The Tryptanthrin Uptake Transporters In Caco-2 Cell Model And The Establishment Of Caco-2 Model For Short-term Culture Method

Tryptanthrin, an indole quinazoline alkaloid with multiple medical activities, has been recently under preclinical development as an anti-tuberculosis and anti-tumor drug. The aims of this study are to characterize the intestinal transport of tryptanthrin in Caco-2 cells, to determine whether P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) are involved in the absorption and transport of tryptanthrin, and to evaluate the potential influence of tryptanthrin on the function of P-gp and MRP2. Transport assays of tryptanthrin were performed in Caco-2 monolayers with the supplement of P-gp and MRP2 inhibitors. Transport assays of P-gp and MRP2 substrates were also performed in the presence of tryptanthrin. The effect of tryptanthrin on the expression of P-gp and MRP2 was analyzed by reverse transcriptase-PCR. Both absorption and secretion of tryptanthrin were concentration-independent at a low concentration range of 0.8-20μM. The apparent permeability (Papp) for the apical (AP) to basolateral (BL) was 6.138±0.291×10-5. The ratio of Papp (BLâ†'AP) to Papp(APâ†'BL) was 0.77, suggesting greater permeability in the absorptive direction. Both the P-gp inhibitor, verapamil, and the MRP2 inhibitor, glibenclamide, didn't affect the efflux transport of tryptanthrin. The efflux transport of the P-gp substrate, digoxin, and the MRP2 substrate, pravastatin sodium, decreased when tryptanthrin was present. However, tryptanthrin didn't change the expression of P-gp and MRP2. Overall, tryptanthrin was well absorbed across the Caco-2 monolayers, and its transepithelial transports were dominated by passive diffusion. Tryptanthrin was not a substrate, but a potential inhibitor of P-gp or MRP2.Caco-2 cells (human colonic cell line), is a widely accepted in vitro model for drug absorption and metabolism studies. The standard 21-day culture system had a high requirement of time and material due to the long culture period. The aim of this study was to develop new protocol for a short-term Caco-2 monolayer culture system with butyrate acid. In our study, Caco-2 cell monolayer model was established with different culture systems and evaluated by morphology feature using inverted microscope and transepithelial electrical resistance (TEER) assay. Additionally, the model was further tested for the activity of alkaline phosphatase and the apparent permeability (Papp) of standard compound fluorescein. Meanwhile, the RNA level of transporters P-gp and MRP2 was examined. Caco-2 cell monolayer established with the short-term culture system showed good integrality of cell monolayer, cell differentiation (reflected by TEER, expression of alkaline phosphatase and cell monolayer morphology), and the Papp value of standard compound fluorescein in the established Caco-2 cell model was satisfactory, too. In conclusion, the short-term established Caco-2 monolayer can be used to study the intestinal absorption of orally administrated chemical components and their absorption mechanism.