Therapy model for advanced intracerebral B16 mouse melanoma using radiation therapy combined with immunotherapy

A reproducible therapy model for advanced intracerebral B16 melanoma is reported. Implanted tumors (D0), suppressed by a single 15 Gy radiosurgical dose of 100 kVp X-rays (D8), were further suppressed by a single ip injection of a Treg-depleting mAb given 2 days prior to the initiation (D9) of four weekly then eight bi-monthly sc injections of GMCSF-transfected, mitotically disabled B16 cells. The trends of seven independent experiments were similar to the combined result: The median (days) [SD/total N] of survival went from 15[1.09/62] (no treatment control) to 35.8[8.8/58] (radiation therapy only) to 52.5[13.5/57] (radiation therapy plus immunotherapy). Within 2 weeks after immunization, tumors in mice receiving radiation therapy plus immunotherapy were significantly smaller than tumors in mice treated only with radiosurgery. Splenocytes and lymph node cells from immunized mice showed increased interferon γ production when cultured with syngeneic tumor cells. We suggest that our model will be useful for the development and testing of novel combination therapies for brain tumors.     

Inhibition of neuronal nitric oxide synthase activity promotes migration of human‐induced pluripotent stem cell‐derived neural stem cells toward cancer cells

The breakthrough in derivation of human‐induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC‐derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC‐NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non‐migratory hiPSC‐NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down‐regulated in migratory hiPSC‐NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC‐NSCs toward cancer cells, and that inhibition of its activity or down‐regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell‐mediated cancer therapy.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Purification and Properties of a Proteolytic Enzyme,Cathepsin D,from Chicken Muscle

Cathepsin D was isolated from crude extract of chicken muscle by the purification procedures of acid- and heat-treatments, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 3700 fold and homogeneous in disc-electrophoretic analysis. The molecular weight was found to be about 36,000 and the isoelectric point to be pH 7.3. The best substrate for this enzyme was 6 m urea-denatured casein, and its activity was maximal at pH 3.5 and 40°C. This enzyme was most stable between pH 4 and 5, and its stability was affected by cupric ion. The enzyme activity was markedly inhibited by sodium laurylsulfate and oxidizing agents such as potassium permanganate, N-bromosuccinimide and iodine, and was slightly activated by hydrogen peroxide. The purified cathepsin D was found to be fairly similar to the acid protease from lotus seed, previously reported by the authors.     

The Role of TLR2, TLR4 and CD14 Genetic Polymorphisms in Gastric Carcinogenesis: A Case-Control Study and Meta-Analysis

Background

In addition to Helicobacter pylori infection, host genetic factors contribute to gastric cancer (GC). Recognition of H. pylori is known to involve Toll-like receptors (TLR), which subsequently leads to activation of NF-κB. Thus, the overall aim of this study was to estimate for the first time the pooled effect size of polymorphisms in TLR2, TLR4 and CD14 on GC development through a meta-analysis.

Methods

A case-control study comprising 284 ethnic Chinese individuals (70 non-cardia GC cases and 214 functional dyspepsia controls) was conducted for the genotyping of TLR2 -196 to -174del, CD14 -260 C/T and TLR4 rs11536889 using PCR, RT-PCR and mass spectrometry. Case-control studies of TLR2, TLR4 and CD14 polymorphisms and GC were searched up to June 2012. Pooled odds ratios and 95% confidence intervals were obtained by means of the random effects model.

Results

In our ethnic Chinese case-control study, the TLR4 rs11536889 C allele increased the risk of GC (OR: 1.89, 95%CI: 1.23–2.92) while the CD14 -260 T allele was protective (OR: 0.62, 95%CI: 0.42–0.91). TLR2 -196 to -174 increased the risk of GC only in H. pylori-infected individuals (OR: 3.10, 95%CI: 1.27–7.60). In the meta-analysis, TLR4 Asp299Gly showed borderline results in the general analysis (pooled OR: 1.58, 95%CI: 0.98–2.60), nevertheless, stratified analysis by ethnicity showed that the mutant allele was a definitive risk factor for GC in Western populations (pooled OR: 1.87, 95%CI: 1.31–2.65). There was a potential association between the TLR2 -196 to -174 deletion allele and GC in Japanese (pooled OR: 1.18, 95%CI: 0.96–1.45). TLR4 Thr399Ile did not provide significant results.

Conclusions

TLR4 rs11536889 and CD14 -260 C/T are associated with non-cardia GC in Chinese. Based on our meta-analysis, the TLR signalling pathway is involved in gastric carcinogenesis, TLR4 Asp299Gly and TLR2 -196 to -174del showing associations with GC in an ethnic-specific manner.     

Spectroscopic (UV/VIS,Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG₁₀₀), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG₁₀₀ to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG₁₀₀ of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG₁₀₀ after exposure to the magnetic field.     

Development of a risk scoring system for patients with papillary thyroid cancer

As the importance of personalized therapeutics in aggressive papillary thyroid cancer (PTC) increases, accurate risk stratification is required. To develop a novel prognostic scoring system for patients with PTC (n = 455), we used mRNA expression and clinical data from The Cancer Genome Atlas. We performed variable selection using Network‐Regularized high‐dimensional Cox‐regression with gene network from pathway databases. The risk score was calculated using a linear combination of regression coefficients and mRNA expressions. The risk score and clinical variables were assessed by several survival analyses. The risk score showed high discriminatory power for the prediction of event‐free survival as well as the presence of metastasis. In multivariate analysis, the risk score and presence of metastasis were significant risk factors among the clinical variables that were examined together. In the current study, we developed a risk scoring system that will help to identify suitable therapeutic options for PTC.