HSPA6, a novel prognostic and therapeutic biomarker, associated with Ming classification in gastric cancer.

OBJECTIVE: This study aimed to explore the clinical relevance of heat shock protein family A member 6 (HSPA6) in gastric cancer (GC) and its effect on GC cell proliferation. METHODS: HSPA6 mRNA and protein levels were analyzed by bioinformatics, RT-qPCR, western blot and immunohistochemistry. HSPA6 was correlated with clinicopathological variables by the Chi-square test. Kaplan-Meier survival analysis and the univariate and multivariate Cox models were used to assess the prognostic value of HSPA6. Nomogram was used to predict overall survival in patients with GC. Knockdown or over-expression of HSPA6 in GC cell lines was constructed by lentiviral transduction. EdU and CCK-8 assay were used to detect cell proliferation. In vivo mouse tumor models were performed to evaluate the effects of HSPA6 on GC growth. RESULTS: HSPA6 were significantly upregulated in the GC tissues compared to the normal stomach epithelium and were associated with Ming classification (p < 0.001) and tumor size (p = 0.002). Patients with high expression of HSPA6 showed worse survival compared to the low expression group. HSPA6 was identified to be an independent prognostic biomarker for GC. HSPA6 was functionally annotated with the cell cycle, G2M checkpoint and Hippo pathway. Knockdown of HSPA6 suppressed XGC-1 cell proliferation both in vitro and in vivo. Overexpression of HSPA6 in AGS cells increased proliferation rates, increased the levels of cyclinB1 and YAP and decreased that of phosphorylated YAP. HSPA6 knockdown in the NUGC2 cells had the opposite effect. CONCLUSIONS: HSPA6 promotes GC proliferation by the Hippo pathway, as a novel prognostic biomarker and potential therapeutic target.

Proteomic signatures of infiltrative gastric cancer by proteomic and bioinformatic analysis.

BACKGROUND: Proteomic signatures of Ming's infiltrative gastric cancer (IGC) remain unknown. AIM: To elucidate the molecular characteristics of IGC at the proteomics level. METHODS: Twelve pairs of IGC and adjacent normal tissues were collected and their proteomes were analyzed by high performance liquid chromatography tandem mass spectrometry. The identified peptides were sequenced de novo and matched against the SwissProt database using Maxquant software. The differentially expressed proteins (DEPs) were screened using |log2(Fold change)| > 1 and P-adj < 0.01 as the thresholds. The expression levels of selected proteins were verified by Western blotting. The interaction network of the DEPs was constructed with the STRING database and visualized using Cytoscape with cytoHubba software. The DEPs were functionally annotated using clusterProfiler, STRING and DAVID for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. P < 0.05 was considered statistically significant. RESULTS: A total of 7361 DEPs were identified, of which 94 were significantly up-regulated and 223 were significantly down-regulated in IGC relative to normal gastric tissues. The top 10 up-regulated proteins were MRTO4, BOP1, PES1, WDR12, BRIX1, NOP2, POLR1C, NOC2L, MYBBP1A and TSR1, and the top 10 down-regulated proteins were NDUFS8, NDUFS6, NDUFA8, NDUFA5, NDUFC2, NDUFB8, NDUFB5, NDUFB9, UQCRC2 and UQCRC1. The up-regulated proteins were enriched for 9 biological processes including DNA replication, ribosome biogenesis and initiation of DNA replication, and the cellular component MCM complex. Among the down-regulated proteins, 17 biological processes were enriched, including glucose metabolism, pyruvic acid metabolism and fatty acid ß-oxidation. In addition, the mitochondrial inner membrane, mitochondrial matrix and mitochondrial proton transport ATP synthase complex were among the 6 enriched cellular components, and 11 molecular functions including reduced nicotinamide adenine dinucleotide dehydrogenase activity, acyl-CoA dehydrogenase activity and nicotinamide adenine dinucleotide binding were also enriched. The significant KEGG pathways for the up-regulated proteins were DNA replication, cell cycle and mismatch repair, whereas 18 pathways including oxidative phosphorylation, fatty acid degradation and phenylalanine metabolism were significantly enriched among the down-regulated proteins. CONCLUSION: The proteins involved in cell cycle regulation, DNA replication and mismatch repair, and metabolism were significantly altered in IGC, and the proteomic profile may enable the discovery of novel biomarkers.

Carfilzomib Inhibits Constitutive NF-κB Activation in Mantle Cell Lymphoma B Cells and Leads to the Induction of Apoptosis.

Mantle cell lymphoma (MCL) remains incurable and new treatments are needed, especially in the relapsed/refractory setting. We therefore investigated the effects of carfilzomib, a novel, long-acting, second-generation proteasome inhibitor, in MCL cells. Eight established MCL cell lines and freshly isolated primary MCL cells were treated with carfilzomib. Cell proliferation was assessed by a 3H-thymidine incorporation assay. Cell apoptosis was evaluated by flow cytometry with annexin V and propidium iodide. Electrophoresis mobility shift (EMSA), Western blot, and luciferase assays were used to analyze NF-κB activation and related signaling proteins. Carfilzomib inhibited growth and induced apoptosis in both established MCL cell lines and freshly isolated primary MCL cells in a dose-dependent manner. In contrast, carfilzomib was less toxic to normal peripheral blood mononuclear cells from healthy individuals. The carfilzomib-induced apoptosis of MCL cells occurred in a caspase-dependent manner through both intrinsic and extrinsic caspase pathways. In addition, carfilzomib inhibited constitutive activation of the NF-κB signaling cascade, both in MCL cell lines and primary MCL cells, by completely blocking the phosphorylation of IκBα. Our results demonstrate that carfilzomib can induce growth arrest and apoptosis in MCL cells and that the mechanism may involve the NF-κB signaling pathway.

Effects of chronic tramadol exposure on the zebrafish brain: a proteomic study.

Tramadol hydrochloride (TH), has become the most prescribed opioid worldwide. However, its neurotoxicity and abuse potential are not well documented. In the present study, TH administration induced abnormal behavior and body and brain mean weight loss. Two principal metabolites O- and N-desmethyltramadol were detected in the brain tissue, and N-desmethyltramadol was the main metabolite produced. A total of 30 differential protein spots were identified using semi-quantitative 2D-PAGE and proteomic analyses, and classified into 13 categories, in which subtypes of 14-3-3 proteins, creatine kinase, ATP synthase beta chain, and tubulin were identified at the separated location on the gels 3, 3, 4, and 11 times respectively. Many TH responsive proteins have functions related to oxidative stress, including 14-3-3 proteins, creatine kinase BB, ubiquitin carboxy-terminal hydrolase L-1, ATP synthase, synaptosome-associated protein, tubulin and actin. Irrespective of oxidative damage, other pathways affected include apoptosis, energy metabolism, signal disorders, and cytoskeletal structure. Ultrastructural observation of mitochondria showed a series of morphological changes in the case of TH exposure.

A study of sympathetic skin response to the damage of autonomic nerves function in patients with chronic N-hexane poisoning / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the sympathetic skin response (SSR) to the effects of N-hexane on autonomic nerves function in patients with chronic N-hexane poisoning.</p><p><b>METHODS</b>The subjects in present study included 30 controls and 37 cases with chronic N-hexane poisoning. Also 37 patients were divided into 3 subgroups (mild, moderate and severe poisoning) according to diagnostic criteria of occupational diseases. All subjects were examined by SSR test and nerve conduction velocity (NCV) test. All patients were reexamined by SSR and NCV every 1 ∼ 2 months. The differences in SSR parameters (latency, amplitude) among groups were observed. In the severe poisoning subgroup, the changes of SSR and NCV parameters (conduction velocity, amplitude) in different poisoning stages were observed.</p><p><b>RESULTS</b>There were significant differences in SSR latency of upper extremity among groups and the significant differences in SSR amplitude of upper and lower extremity among groups (P < 0.05). No significant differences in SSR parameters were found between the adjacent groups (P > 0.05). There were significant differences in SSR latency of upper extremity during different periods and the significant differences in SSR amplitude of upper and lower extremity during different periods among all groups (P < 0.05). The change of SSR parameters consistent with that in NCV. The longest SSR latency of upper extremity and the smallest SSR amplitudes of upper and lower extremity appears 1 - 2 months earlier than that of the smallest action potential amplitude.</p><p><b>CONCLUSION</b>The damage of autonomic nerves induced by N-hexane increased with poisoning progresses. The damage of autonomic nerves corresponded with the damage of myelin sheath of large myelinated nerves, but which appeared 1 - 2 months earlier than the damage of axon of large myelinated nerves. SSR test may serve as a method to detect the damage of autonomic nerves function in patients with chronic N-hexane poisoning.</p>

Mineral oil-, glycerol-, and Vaseline-coated plates as matrix-assisted laser desorption/ionization sample supports for high-throughput peptide analysis.

A novel protocol for rapid and high-quality sample preparation prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed by coating bare stainless steel plates with one of three adhesives: mineral oil, glycerol, or Vaseline. The advantages of these three adhesive coats are that they take little time to both prepare and wipe away, hold the matrices to prevent them from flying from the support, reduce the background matrix, and affect neither the resolution of the peptide peaks nor the accuracy of their determined molecular masses. Consequently, the signal intensity, detection limit, and tolerance of the analytes to contaminants on the three adhesive-coated plates are improved. In the two strategies of on-plate desalting and concentration of the peptide mixture, all three adhesives reduced the loss of peptides, especially in the case of larger molecular mass peptides. The microscope and stereomicroscope images of the deposited droplets showed that after dropping onto the adhesive coats, the droplets formed a reduced spot size, were more homogeneous, and showed sticky crystallization. Therefore, this is an easy-to-use, reproducible, highly sensitive, tolerant (to salts), and high-throughput method of peptide sample preparation for MALDI-TOF MS analysis.