Effect of green tea extract on expression of proteins involved in drug transport and metabolism and on the expression and secretion of the chemokine interleukin-8 in intestinal cell lines

Pharmacokinetic interactions often occur as a result of changes in the functional expression of drug-metabolising and transporting proteins. In recent years, interactions of herbal medicines with synthetic drugs have come into focus and drug-interactions for more than 150 herbal medicines have been reported including the most prominent hypericum. Importantly, also dietary drug-food interactions have been observed, grapefruit juice presenting the most famous herein.
Systemic elimination and uptake of xenobiotics is regulated, predominantly in the liver but also in other extrahepatic tissues including the gut, by their biotransformation and excretion. In the metabolic conversion of drugs the cytochrome p450 (CYP) enzyme family is the major catalyst of phase I drug biotransformation reactions. The isoenzymes most commonly involved in drug metabolism include CYP3A4, which is also the most abundantly expressed CYP, and CYP1A2. The absorption, distribution, and excretion of endogenous and ingested substances is mediated by membrane transporter proteins. In this context, the transporters P-glycoprotein (P-gp) and multidrug-resistance associated protein 2 (MRP2), both located in the apical membrane of enterocytes, exert a key role in the gastrointestinal tract. The interplay of CYP3A4 and P-gp in limiting oral drug availability is emphasised by their striking overlaps of substrates and inhibitors. Overexpression of particular drug transporters can even lead to the phenotype of drug resistance, which is often observed in cancer patients. However, therapy resistance has also been observed in other diseases such as inflammatory bowel disease (IBD).
Green tea (Camellia sinensis (L.) O. KUNTZE, fam. Theaceae) is one of the most popular beverages in the world and has been reported to exert beneficial effects on several life-style related diseases. These have been ascribed to several potential activities of green tea or single constituents thereof, such as antiinflammatory and anticancer activities. However, a potential modulation of drug metabolism by green tea described in the literature remains controversial.
The aim of the thesis was to investigate in vitro the influence of a commercially available green tea extract (GTE) on the gastrointestinal drug metabolism and transport. These results might be of therapeutical relevance and/or useful in the prediction of the outcome of future clinical trials. Therefore, the effect of GTE on the expression of different CYPs and membrane transport proteins was assessed in two established intestinal cell lines, LS-180 and Caco-2 cells. Additionally, the influence of GTE on metabolic or transport activity was also investigated in vitro.
Green tea has been reported to modulate the generation of reactive oxygen species (ROS) under cell culture conditions. This effect may lead to the generation of artefacts, especially in mRNA induction experiments. In view of this, preliminary experiments excluded an influence of GTE on hydrogen peroxide concentrations in the medium (Chapter 2).
The effect of GTE, or constituents thereof, on the mRNA expression of the two efflux pumps P-gp and MRP2 was investigated in LS-180 cells (Chapter 3). mRNA expression levels were determined by quantitative RT-PCR. Due to their location in the apical membrane of enterocytes, these transporters exert the first obstacle in the uptake of orally ingested xenobiotics. At low concentrations GTE was found not to modulate the mRNA expression of P-gp or MRP2. Functional assays using a cell line stably overexpressing human MRP2 (MDCK-MRP2) showed an inhibition of the extrusion of methotrexate, a MRP2 substrate, by GTE at high concentrations (Chapter 3). In concentrations corresponding to their content in GTE, none of the green tea constituents tested did exert this inhibitory activity on MRP2 function.
In two intestinal cell lines, Caco-2 and LS-180, the effect of GTE on CYP1A1 and CYP1A2 mRNA expression was assessed using quantitative RT-PCR (Chapter 4). CYP1A2 mRNA expression was inducible by GTE in a dose-dependent manner in both cell lines. GTE influenced CYP1A1 expression differentially in these cell lines. mRNA expression of CYP1A1 was only induced in the Caco-2 cell line. These data were confirmed on the protein level by western blot experiments. No effect of GTE on CYP3A4 mRNA expression levels was observed in LS-180 cells. However, GTE showed a dose-dependent inhibition of CYP1A2 and CYP3A4 metabolic activity in vitro using luminescent substrates.
These data demonstrate that dietary substances might have an impact on therapeutic effectiveness by a possible influence on drug uptake and metabolism. Additionally, the chemopreventive character implicated for GTE might be explained by an inhibition of the generation of carcinogenic intermediates by CYP1A2.
The gut mucosa represents a site of active immunological activity potentially involved in the initiation of an immune response to inflammatory stimuli. Intestinal epithelial cells are capable to secrete proinflammatory mediators like chemokines and cytokines in response to such stimulants. Chemokines are involved in the regulation of leukocyte migration from the blood into the inflamed tissue and play an important role in the generation of the respiratory burst in these cells. A prominent member of the chemokines is interleukin (IL)-8, which acts on neutrophils. An increased expression of this chemokine has been observed in enterocytes in inflammatory bowel diseases and in several gastrointestinal cancers.
Antiinflammatory and chemopreventive activities have been described for green tea in the literature. Therefore, in this thesis we wanted to clarify the effect of GTE on the potential of enterocytes to produce proinflammatory mediators. Due to inconclusive results concerning different cytokines or enzymes involved in arachidonic acid metabolism (Chapter 6.3), the focus was laid on IL-8 expression and secretion in Caco-2 cells (Chapter 6.1). Induction of IL-8 mRNA expression by GTE at highest dose was shown by quantitative RT-PCR. While this lead to an increase of intracellular IL-8 protein concentration, the excretion of IL-8 was specifically inhibited by GTE as demonstrated by EIA (Enzyme-linked immunosorbent assay) detection. GTE dose-dependently inhibited the induction of IL-8 secretion by the proinflammatory stimulus IL-1β. However, these results give evidence for an anti-inflammatory activity of GTE in enterocytes, which might result in a decreased infiltration of neutrophils in the inflamed tissue. Thus, GTE might be useful for acute treatment of intestinal inflammation.