Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Abstract

Caco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

Analyzing the metabolic fate of oral administration drugs: A review and state-of-the-art roadmap

“…Specifically, CYP3A4 and CPR genes were cloned into HAC vectors in CHO cells using the Cre-loxP system, and then CYP3A4-CPR-HAC was transferred to Caco-2 cells by chromosomal transfer technology (Ohta et al, 2020). PiggyBac transposon isolated from Trichoplusiani also serves as a tool to overexpress CYP3A4 in Caco-2 cells (Ichikawa et al, 2021). pPB-TRE3G-CYP3A4 and piggyBac transposase vectors were co-transfected into Caco-2 cells and subjected to immunofluorescence analysis (Figure 2).…”