miR-21 Targets ASPP2 to Inhibit Apoptosis via CHOP-Mediated Signaling in Helicobacter pylori-Infected Gastric Cancer Cells.

Helicobacter pylori (H. pylori) infection affects cell survival pathways, including apoptosis and proliferation in host cells, and disruption of this balance is the key event in the development of H. pylori-induced gastric cancer (HPGC). H. pylori infection induces alterations in microRNAs expression that may be involved in GC development. Bioinformatic analysis showed that microRNA-21 (miR-21) is significantly upregulated in HPGC. Furthermore, quantitative proteomics and in silico prediction were employed to identify potential targets of miR-21. Following functional enrichment and clustered interaction network analyses, five candidates of miR-21 targets, PDCD4, ASPP2, DAXX, PIK3R1, and MAP3K1, were found across three functional clusters in association with cell death and survival, cellular movement, and cellular growth and proliferation. ASPP2 is inhibited by H. pylori-induced miR-21 overexpression. Moreover, ASPP2 levels are inversely correlated with miR-21 levels in HPGC tumor tissues. Thus, ASPP2 was identified as a miR-21 target in HPGC. Here, we observed that H. pylori-induced ASPP2 suppression enhances resistance to apoptosis in GC cells using apoptosis assays. Using protein interaction network and coimmunoprecipitation assay, we identified CHOP as a direct mediator of the ASPP2 proapoptotic activity in H. pylori-infected GC cells. Mechanistically, ASPP2 suppression promotes p300-mediated CHOP degradation, in turn inhibiting CHOP-mediated transcription of Noxa, Bak, and suppression of Bcl-2 to enact antiapoptosis in the GC cells after H. pylori infection. Clinicopathological analysis revealed correlations between decreased ASPP2 expression and higher HPGC risk and poor prognosis. In summary, the discovery of H. pylori-induced antiapoptosis via miR-21-mediated suppression of ASPP2/CHOP-mediated signaling provides a novel perspective for developing HPGC management and treatment.

S100A8, S100A9 and S100A8/A9 heterodimer as novel cachexigenic factors for pancreatic cancer-induced cachexia.

BACKGROUND: Cancer cachexia, occurring in ~ 80% pancreatic cancer (PC) patients overall, is a paraneoplastic syndrome mediated by cancer-induced systemic inflammation and characterized by weight loss and skeletal muscle wasting. Identifying clinically relevant PC-derived pro-inflammatory factors with cachexigenic potential may provide novel insights and therapeutic strategies. METHODS: Pro-inflammatory factors with cachexigenic potential in PC were identified by bioinformatic analysis. The abilities of selected candidate factors in inducing skeletal muscle atrophy were investigated. Expression levels of candidate factors in tumors and sera was compared between PC patients with and without cachexia. Associations between serum levels of the candidates and weight loss were assessed in PC patients. RESULTS: S100A8, S100A9, and S100A8/A9 were identified and shown to induce C2C12 myotube atrophy. Tumors of PC patients with cachexia had markedly elevated expression of S100A8 (P = 0.003) and S100A9 (P < 0.001). PC patients with cachexia had significantly higher serum levels of S100A8, S100A9 and S100A8/A9. Serum levels of these factors positively correlated with percentage of weight loss [correlation coefficient: S100A8: 0.33 (P < 0.001); S100A9: 0.30 (P < 0.001); S100A8/A9: 0.24 (P = 0.004)] and independently predicted the occurrence of cachexia [adjusted odds ratio (95% confidence interval) per 1ng/ml increase: S100A8 1.11 (1.02-1.21), P = 0.014; S100A9 1.10 (1.04-1.16), P = 0.001; per 1 µg/ml increase: S100A8/A9 1.04 (1.01-1.06), P = 0.009]. CONCLUSIONS: Atrophic effects of S100A8, S100A9, and S100A8/A9 indicated them as potential pathogenic factors of PC-induced cachexia. In addition, the correlation with the degree of weight loss and prediction of cachexia in PC patients implicated their potential utility in the diagnosis of PC-induced cachexia.

Aberrant Upregulation of Indoleamine 2,3-Dioxygenase 1 Promotes Proliferation and Metastasis of Hepatocellular Carcinoma Cells via Coordinated Activation of AhR and ß-Catenin Signaling.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide. Chronic liver inflammation due to hepatitis virus infection and other major effectors is a major risk factor of HCC. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme enzyme highly expressed upon stimulation with proinflammatory cytokines such as interferon-γ (IFN-γ), is activated to modulate the tumor microenvironment and potentially crucial in the development of certain cancer types. Earlier studies have majorly reported an immunomodulatory function of IDO1. However, the specific role of IDO1 in cancer cells, particularly HCC, remains to be clarified. Analysis of The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) dataset in the current study revealed a significant correlation between IDO1 expression and HCC. We further established inducible IDO1-expressing cell models by coupling lentivirus-mediated knockdown and IFN-γ induction of IDO1 in normal and HCC cells. In functional assays, proliferation and motility-related functions of HCC cells were compromised upon suppression of IDO1, which may partially be rescued by its enzymatic product, kynurenine (KYN), while normal hepatocytes were not affected. Aryl hydrocarbon receptor (AhR), a reported endogenous KYN receptor, is suggested to participate in tumorigenesis. In mechanistic studies, IDO1 activation promoted both AhR and ß-catenin activity and nuclear translocation. Immunofluorescence staining and co-immunoprecipitation assays further disclosed interactions between AhR and ß-catenin. In addition, we identified a Src-PTEN-PI3K/Akt-GSK-3ß axis involved in ß-catenin stabilization and activation following IDO1-mediated AhR activation. IDO1-induced AhR and ß-catenin modulated the expression of proliferation- and EMT-related genes to facilitate growth and metastasis of HCC cells. Our collective findings provide a mechanistic basis for the design of more efficacious IDO1-targeted therapy for HCC.

Y-Box Binding Protein-1 Promotes Epithelial-Mesenchymal Transition in Sorafenib-Resistant Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is one of the most common cancer types worldwide. In cases of advanced-stage disease, sorafenib is considered the treatment of choice. However, resistance to sorafenib remains a major obstacle for effective clinical application. Based on integrated phosphoproteomic and The Cancer Genome Atlas (TCGA) data, we identified a transcription factor, Y-box binding protein-1 (YB-1), with elevated phosphorylation of Ser102 in sorafenib-resistant HuH-7R cells. Phosphoinositide-3-kinase (PI3K) and protein kinase B (AKT) were activated by sorafenib, which, in turn, increased the phosphorylation level of YB-1. In functional analyses, knockdown of YB-1 led to decreased cell migration and invasion in vitro. At the molecular level, inhibition of YB-1 induced suppression of zinc-finger protein SNAI1 (Snail), twist-related protein 1 (Twist1), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin levels, implying a role of YB-1 in the epithelial-mesenchymal transition (EMT) process in HuH-7R cells. Additionally, YB-1 contributes to morphological alterations resulting from F-actin rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance.

Quantitative phosphoproteomic analysis identifies the potential therapeutic target EphA2 for overcoming sorafenib resistance in hepatocellular carcinoma cells.

Limited therapeutic options are available for advanced-stage hepatocellular carcinoma owing to its poor diagnosis. Drug resistance to sorafenib, the only available targeted agent, is commonly reported. The comprehensive elucidation of the mechanisms underlying sorafenib resistance may thus aid in the development of more efficacious therapeutic agents. To clarify the signaling changes contributing to resistance, we applied quantitative phosphoproteomics to analyze the differential phosphorylation changes between parental and sorafenib-resistant HuH-7 cells. Consequently, an average of ~1500 differential phosphoproteins were identified and quantified, among which 533 were significantly upregulated in resistant cells. Further bioinformatic integration via functional categorization annotation, pathway enrichment and interaction linkage analysis led to the discovery of alterations in pathways associated with cell adhesion and motility, cell survival and cell growth and the identification of a novel target, EphA2, in resistant HuH-7R cells. In vitro functional analysis indicated that the suppression of EphA2 function impairs cell proliferation and motility and, most importantly, overcomes sorafenib resistance. The attenuation of sorafenib resistance may be achieved prior to its development through the modulation of EphA2 and the subsequent inhibition of Akt activity. Binding analyses and in silico modeling revealed a ligand mimic lead compound, prazosin, that could abate the ligand-independent oncogenic activity of EphA2. Finally, data obtained from in vivo animal models verified that the simultaneous inhibition of EphA2 with sorafenib treatment can effectively overcome sorafenib resistance and extend the projected survival of resistant tumor-bearing mice. Thus our findings regarding the targeting of EphA2 may provide an effective approach for overcoming sorafenib resistance and may contribute to the management of advanced hepatocellular carcinoma.

Chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells.

Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy.

Mechanisms controlling the effects of bevacizumab (avastin) on the inhibition of early but not late formed corneal neovascularization.

PURPOSE: To evaluate the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab on the inhibition of corneal neovascularization (NV). METHODS: Corneal NV was induced by closed eye contact lens wear followed by a silk suture tarsorrhaphy in rabbits. Weekly subconjunctival injections of bevacizumab (5.0 mg) for 1 month were started immediately (early treatment group) or 1 month after induction of corneal NV with continuous induction (late treatment group). The severity of corneal NV was evaluated. Immunostaining was used to evaluate the intracorneal diffusion of bevacizumab, and the existence of pericytes and smooth muscle cells around the NV. The expression of AM-3K, an anti-macrophage antibody, vascular endothelial growth factor (VEGF) with its receptors (VEGFR1 and VEGFR2), and vascular endothelial apoptosis were also evaluated. Western blot analysis was performed to quantify the expression level of VEGF, VEGFR1 and VEGFR2 on corneal epithelium and stroma in different groups. RESULTS: Early treatment with bevacizumab inhibited corneal NV more significantly than late treatment. Intracorneal diffusion of bevacizumab was not different among different groups. Immunostaining showed pericytes and smooth muscle cells around newly formed vessels as early as 2 weeks after induction. Immunostaining and Western blot analysis showed that VEGF, VEGFR1, and VEGFR2 on corneal stroma increased significantly in no treatment groups and late treatment groups, but not in early treatment group. Bevacizumab significantly inhibited macrophage infiltration in the early but not late treatment group. Sporadic vascular endothelial apoptosis was found at 4 weeks in the late but not early treatment group. CONCLUSIONS: Early but not late injection of bevacizumab inhibited corneal NV. Late injection of bevacizumab did not alter macrophage infiltration, and can't inhibit the expression of VEGF, VEGFR1, and VEGFR2 on corneal vessels. The inhibition of corneal NV in early treatment group does not occur via vascular endothelial apoptosis.

Development of an AlphaLISA assay to quantify serum core-fucosylated E-cadherin as a metastatic lung adenocarcinoma biomarker.

Lung cancer is the leading cause of cancer-associated deaths worldwide. Non-small cell lung cancer is a heterogeneous condition with variability in prognosis and in individual response to treatment. Thus, the identification of patients with a high risk of metastasis or relapse after surgery would allow better management. There is increasing evidence that glycosylation plays a significant role in biological processes including oncogenic transformation and metastasis. We set up a platform to screen and identify serum glycoproteins as metastasis biomarkers of lung cancer. Concanavalin A affinity chromatography was used to enrich glycoproteins from pooled serum of lung adenocarcinoma patients. The captured glycoproteins were separated with 2-D DIGE combined with nano-LC-MS/MS and identified by database searching. Some differentially expressed cancer-related glycoproteins, such as α-1-antitrypsin, complement C3c, haptoglobin, and E-cadherin, were identified. These glycoproteins were evaluated by Western blotting and Aleuria aurantia lectin staining and several, including E-cadherin, showed increased core-fucosylation during lung cancer progression. We then measured the fucosylation index (FI) of E-cadherin in 154 lung adenocarcinoma patients. In addition, a homogeneous proximity-based AlphaLISA assay to measure the FI of E-cadherin was established. The present study indicates that the FI of E-cadherin could be a potential prognostic marker of metastatic lung adenocarcinoma.

Electric energy generator.

This study reports an extremely cost-effective mechanism for converting wind energy into electric energy using piezoelectric bimorph actuators at small scale. The total dimensions of the electric energy generator are 5.08 x 11.6 x 7.7 cm3. The rectangular, box-shaped body of the overall structure is made using 3.2-mm thick plastic. Slits are made on two opposite faces of the box so that two columns and six rows of bimorph actuators can be inserted. Each row of bimorph actuators is separated from each other by a gap of 6 mm, and the two columns of bimorphs are separated from each other by a gap of 6.35 mm. In between the two columns, a cylindrical rod is inserted consisting of six rectangular hooks. The hooks are positioned in such a way that each of them just touches the two bimorphs on either side in a particular row. As the wind flows across the generator, it creates a rotary motion on the attached fan that is converted into vertical motion of the cylindrical rod using the cam-shaft mechanism. This vertical motion of the cylindrical rod creates oscillating stress on the bimorphs due to attached hooks. The bimorphs produce output voltage proportional to the applied oscillating stress through piezoelectric effect. The prototype fabricated in this study was found to generate 1.2 mW power at a wind speed of 12 mph across the load of 1.7 komega.