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.

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.