Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells

Abstract

Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.

Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics

“…Spiked digests were enriched and then analyzed by shotgun proteomics, using nanoHPLC-MS/MS on an Orbitrap Elite mass spectrometer (Thermo Scientific), as described previously . Spectra were matched to SwissProt database for bovine taxonomy (6003 entries) for peptide and protein identification using MaxQuant (v1.6.3.4), as previously described . Details are reported in the Supporting Information section 2 (Shotgun-Proteomics Analysis and Peptide Identification).…”

The Fight against Cancer by Microgravity: The Multicellular Spheroid as a Metastasis Model

“…Proteomics analyses of human colon adenocarcinoma Caco-2 cells exposed to a 2D clinostat revealed 38 and 26 proteins differently regulated by s-µ g after 48 and 72 h. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes, and localization. S-µ g -induced changes in the transcriptional regulation of the NF-kB pathway and CYP27A1 were detectable [ 144 ].…”

Physiological adaptations affecting drug pharmacokinetics in space: what do we really know? A critical review of the literature

“…As far as metabolism is concerned, alterations in the composition of intestinal microflora, and differences in hepatic blood flow velocity and first‐pass hepatic metabolism also play a role in drug absorption and bioavailability. In experimental studies under microgravity conditions, significant changes have been reported in the expression profile of multiple proteins in Caco‐2 cells, a cell line used as a model to evaluate drug permeability and, therefore, absorption of drugs through the intestinal wall (La Barbera et al, 2017). Furthermore, decreased activity of lipid‐hydrolysing enzymes, activation of proteolytic enzymes and impaired hepatic secretion of the biliary lipid complex have been reported in spaceflight experiments (Smirnov, 1986).…”