Are Compounds Membrane-Associated or Present in the Cytosol? A Study Using Polyphenols in a Colon Carcinoma Cell Line Model

Abstract

Purpose We investigated the cytosolic and membrane-associated contents of polyphenols after 4 hours of incubation (50 μM of each polyphenol) in the colon carcinoma cell line T84 using a novel, rapid, and convenient method based on permeabilization of the cell membrane using digitonin. The colon carcinoma cell line was used to investigate the intestinal uptake of polyphenols present in apple products. Recent Findings The results showed that hydroxycinnamic acids (caffeic and 5-caffeoylquinic acid) were only detected in the cytosolic fractions. In contrast, 0.3 to 8.2% of the initial concentrations (50 μM) of the flavonoids phloretin, quercetin, phloretin 2′-O-glucoside, and quercetin 3-O-rhamnoside were found in the membrane-associated fractions. In the cytosolic fractions, 0.2–2.9% of these compounds were detected, corresponding to 25 to 40% of the total cell-associated (cytosolic plus membrane-associated fractions) polyphenol content. Summary Our results showed that after uptake, polyphenols were present in the cytosolic fraction of the cells as well as associated with the cell membrane. The presented method provides a useful in vitro tool for determining biologically active compounds in cellular fractions.

Toward Realistic Dosimetry In Vitro: Determining Effective Concentrations of Test Substances in Cell Culture and Their Prediction by an In Silico Mass Balance Model

“…For many purposes, C Cell may be a sufficient proxy and refinements by, e.g., using free C Cytosol may only yield improvements within the experimental error of measuring the concentrations. The current study indicates that two methods provide comparable concentrations: (i) trypsinization and a following disruption of cells by thawing and freezing cycles and (ii) lysis with a digitonin solution . This needs to be verified by further studies, including controls with buffer, and addressing the possible wash out effect.…”

“…The current study indicates that two methods provide comparable concentrations: (i) trypsinization and a following disruption of cells by thawing and freezing cycles 12 and (ii) lysis with a digitonin solution. 46 This needs to be verified by further studies, including controls with buffer, and addressing the possible wash out effect.…”

“…Several methods have been described to prepare samples for the measurement of cell-associated chemical concentrations. These include using detergents, , freezing and thawing cycles, ultrasonication, , and liquid homogenization . One of the consequences of each method is the resulting sample may or may not contain plasma membranes together with chemicals that may have bound to the outside of the cells.…”

“…For potential differentiation between the intracellular and membrane-bound test chemical, an additional experiment was performed with APAP, CAF, FLU, and KET as model compounds adapted from Deusser et al 46 and Kaiser et al 47 Balb/c 3T3 cells were treated with the same concentrations of APAP, CAF, FLU, and KET for 48 h as described in the previous section. After 48 h of incubation, the culture medium was removed, and the cell layer washed twice with 10 mL of PBS.…”

Review: Harmonised in vitro digestion and the Ussing chamber for investigating the effects of polyphenols on intestinal physiology in monogastrics and ruminants

“…Cell line/Tissue References Cell lines T84 Schuier et al, 2005;Bergmann et al, 2009;Rogoll et al, 2010;Deusser et al, 2020Caco-2 Steinert et al, 2008Scherbl et al, 2014;Biolley et Abbreviation: SI, small intestine (both jejunum and ileum).…”