma is one of the most malignant brain cancer, thus, establishing an effective therapy is paramount. Our previous results indicate that dendritic cell-derived factor (DCF1) is an attractive candidate for therapy against Glioblastoma, since its overexpression in Glioblastoma U251 cells leads to apoptosis. However, the delivery approach limits its clinical application, in this paper, we expressed TAT-DCF1 fusion protein in E.coli in order to surmount its current delivery problems. METHODS: The coding sequences of the different domains of DCF1 (full length, cytoplasmic, extracellular, 19-amino acid), together with the N-terminal transactivator of transcription (TAT) sequence, were amplified and subcloned into the bacterial expression vector pET30a(+) in order to produce (His)6-tagged fusion proteins. Coomassie blue-stained SDS-PAGE and Western blotting identification showed that purity of the fusion proteins. RESULTS: Immunofluorescence and flow cytometry show that U251 cells were efficiently transduced with the fusion proteins. Cell viability, proliferation, and migration assays suggest that the complete TAT-DCF1 fusion protein significantly decreased U251 proliferation and migration. Flow cytometry further reveals that TAT-DCF1 triggered cellular apoptosis. CONCLUSIONS: In conclusion, these findings suggest that the TAT-DCF1 fusion protein was efficiently transduced into Glioblastoma U251 cells and induced the antitumor effect and support further investigation into specific targeting and side effects of TAT-DCF1 during drug delivery.

[Association between development of hypertension and nutrition in school-age children in Fengdu County of Chongqing, China].

OBJECTIVE: To investigate the association between the development of hypertension and nutrition in school-age children in Fengdu County of Chongqing, China. METHODS: A total of 8 033 children from 2 public primary schools in Fengdu County of Chongqing, whose registered residence was in the subdistricts where the two schools were located, were selected as study subjects using cluster random sampling. Body height, body weight, and blood pressure were measured, and the semi-quantitative food frequency questionnaire was used for dietary survey. The association between body mass index (BMI), dietary nutrients, and the development of hypertension in children was analyzed. RESULTS: A total of 7 538 children were enrolled for analysis. The detection rates of obesity, overweight, and hypertension were 9.11%, 12.27%, and 11.83% respectively. In children with obesity and overweight, the detection rate of hypertension was 33.62% and 17.84% respectively, 4.02 and 2.13 times that in normal children. The multivariate logistic stepwise regression analysis revealed that increased intake of calcium and sodium increased the risk of hypertension (OR=1.003 and 1.002 respectively), while the increased iron intake and calcium intake per unit body weight reduced the risk of hypertension (OR=0.979 and 0.926 respectively). CONCLUSIONS: The prevalence of hypertension and obesity in school-age children in Fengdu County of Chongqing is high. BMI and dietary nutrients are closely associated with the development of hypertension in children. Active control of body weight, adjustment of dietary structure, and limitation of sodium intake should be adopted to reduce the development of hypertension in school-age children.

Cellulase hydrolysis of rice straw and inactivation of endoglucanase in urea solution.

In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k(+0) > k'(+0), indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected.

Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9.

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 degrees C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 degrees C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 degrees C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis-Menten kinetics with constant K(m) of 5.0 mg/mL at pH 4.0 and 50 degrees C. The enzyme activity was stimulated by Fe(2+) and Mn(2+) but inhibited by Cd(2+), Pb(2+), and Cu(2+). The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

[Inhibition of postharvest decay and induction of defensive enzymes by pure oxygen in Chinese bayberry fruit].

Chinese bayberry fruits were stored in air (control) or pure oxygen atmosphere for up to 12 days at 5 degrees C to investigate the effects of high oxygen on decay control and its relation to the induction of defensive enzyme activities. The results showed that exposure of Chinese bayberry to pure oxygen significantly prevented fruit decay. At the end of the storage period, the decay index of fruits exposed to pure oxygen was only 17% while that of control fruits reached 54% (Fig.1). Pure oxygen caused a significant increase in chitinase and beta-1,3-glucanase activities which reached a peak on the 6th day of storage (Fig.2). Phenylalanine ammonium-lyase (Fig.3A) and peroxidase (Fig.4) activities as well as total phenolic content (Fig.3B) increased more quickly and stayed at significantly higher levels in fruits exposed to pure oxygen during storage than the control fruits. These results suggest that the inhibition of postharvest fruit decay by high oxygen was related to the induction of defensive enzyme activities. The induced disease resistance may be involved in the mechanisms by which high oxygen treatment inhibited fruit decay in Chinese bayberry.