Uptake And Transportation Characteristics Of EGCG Across Caco-2 Monolayers

(-)-epigallocatechin-3-gallate (EGCG) is the most abundant constituents (more than 50%) in tea polyphenol, which is responsible for the health benefits associated with the consumption of green tea. EGCG has been showed to have various compelling biological activities in in vitro and in animal models, which include antioxidant, antitumor, anti-inflammatory, anti-radiation, anti-aging, and improving liver function. On the other hand, studies in pharmacokinetics showed that EGCG has poor bioavailability and very low absorption in vivo. The cellular and molecular mechanisms underlying EGCG absorption in vivo are so far not completely understood.Caco-2 cell line is one of the most popular cell models for elucidating the cellular uptake and transportation properties of a drug in vitro. The Caco-2 cell line originates, from human colon adenocarcinoma. Under appropriate culturing conditions, Caco-2 cells could form differentiated monolayers on the porous filters with many properties, such as the expression of the tight junctions, microvillus and the brush border enzymes, identical to the normal intestinal epithelium.In this study, we used the Caco-2 monolayers to characterize the cellular transportation behaviour of EGCG and its functional interaction with P-glycoprotein and the other multi-drug resistance proteins that expressed on the cell membrane. The results should help explaining why the bioavailability of EGCG is low in vivo and might also give insights into the understanding of molecular mechanisms that lead to a poor cellular absorption of EGCG.1. Establishment and validation of Caco-2 monolayersCaco-2 cells were seeded into Transwell inserts at a density of 3 X 105 cells/cm2 and cultured for 21 days. A TEER value of 345Ωs·cm2 or above was used to represent a well establishment of Caco-2 monolayers. After culturing for 21 days, remarkable microvillus at the apical surface and tight junctions between cells of the Caco-2 monolayers were observed. The Papp values of the positive marker Warfarin and the negative marker Lucifer yellow across Caco-2 monolayers were 1.98×10-5 cm/s and 5.1 X 10-7 cm/s, respectively, including that the Caco-2 monolayers of integrity were formed.To validate the functional properties of the established Caco-2 monolayers, we used Verapamil, a special inhibitor of P-glycoprotein, to examine the expression of P-glycoprotein on the cell membranes. The data showed that Verapamil could enhance the transportation of Rhodamine-123, the substrate of P-glycoprotein, across the Caco-2 monolayers from AP side to BL side. This indicated that the Caco-2 monolayers expressed P-glycoprotein at the apical surface, and P-glycoprotein was playing a role in mediating transcellular transportation.The permeabilities of several reference compounds, which are known absorption in human, upon the cell model were also measured. We found that the permeabilities of these compounds were highly correlated with their absorption values documented for human being with correlation coefficient was 0.806(P=0.003), indicating that data delivered from this in vitro cell model could be used to certain degree to predict the absorption behaviour of a compound in vivo. 2. Absorption and transportation of EGCG at Caco-2 monolayersThe influx amount of EGCG from AP side to BL side of Caco-2 monolayers were significantly correlated with the EGCG concentrations between 100-400μg/mL (r2=0.9907), suggesting that EGCG was uptaked and permeated across the cell membrane in a passive diffusion manner.Meanwhile, Papp values of EGCG from AP side to BL side of the monolayers was less than 10-6cm/s, indicating that cellular absorption of EGCG was poor.3. Interactions of EGCG with the efflux transporters (P-gp and MRP2)To assess possible molecular mechanism underlying the poor uptake and permeability of EGCG, we used two P-glycoprotein inhibitors, Verapamil or Cyclosporin A, or the MRP2 inhibitor, MK-571, to co-treat the cells with EGCG. The results showed that the P-glycoprotein inhibitors could apparently enhance the absorption and increase the Papp values of EGCG from AP side to BL side of the Caco-2 monolayers, while the opposite transportation of EGCG(from BL side to AP side) was reduced. The role of MK-571 on the cellular permeability of EGCG was even stronger. The results suggest that there were functional interactions of EGCG with the efflux transporters on the cell membrane, and furthermore, EGCG might function as the substrate of both P-gp and MRP2. On the other hand, we also assessed the effect of EGCG on the permeability of Rhodamine-123, a known P-glycoprotein substrate, across the cell membrane. The data showed that EGCG could increase the Papp value of Rhodamine-123 from AP side to BL side on the monolayers, suggesting that there is also an inhibitory role of EGCG on the efflux function of P-glycoprotein.