Development of a Cell-Based Assay for Identifying KCa3.1 Inhibitors Using Intestinal Epithelial Cell Lines

2020 ◽  
pp. 247255522095066
Author(s):  
Chanon Jakakul ◽  
Phongthon Kanjanasirirat ◽  
Chatchai Muanprasat
Keyword(s):  
Epithelial Cell ◽  
High Throughput Screening ◽  
Intestinal Epithelial Cell ◽  
Therapeutic Potential ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Screening Assay ◽  
Epithelial Cell Lines ◽  
A Cell ◽  
Screening Platform

Inhibition of the KCa3.1 potassium channel has therapeutic potential in a variety of human diseases, including inflammation-associated disorders and cancers. However, KCa3.1 inhibitors with high therapeutic promise are currently not available. This study aimed to establish a screening assay for identifying inhibitors of KCa3.1 in native cells and from library compounds derived from natural products in Thailand. The screening platform was successfully developed based on a thallium flux assay in intestinal epithelial (T84) cells with a Z′ factor of 0.52. The screening of 1352 compounds and functional validation using electrophysiological analyses identified 8 compounds as novel KCa3.1 inhibitors with IC50 values ranging from 0.14 to 6.57 µM. These results indicate that the assay developed is of excellent quality for high-throughput screening and capable of identifying KCa3.1 inhibitors. This assay may be useful in identifying novel KCa3.1 inhibitors that may have therapeutic potential for inflammation-associated disorders and cancers.

scholarly journals Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium

1997 ◽  
Vol 273 (6) ◽  
pp. G1349-G1358 ◽  
Author(s):  
Dana J. Philpott ◽  
Cameron A. Ackerley ◽  
Amanda J. Kiliaan ◽  
Mohamed A. Karmali ◽  
Mary H. Perdue ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Barrier Function ◽  
Pathogenic Bacteria ◽  
Intestinal Epithelial Cell ◽  
Cell Function ◽  
Bacterial Toxin ◽  
General Mechanism ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
T84 Cells

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 μg/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 ± 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 ± 7 and 38 ± 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.

scholarly journals Lipopolysaccharide Activates Distinct Signaling Pathways in Intestinal Epithelial Cell Lines Expressing Toll-Like Receptors

2000 ◽  
Vol 164 (2) ◽  
pp. 966-972 ◽  
Author(s):  
Elke Cario ◽  
Ian M. Rosenberg ◽  
Steven L. Brandwein ◽  
Paul L. Beck ◽  
Hans-Christian Reinecker ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial ◽  
Toll Like Receptors ◽  
Epithelial Cell Lines
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