scholarly journals Proteomic Characterization of Colorectal Cancer Cells versus Normal-Derived Colon Mucosa Cells: Approaching Identification of Novel Diagnostic Protein Biomarkers in Colorectal Cancer

2020 ◽  
Vol 21 (10) ◽  
pp. 3466 ◽  
Author(s):  
Maja Ludvigsen ◽  
Louise Thorlacius-Ussing ◽  
Henrik Vorum ◽  
Mary Pat Moyer ◽  
Mogens Tornby Stender ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Line ◽  
Polyacrylamide Gel Electrophoresis ◽  
Primary Adenocarcinoma ◽  
Cellular Model ◽  
Colon Mucosa ◽  
Cellular Models ◽  
Proteomic Approach ◽  
Diagnostic Potential

In the western world, colorectal cancer (CRC) is the third most common cause of cancer-related deaths. Survival is closely related to the stage of cancer at diagnosis striking the clinical need for biomarkers capable of early detection. To search for possible biological parameters for early diagnosis of CRC we evaluated protein expression for three CREC (acronym: Cab45, reticulocalbin, ERC-55, calumenin) proteins: reticulocalbin, calumenin, and ERC-55 in a cellular model consisting of a normal derived colon mucosa cell line, NCM460, and a primary adenocarcinoma cell line of the colon, SW480. Furthermore, this cellular model was analyzed by a top-down proteomic approach, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for novel putative diagnostic markers by identification of differentially expressed proteins between the two cell lines. A different colorectal carcinoma cell line, HCT 116, was used in a bottom-up proteomic approach with label-free quantification (LFQ) LC–MS/MS. The two cellular models gave sets of putative diagnostic CRC biomarkers. Various of these novel putative markers were verified with increased expression in CRC patient neoplastic tissue compared to the expression in a non-involved part of the colon, including reticulocalbin, calumenin, S100A6 and protein SET. Characterization of these novel identified biological features for CRC patients may have diagnostic potential and therapeutic relevance in this malignancy characterized by a still unmet clinical need.

scholarly journals The structure of the oligosaccharides of alpha3beta1 integrin from human ureter epithelium (HCV29) cell line.

2002 ◽  
Vol 49 (2) ◽  
pp. 491-500 ◽  
Author(s):  
Anna Lityńska ◽  
Ewa Pocheć ◽  
Dorota Hoja-Lukowicz ◽  
Elzbieta Kremser ◽  
Piotr Laidler ◽  
...  
Keyword(s):  
Cell Line ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Beta Subunits ◽  
Ionization Mass ◽  
Complex Type ◽  
Peptide Characterization ◽  
Alpha3beta1 Integrin ◽  
Human Ureter

There is a growing line of evidence that glycosylation of alpha and beta subunits is important for the function of integrins. Integrin alpha3beta1, from human ureter epithelium cell-line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin alpha3beta1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.

scholarly journals Proteomic Characterization of Colorectal Cancer Tissue from Patients Identifies Novel Putative Protein Biomarkers

2021 ◽  
Vol 43 (2) ◽  
pp. 1043-1056
Author(s):  
Maja Ludvigsen ◽  
Louise Thorlacius-Ussing ◽  
Henrik Vorum ◽  
Mogens Tornby Stender ◽  
Ole Thorlacius-Ussing ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Expression Patterns ◽  
Cancer Tissue ◽  
Protein Biomarkers ◽  
Differential Protein Expression ◽  
Two Dimensional Gel Electrophoresis ◽  
One Dimensional ◽  
Diagnostic Potential ◽  
Liquid Chromatography Tandem Mass

Colorectal cancer (CRC) is one of the leading causes of cancer-related death over the world. There is a great need for biomarkers capable of early detection and as targets for treatment. Differential protein expression was investigated with two-dimensional gel electrophoresis (2D-PAGE) followed by identification with liquid chromatography–tandem mass spectrometry (LC-MS/MS) in CRC patient tissue from (i) the peripheral part of the tumor, (ii) the central part of the tumor as well as from (iii) a non-involved part of the colorectal tissue. The expression patterns of six identified proteins were further evaluated by one-dimensional Western blot (1D-WB) analysis of the CRC tissue. Proteins that were perturbed in expression level in the peripheral or in the central part of the tumor as compared with the non-involved part included S100A11, HNRNPF, HNRNPH1 or HNRNPH2, GSTP1, PKM and FABP1. These identified markers may have future diagnostic potential or may be novel treatment targets after further evaluation in larger patient cohorts.

scholarly journals Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis

2020 ◽  
Vol 11 ◽  
Author(s):  
Azzurra Sargenti ◽  
Francesco Musmeci ◽  
Francesco Bacchi ◽  
Cecilia Delprete ◽  
Domenico Andrea Cristaldi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Culture ◽  
Cell Viability ◽  
Cell Line ◽  
Nk Cell ◽  
3D Cell Culture ◽  
Physical Characterization ◽  
Tumor Spheroids

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

scholarly journals A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1838
Author(s):  
Laura Darie-Ion ◽  
Madhuri Jayathirtha ◽  
Gabriela Elena Hitruc ◽  
Marius-Mihai Zaharia ◽  
Robert Vasile Gradinaru ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Industrial Applications ◽  
Experimental Conditions ◽  
Proteomic Approach ◽  
Sds Page ◽  
Pharmaceutical Industries

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

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