evaluating-cell-death-using-cell-free-supernatant-probiotics-three

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Korea Research Institute of Standards and Science (KRISS)

Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.

Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome.

We introduce a simple online O-labeling protocol for protein samples that uses a parallelizing microbore hollow fiber enzyme reactor (mHFER) as an alternative tool for online proteolytic digestion. Online O-labeling is performed by separately attaching two mHFERs in parallel to a 10-port switching valve with a high-pressure syringe pump and two syringes containing O- or O-water. O-/O-labeled peptides are formed in this manner and simultaneously analyzed online using nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) without any residual trypsin activity. The usefulness of a parallel mHFER platform (P-mHFER) in O-labeling was tested using both cytochrome C and alpha-1-acid-glycoprotein to verify the incorporation level of two O atoms into tryptic peptides and to provide a quantitative assessment with varied mixing ratios. Additionally, our O-labeling approach was used to study the serum N-glycoproteome from lung cancer patients and controls to evaluate the applicability of lectin-based quantitative N-glycoproteomics. We successfully quantified 76 peptides (from 62 N-glycoproteins). Nineteen of these peptides from lung cancer serum were up-/down-regulated at least 2.5-fold compared to controls. As a result, the P-mHFER-based online O-labeling platform presented here can be a simple and reproducible way to allow quantitative proteomic analysis of diverse proteome samples.

Development of a parallel microbore hollow fiber enzyme reactor platform for online O-labeling: Application to lectin-specific lung cancer N-glycoproteome.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using  for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of  cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.

Characterization of the Anti-Cancer Activity of the Probiotic Bacterium  Using 2D vs. 3D Culture in Colorectal Cancer Cells.

The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

Article	Year
Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome. Scientific reports\| PubMed ID: 30185973	2018
Development of a parallel microbore hollow fiber enzyme reactor platform for online O-labeling: Application to lectin-specific lung cancer N-glycoproteome. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\| PubMed ID: 30292950	2018
Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Using 2D vs. 3D Culture in Colorectal Cancer Cells. Biomolecules\| PubMed ID: 31581581	2019
iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS. Scientific reports\| PubMed ID: 31727937	2019

Dukjin Kang

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