Label-Free Quantitative Proteomics Combined with Biological Validation Reveals Activation of Wnt/β-Catenin Pathway Contributing to Trastuzumab Resistance in Gastric Cancer

Resistance to trastuzumab, which specifically target HER2-positive breast and gastric cancer, can develop ultimately in cancer patients. However, the underlying mechanisms of resistance in gastric cancer have not been fully elucidated. Here, we established trastuzumab-resistant MKN45 and NCI N87 gastric cancer sublines from their parental cells. The resistant cells exhibited characteristics of epithelial-mesenchymal transition (EMT) and acquired higher migratory and invasive capacities. To exploit the activated pathways and develop new strategies to overcome trastuzumab resistance, we investigated MKN45 and MKN45/R cells via label-free quantitative proteomics, and found pathways that were altered significantly in MKN45/R cells, with the Wnt/β-catenin pathway being the most significant. We further confirmed the activation of this pathway by detecting its key molecules in MKN45/R and NCI N87/R cells via Western blot, in which Wnt3A, FZD6, and CTNNB1 increased, whereas GSK-3β decreased, manifesting the activation of the Wnt/β-catenin pathway. Correspondingly, inhibition of Wnt/β-catenin pathway by ICG-001, a specific Wnt/β-catenin inhibitor, preferentially reduced proliferation and invasion of trastuzumab-resistant cells and reversed EMT. Concurringly, CTNNB1 knockdown in stable cell lines potently sensitized cells to trastuzumab and induced more apoptosis. Taken together, our study demonstrates that the Wnt/β-catenin pathway mediates trastuzumab resistance, and the combination of Wnt/β-catenin inhibitors with trastuzumab may be an effective treatment option

Flexure Performance of Externally Bonded CFRP Plates-Strengthened Reinforced Concrete Members

This paper investigates the flexural behavior of CFRP plate-strengthened concrete structures. Specimens of the CFRP plate-reinforced beam were designed and tested by the four-point flexural test. The load-deflection relationship, failure modes, and crack propagation were analyzed. The results showed that the postcracking stiffness and bearing capacity of the test beams can be improved by the additional anchoring measures for CFRP strengthening. The relationship between flexural moment and curvature was analyzed by introducing a MATLAB program. The calculation model between curvature, flexural moment, and stiffness was derived for the CFRP plate-strengthened structure. The recommended calculation model was applied in the analysis of deflection, and the theoretical values were compared with the test results

Long non-coding RNA colon cancer-associated transcript 1-Vimentin axis promoting the migration and invasion of HeLa cells

Abstract. Background:. Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1–protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1–protein interaction mediated tumor metastasis. Methods:. CCAT1–proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1–Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells. Results:. RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin (VIM) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein. Conclusion:. CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities

Plasma High-Mannose and Complex/Hybrid N-Glycans Are Associated with Hypercholesterolemia in Humans and Rabbits.

N-glycans play important roles in various pathophysiological processes and can be used as clinical diagnosis markers. However, plasma N-glycans change and their pathophysiological significance in the setting of hypercholesterolemia, a major risk factor for atherosclerosis, is unknown. Here, we collected plasma from both hypercholesterolemic patients and cholesterol-fed hypercholesterolemic rabbits, and determined the changes in the whole-plasma N-glycan profile by electrospray ionization mass spectrometry. We found that both the hypercholesterolemic patients and rabbits showed a dramatic change in their plasma glycan profile. Compared with healthy subjects, the hypercholesterolemic patients exhibited higher plasma levels of a cluster of high-mannose and complex/hybrid N-glycans (mainly including undecorated or sialylated glycans), whereas only a few fucosylated or fucosylated and sialylated N-glycans were increased. Additionally, cholesterol-fed hypercholesterolemic rabbits also displayed increased plasma levels of high-mannose in addition to high complex/hybrid N-glycan levels. The whole-plasma glycan profiles revealed that the plasma N-glycan levels were correlated with the plasma cholesterol levels, implying that N-glycans may be a target for treatment of hypercholesterolemia

Simplified Quantitative Glycomics Using the Stable Isotope Label Girard’s Reagent P by Electrospray Ionization Mass Spectrometry

Fast, sensitive, and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features nonreductive isotopic labeling of reducing glycans with either nondeuterated (<i>d</i>₀-) or deuterated (<i>d</i>₅-) Girard’s reagent P (GP) without salts introduced and simplified mass spectrometric profiles of <i>d</i>₀- and <i>d</i>₅-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility, and accuracy of data by the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral <i>N</i>-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization

Radiogenomic analysis of ultrasound phenotypic features coupled to proteomes predicts metastatic risk in primary prostate cancer

Abstract Background Primary prostate cancer with metastasis has a poor prognosis, so assessing its risk of metastasis is essential. Methods This study combined comprehensive ultrasound features with tissue proteomic analysis to obtain biomarkers and practical diagnostic image features that signify prostate cancer metastasis. Results In this study, 17 ultrasound image features of benign prostatic hyperplasia (BPH), primary prostate cancer without metastasis (PPCWOM), and primary prostate cancer with metastasis (PPCWM) were comprehensively analyzed and combined with the corresponding tissue proteome data to perform weighted gene co-expression network analysis (WGCNA), which resulted in two modules highly correlated with the ultrasound phenotype. We screened proteins with temporal expression trends based on the progression of the disease from BPH to PPCWOM and ultimately to PPCWM from two modules and obtained a protein that can promote prostate cancer metastasis. Subsequently, four ultrasound image features significantly associated with the metastatic biomarker HNRNPC (Heterogeneous nuclear ribonucleoprotein C) were identified by analyzing the correlation between the protein and ultrasound image features. The biomarker HNRNPC showed a significant difference in the five-year survival rate of prostate cancer patients (p < 0.0053). On the other hand, we validated the diagnostic efficiency of the four ultrasound image features in clinical data from 112 patients with PPCWOM and 150 patients with PPCWM, obtaining a combined diagnostic AUC of 0.904. In summary, using ultrasound imaging features for predicting whether prostate cancer is metastatic has many applications. Conclusion The above study reveals noninvasive ultrasound image biomarkers and their underlying biological significance, which provide a basis for early diagnosis, treatment, and prognosis of primary prostate cancer with metastasis

Additional file 1 of Radiogenomic analysis of ultrasound phenotypic features coupled to proteomes predicts metastatic risk in primary prostate cancer

Supplementary Material

Information of healthy subjects and hypercholesterolemic patients.

<p>Information of healthy subjects and hypercholesterolemic patients.</p

ESI-MS profiles of the plasma N-glycans released from humans.

<p>Representative N-glycan profiles of the whole plasma from either healthy (A and C) or hypercholesterolemic subjects (B and D). A and B show mass spectra of N-glycans in positive-ion mode; and C and D show mass spectra in negative-ion mode. Putative glycan compositions were presented on the basis of the MS/MS analysis. Structural formulas: blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; red triangle, fucose; purple diamond, N-acetylneuraminic acid.</p