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Bacillus amyloliquefaciens is a probiotic for animals. A strain of B. amyloliquefaciens designated amy-1 was isolated from soil, and the exopolysaccharides (EPSs) of the strain were characterized in terms of their effect on glycemic control. The EPSs were composed of mannose, glucose, and galactose, with the major components being polymers larger than 1000 kDa as revealed by size-exclusion high-performance liquid chromatography. The EPSs reduced the elevation of blood glucose in mice on oral glucose tolerance tests. The hypoglycemic effect was still apparent when glucose was administered through intraperitoneal injection. Further investigation revealed that the EPSs stimulated glucagon-like peptide 1 (GLP-1) secretion from enteroendocrine cells in vitro and increased plasma GLP-1 level in vivo. Moreover, the EPSs promoted the glucose consumption of a liver cell line and an intestinal epithelial cell line. Therefore, the interaction between EPSs and intestinal tissues at least partially contributed to their hypoglycemic effect. The enhanced glucose uptake of cells was likely mediated by the activation of phosphatidylinositol-3-kinase and Akt and was independent of insulin receptor substrate and AMP-activated protein kinase. These findings suggest that EPSs likely involve in the hypoglycemic functions of probiotics and are potential new agents for glycemic control.

Inflammation contributes to the development and progression of cancer. Interleukin-17 (IL-17) is an inflammatory cytokine that functions in inflammation and cancer, as well as several other cellular processes. In this study, we investigated the roles and the prognostic value of IL-17 and the IL-17 receptor (IL-17R) in lung cancer. Gene expression microarray analysis followed by Kaplan-Meier survival curve showed that IL-17B was associated with poor patient survival, and IL-17B receptor (IL-17RB) was up-regulated in lung cancer tissue compared with normal tissue. Expression of IL-17RB was associated with lymph node metastasis and distant metastasis, as well as poor patient survival. IL-17RB overexpression significantly increased cancer cell invasion/migration and metastasis in vitro and in vivo. IL-17RB induced ERK phosphorylation, resulting in GSK3β inactivation and leading to β-catenin up-regulation. IL-17RB also participated in IL-17B synthesis via the ERK pathway. IL-17RB activation is required for IL-17B-mediated ERK phosphorylation. Taken together, IL-17B-IL-17RB signaling and ERK participate in a positive feedback loop that enhances invasion/migration ability in lung cancer cell lines. IL-17RB may therefore serve as an independent prognostic factor and a therapeutic target for lung cancer.

Background The β-nitrostyrene family possesses anticancer properties. In this study, β-nitrostyrene derivative CYT-Rx20 (3'-hydroxy-4'-methoxy-β-methyl-β-nitrostyrene) was synthesized and investigated its anticancer activity in oral cancer. Methods Anticancer activity of CYT-Rx20 and the underlying mechanisms were analyzed using cell viability assay, reactive oxygen species (ROS) generation assay, fluorescence-activated cell sorter analysis, annexin V staining, comet assay, glutathione (GSH)/glutathione disulfide (GSSG) ratio, immunoblotting, soft agar assay, nude mice xenograft study, and immunohistochemistry. Results CYT-Rx20-induced cell apoptosis via ROS generation and mitochondrial membrane potential reduction, associated with release of mitochondrial cytochrome C to cytosol and activation of downstream caspases and poly ADP-ribose polymerase (PARP). Furthermore, CYT-Rx20 induced mitochondrial ROS accumulation and mitochondrial dysfunction, followed by GSH downregulation. CYT-Rx20-induced cell apoptosis, ROS generation, and DNA damage were reversed by thiol antioxidants. In nude mice, CYT-Rx20 inhibited oral tumor growth accompanied by increased expression of γH2AX, GSH reductase, and cleaved-caspase-3. Conclusion CYT-Rx20 has the potential to be further developed into an antioral cancer drug clinically. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1055-1064, 2017.

Primary spontaneous pneumothorax (PSP) is a common benign disorder. However, unpredictable recurrence is a major concern for most patients. The aim of the present study was to assess the role of matrix metalloproteinase-2 (MMP-2) and MMP-9 in alveolar macrophages of patients with PSP and its relationship with recurrence.Ninety-two patients who received needlescopic video-assisted thoracoscopic surgery (NVATS) wedge resection of lung with identifiable blebs for PSP were enrolled for the study. Immunohistochemistry was performed to evaluate the expression of MMP-2 and MMP-9 in lung tissues of patients with PSP. The result was correlated with clinicopathological variables and recurrence rates by the chi-square test. The value of MMP-2 and MMP-9 for overall recurrence was evaluated by univariate and multivariable Cox regression analyses.The MMP-2 and MMP-9 staining was predominantly observed in alveolar macrophages of patients with PSP. We found that MMP-2 (recurrence: P0.001; smoking status: P=0.029) and MMP-9 (recurrence: P=0.001; smoking status: P=0.045) expression in PSP, especially male patients, was significantly correlated with recurrence and smoking status. In the multivariate analyses, MMP-2 [hazard ratio (HR) =2.83; 95% confidence interval (CI): 1.37-5.85, P=0.005) and MMP-9 (HR =2.25; 95% CI: 1.19-4.24, P=0.013) were statistically significant risk factors for overall recurrence in PSP patients.High expression levels of MMP-2 and MMP-9 showed a positive correlation with recurrence in PSP patients. Further studies are required to test whether inhibition of MMP-2 and MMP-9 expression renders a promising approach for reducing the risk of PSP recurrence in the future.

Acute lung injury (ALI) is a life-threatening syndrome characterized by acute and severe hypoxemic respiratory failure. Visfatin, which is known as an obesity-related cytokine with pro-inflammatory activities, plays a role in regulation of inflammatory cytokines. The mechanisms of ALI remain unclear in critically ill patients. Survival in ALI patients appear to be influenced by the stress generated by mechanical ventilation and by ALI-associated factors that initiate the inflammatory response. The objective for this study was to understand the mechanisms of how visfatin regulates inflammatory cytokines and promotes ALI. The expression of visfatin was evaluated in ALI patients and mouse sepsis models. Moreover, the underlying mechanisms were investigated using human bronchial epithelial cell lines, BEAS-2B and NL-20. An increase of serum visfatin was discovered in ALI patients compared to normal controls. Results from hematoxylin and eosin (H&amp, E) and immunohistochemistry staining also showed that visfatin protein was upregulated in mouse sepsis models. Moreover, lipopolysaccharide (LPS) induced visfatin expression, activated the STAT3/NF&kappa, B pathway, and increased the expression of pro-inflammatory cytokines, including IL1-&beta, IL-6, and TNF-&alpha, in human bronchial epithelial cell lines NL-20 and BEAS-2B. Co-treatment of visfatin inhibitor FK866 reversed the activation of the STAT3/NF&kappa, B pathway and the increase of pro-inflammatory cytokines induced by LPS. Our study provides new evidence for the involvement of visfatin and down-stream events in acute lung injury. Further studies are required to confirm whether the anti-visfatin approaches can improve ALI patient survival by alleviating the pro-inflammatory process.

Rad51 is a protein which plays a vital role in DNA double-strand break repair and maintenance of telomeres. However, the underlying mechanism for its action in esophageal squamous cell carcinoma (ESCC) remains unclear. Eighty-seven patients with ESCC were enrolled in this study. Expression of Rad51 in ESCC was determined by immunohistochemistry and correlated with clinicopathological variables by Chi square test. The role of Rad51 in patient survival was determined by Kaplan–Meier estimates. The effects of Rad51 knockdown and overexpression on esophageal cancer growth, migration, and invasion were examined using TE8, CE81T, and KYSE70 cells. The mechanisms involved were also analyzed. Nude mice models were used for assessment of tumor growth. Rad51 staining was predominantly observed in ESCC patients. ESCC patients with high Rad51 expression had significantly decreased survival (P

Exposure to arsenic is known to be a risk factor for various types of cancer. Apart from its carcinogenic activity, arsenic also shows promoting effects on angiogenesis, a crucial process for tumor growth. Yet, the mechanism underlying arsenic-induced angiogenesis is not fully understood. In this study, we aimed at investigating the involvement of inhibitor of DNA binding 1 (Id1) and the associated signal molecules in the arsenic-mediated angiogenesis. Our initial screening revealed that treatment with low concentrations of arsenic (0.5–1 μM) led to multiple cellular responses, including enhanced endothelial cell viability and angiogenic activity as well as increased protein expression of Id1. The arsenic-induced angiogenesis was suppressed in the Id1-knocked down cells compared to that in control cells. Furthermore, arsenic-induced Id1 expression and angiogenic activity were regulated by PI3K/Akt, NF-κB, and nitric oxide synthase (NOS) signaling. In summary, our current data demonstrate for the first time that Id1 mediates the arsenic-promoted angiogenesis, and Id1 may be regarded as an antiangiogenesis target for treatment of arsenic-associated cancer.

Adjuvant chemotherapy is commonly given to surgically treated non-small-cell lung cancer (NSCLC) patients. However, the prerequisite for chemotherapy needs to be scrutinized in order to maximize the benefits to patients. In this study, we observed that NSCLC cells with high Id1 protein expression were vulnerable to the treatment of paclitaxel and cisplatin. In addition, paclitaxel and cisplatin caused Id1 protein degradation through ubiquitination. In the nude mice xenograft model, the tumor growth was reduced to a large degree in the Id1-overexpressing group upon treatment with paclitaxel and cisplatin. Furthermore, immunohistochemical staining for Id1 followed by Kaplan-Meier survival analysis showed that surgically treated NSCLC patients with high Id1 expression in primary tumor tissues had better disease-free and overall survivals after adjuvant paclitaxel and cisplatin chemotherapy. In summary, our current data suggest that Id1, a generally negative prognostic factor, predicts a favorable prognosis in the case of surgically treated NSCLC patients receiving the definitive adjuvant chemotherapy. The distinct role of Id1 reported in this study may arise from the phenomenon of Id1 dependence of NSCLC cells for survival, which renders the cancer cells additionally susceptive to the adjuvant chemotherapy with paclitaxel and cisplatin.

Primary spontaneous pneumothorax (PSP) is a common clinical problem. However, PSP recurrence is still a major concern. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a protective role against oxidative airway diseases. The aim was to investigate the role of Nrf2 in PSP patients and its correlation with recurrence.Eighty-nine patients were enrolled and received wedge resection of lung with identifiable blebs. Nrf2 expression in resected lung tissues was determined by immunohistochemistry (IHC) and correlated with clinicopathological variables. The prognostic value of Nrf2 for incidence-of-recurrence was determined by Kaplan-Meier estimates and the significance of differences was evaluated by the log-rank test.Nrf2 staining was predominantly observed in alveolar macrophages and type II pneumocytes of PSP patients and correlated with recurrence (P0.001 and P=0.001, respectively) and PSP location (macrophages, P=0.013). High Nrf2 expression was correlated with better incidence-of-recurrence (macrophages, P=0.003; type II pneumocytes, P=0.003). Moreover, incidence-of-recurrence was better in patients with higher Nrf2 expression, especially those in the age ≤20, male, and non-smoking groups (macrophages, P=0.009, 0.006, and 0.012; type II pneumocytes, P=0.003, 0.011, and 0.010, respectively).High Nrf2 expression in alveolar macrophages and type II pneumocytes was significantly associated with the decreased recurrence risk and was the independent factor predicting a better incidence-of-recurrence in PSP. Our results suggest that Nrf2 activation in high risk patients may be a potential target for reducing PSP recurrence.

The β-nitrostyrene family possesses anticancer properties. In this study, β-nitrostyrene derivative CYT-Rx20 (3'-hydroxy-4'-methoxy-β-methyl-β-nitrostyrene) was synthesized and investigated its anticancer activity in oral cancer.Anticancer activity of CYT-Rx20 and the underlying mechanisms were analyzed using cell viability assay, reactive oxygen species (ROS) generation assay, fluorescence-activated cell sorter analysis, annexin V staining, comet assay, glutathione (GSH)/glutathione disulfide (GSSG) ratio, immunoblotting, soft agar assay, nude mice xenograft study, and immunohistochemistry.CYT-Rx20-induced cell apoptosis via ROS generation and mitochondrial membrane potential reduction, associated with release of mitochondrial cytochrome C to cytosol and activation of downstream caspases and poly ADP-ribose polymerase (PARP). Furthermore, CYT-Rx20 induced mitochondrial ROS accumulation and mitochondrial dysfunction, followed by GSH downregulation. CYT-Rx20-induced cell apoptosis, ROS generation, and DNA damage were reversed by thiol antioxidants. In nude mice, CYT-Rx20 inhibited oral tumor growth accompanied by increased expression of γH2AX, GSH reductase, and cleaved-caspase-3.CYT-Rx20 has the potential to be further developed into an antioral cancer drug clinically. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1055-1064, 2017.

Aims Members of the β-nitrostyrene family are known to suppress tumor growth, with the underlying mechanisms of β-nitrostyrene remain mostly unclear. Herein, we synthesized a β-nitrostyrene derivative, 3′-hydroxy-4′-methoxy-β-methyl-β-nitrostyrene (CYT-Rx20), and explored its anticancer activities in human lung cancer cells in vitro and in vivo. Main methods Cell viability was measured by XTT assay. Apoptosis was detected by Annexin V/PI staining. Caspase activation was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by flow cytometry. GSH level was detected by ELISA assay. Key findings In this study, we found that CYT-Rx20 significantly reduced cell viability, accompanied by G2/M arrest in lung cancer cells. Increased protein levels of cleaved-caspase families indicated apoptotic cell death upon CYT-Rx20 treatment. Furthermore, increased level of intracellular reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔΨm), glutathione (GSH) depletion and inhibition of GSH reductase were observed after CYT-Rx20 treatment. The effects of CYT-Rx20 on cell viability and the loss of ΔΨm were significantly reversed when cells were pretreated with thiol antioxidants NAC, GSH, or 2-ME. Finally, xenograft animal study demonstrated that CYT-Rx20 significantly suppressed lung tumor growth in vivo. Significance Our data demonstrated that CYT-Rx20 triggered apoptotic cell death in lung cancer cells and suppressed lung tumor growth through GSH depletion, suggesting that CYT-Rx20 may have the potential to be further developed as an anticancer compound for treating lung cancer.

Primary spontaneous pneumothorax (PSP) is a common benign problem. However, PSP recurrence is still a troublesome complication for most patients. This study intended to determine the role of matrix metalloproteinase-2 (MMP-2) and MMP-9 in type II pneumocytes of patients with PSP and its relation with recurrence. Ninety-one patients who had undergone needlescopic video-assisted thoracoscopic surgery wedge resection of lung with identifiable blebs for PSP were included in this study. Immunohistochemical (IHC) staining was used to measure the expression of MMP-2 and MMP-9 in lung tissues of PSP patients. The results were further correlated with clinicopathological parameters and recurrence rates using chi-square or Fisher's exact test. The value of MMP-2 and MMP-9 for overall recurrence was analyzed by univariate and multivariable Cox regression model. IHC data revealed that MMP-2 and MMP-9 staining was predominantly observed in type II pneumocytes of patients with PSP. We found that MMP-2 and MMP-9 expression in PSP, especially male PSP patients, was significantly correlated with recurrence. In the univariate and multivariate analyses, MMP-2 and MMP-9 were statistically significant risk factors for overall recurrence in PSP patients. Therefore, high expression levels of MMP-2 and MMP-9 in type II pneumocytes show a positive correlation with PSP recurrence risk. Further studies are needed to validate whether reduction of MMP-2 and MMP-9 expression may be a promising way for decreasing the risk of PSP recurrence in the future.

The β-nitrostyrene family have been implicated for anti-cancer property. However, the pharmacological role of β-nitrostyrene in esophageal cancer remain unclear. Here, a β-nitrostyrene derivative, CYT-Rx20, was synthesized and assessed for its anti-cancer activities and underlying mechanism in esophageal cancer. CYT-Rx20 induced cytotoxicity in esophageal cancer cells by promoting apoptosis through activation of caspase cascade and poly(ADP-ribose) polymerase (PARP) cleavage. Besides, CYT-Rx20 inhibited esophageal cancer cell migration and invasion by regulating the expression of epithelial to mesenchymal transition (EMT) markers. CYT-Rx20 decreased cell viability and migration through suppression of the PI3K/AKT and STAT3 pathways. Of note, the cytotoxicity and anti-migratory effect of CYT-Rx20 were enhanced by co-treatment with SC79 (AKT activator) or colivelin (STAT3 activator), suggesting the dependency of esophageal cancer cells on AKT and STAT3 for survival and migration, an oncogene addiction phenomenon. In xenograft tumor-bearing mice, CYT-Rx20 significantly reduced tumor growth of the implanted esophageal cancer cells accompanied by decreased Ki-67, phospho-AKT, and phospho-STAT3 expression. In orthotopic esophageal cancer mouse model, decreased tumor growth and lung metastasis with reduced Ki-67 and phospho-STAT3 expression were observed in mice treated with CYT-Rx20. Together, our results suggest that CYT-Rx20 is a potential β-nitrostyrene-based anticancer compound against the tumor growth and metastasis of esophageal cancer.

β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of β2-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of β2-GPI to endothelial cells, we investigated the effect of β2-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of β2-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that β2-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, β2-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, β2-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that β2-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.

β2-GPI (β2-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of the present study was to assess the contribution of β2-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β2-GPI inhibited endothelial cell migration, which was restored by its neutralizing antibody. NF-κB (nuclear factor κB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-κB significantly attenuated the inhibitory effect of β2-GPI on cell migration. Moreover, β2-GPI was found to induce IκBα (inhibitor of NF-κB) phosphorylation and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS [endothelial NO (nitric oxide) synthase] and NO production were all increased by β2-GPI and these effects were remarkably inhibited by NF-κB inhibitors and siRNAs of p65 and p50. Furthermore, β2-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. The findings of the present study provide novel insight into the ability of β2-GPI to inhibit endothelial cell migration predominantly through the NF-κB/eNOS/NO signalling pathway, which indicates a potential direction for clinical therapy in vascular diseases.

β₂-GPI (β₂-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of the present study was to assess the contribution of β₂-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β₂-GPI inhibited endothelial cell migration, which was restored by its neutralizing antibody. NF-κB (nuclear factor κB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-κB significantly attenuated the inhibitory effect of β₂-GPI on cell migration. Moreover, β₂-GPI was found to induce IκBα (inhibitor of NF-κB) phosphorylation and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS [endothelial NO (nitric oxide) synthase] and NO production were all increased by β₂-GPI and these effects were remarkably inhibited by NF-κB inhibitors and siRNAs of p65 and p50. Furthermore, β₂-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. The findings of the present study provide novel insight into the ability of β₂-GPI to inhibit endothelial cell migration predominantly through the NF-κB/eNOS/NO signalling pathway, which indicates a potential direction for clinical therapy in vascular diseases.

β2-Glycoprotein I (β2-GPI), also known as apolipoprotein H, is a plasma glycoprotein with poorly defined gene regulation. The aim of this study was to clarify the role of oxidative stress in β2-GPI gene regulation and determine the essential transcription element regulating β2-GPI expression. We demonstrate that expression of β2-GPI at the protein and mRNA levels was significantly elevated in Huh7 and HepG2 cells treated with 100 µM hydrogen peroxide (H2O2). To address the transcriptional mechanism of H2O2-mediated β2-GPI gene regulation, several promoter constructs were cloned and characterized by deletion assays. A region spanning from −2141 to −1419 (relative to the transcription start site), which contains two activator protein-1 (AP-1) sites (AP1-2 and AP1-3) and one nuclear factor-kappaB (NF-κB) site was found to be the main target site for up-regulation of β2-GPI promoter activity by oxidative stress. In addition, we found that H2O2 stimulation enhanced the nuclear translocation of AP-1 and NF-κB subunits. Using an electrophoretic mobility shift assay, it was confirmed that nuclear protein binding to the AP1-2, AP1-3, and NF-κB sites was increased in Huh7 cells treated with H2O2. Knockdown of the c-Jun, c-Fos, p65, and p50 genes using small interfering RNAs (siRNAs) further confirmed that AP-1 and NF-κB play an essential role in the H2O2-induced β2-GPI expression. Overall, these findings provide new insight suggesting that multiple cis-elements in the β2-GPI promoter work cooperatively to regulate β2-GPI expression in cells under oxidative stress. J. Cell. Biochem. 111: 988–998, 2010. © 2010 Wiley-Liss, Inc.