Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies

Abstract

Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2 cells). Then, we developed Caco-2 cells that stably express CES2 (CES2/Caco-2 cells) and their control Mock/Caco-2 cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2 cells. Furthermore, we also confirmed that CES2/Caco-2 cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2 cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.

Bioengineered human colon organoids within vivo-like complexity and function

“…We next sought to compare our model to human colon carcinoma Caco-2 cells, widely considered the gold standard for assessing oral drug absorption and ADMETox studies 32 . Even though these cells are of colonic origin and share some features with small intestinal enterocytes, their transformed character and genomic instability raise doubts about their use for basic and translational research.…”

“…We next sought to compare our model to human colon carcinoma Caco-2 cells, widely considered the gold standard for assessing oral drug absorption and ADMETox studies 32 .…”

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes

“… 16 – 18 Expression of CES2 in the human intestine is well documented. 19 , 20 To delineate whether CES1 protein is expressed in human intestine, we performed western blot of human small bowel epithelial cell lysates prepared from intestinal resections of patients with inflammatory bowel disease. Both CES1 and CES2 were expressed in these lysates ( Figure 3A ).…”

Establishment of SLC15A1/PEPT1-Knockout Human-Induced Pluripotent Stem Cell Line for Intestinal Drug Absorption Studies

“…The microarray analysis showed that the gene expression levels of carboxylesterase 1 ( CES1 ) were unexpectedly increased by PEPT1 knockout (Figure 3A). It is known that carboxylesterases catalyze the hydrolysis of esters, amides, thioesters, and carbamates 28, 29, 30. In humans, two carboxylesterases, CES1 and CES2, are important mediators of drug metabolism.…”