Transcriptional regulation and impact of ABC-transporters in intestinal cell lines

The intestine has an important role in facilitating and limiting absorption of drugs. Since the intestine forms a barrier to the external, epithelial cells have several properties to protect the body from microorganisms and toxins, and to control uptake of xenobiotics. This barrier system consists of tight-junction proteins which minimise paracellular uptake and on the other hand of membrane transport proteins and metabolic enzymes, which regulate transcellular uptake. Membrane transporters are very important in regulating drug absorption, distribution and extrusion. They are expressed in many tissues and regulate transport of endogenous and exogenous substances. ATP-binding cassette transporters (ABC-transporters) form a huge class of membrane transporters which are involved in drug transport. They exhibit large substrate specificity and effectively regulate drug absorption in the intestine. Induction or inhibition of these transporters may influence drug bioavailability of orally ingested drugs. The aim of the thesis was to investigate the impact of ABC-transporters on intestinal drug absorption. The second purpose was to gain insight into transcriptional regulation of these transporters. Two highly expressed ABC-transporters in the intestine, ABCB1 (P-gp, MDR1) and ABCG2 (BCRP), which have a broad and partly overlapping substrate specificity were of special interest in this thesis. The impact of P-gp and BCRP on transport of substrates, which are transported by both transporters, was investigated in the intestinal cell line Caco-2. A stable cellular in vitro system with single and concomitant knock-down of P-gp and BCRP was established using the method of mRNA silencing. Stable Caco-2 clones with selective, single knock-down of P-gp (siMDR1) and BCRP (siBCRP) and with concomitant knock-down of both transporters (cosilencing) were generated and characterised on the level of mRNA and protein expression. Additional functional characterisation was performed by transport and efflux studies using selective substrates of each transporter (chapter 3.1). Compounds which are substrates of P-gp and BCRP were examined in this Caco-2 cell system by transport studies. The tyrosin-kinase inhibitor imatinib and the anti-retroviral drug abacavir are described to be substrates of both, P-gp and BCRP. Bidirectional transport of imatinib was observed in Caco-2 wild-type and BCRP-silencing clones, but no bidirectional transport was seen in P-gp- and co-silencing clones. Since bidirectional transport means active transport processes, these results might suggest that P-gp possibly compensates transport in clones where only BCRP is silenced but BCRP does not when P-gp is silenced. We therefore concluded that P-gp might play a more important role in intestinal imatinib transport, but disturbing factors such as possible silencing off-target effects of our cell system have to be ruled out in further studies. Transport of abacavir showed a similar tendency as imatinib, but effects were only small and further evaluations have to be performed (chapter 3.2). Regulation of P-gp in the intestinal cell lines Caco-2 and LS180 was investigated. Since budesonide is a frequently used glucocorticoid in inflammatory bowel disease (IBD), we investigated how budesonide influences P-gp expression in these intestinal cell lines. We observed differential effects in the two intestinal cell lines; budesonide showed an induction of P-gp in LS180 cells and a downregulation in Caco-2 cells. Expression levels of nuclear receptors revealed high expression of pregnane X receptor (PXR) only in LS180 cells and exclusive expression of glucocorticoid receptor (GR) in Caco-2 cells. Mifepristone, an anti-glucocorticoid, could not reverse the downregulation of P-gp by budesonide in Caco-2 cells. In PXR-transfected Caco-2 cells the budesonide-mediated downregulation of P-gp was abolished. Furthermore the expression of cytochrome P450 3A4 (CYP3A4), another target gene, was induced in PXR-transfected Caco2 cells after budesonide treatment. Therefore, the induction of P-gp by budesonide in LS180 cells probably is mediated via PXR. However, the mechanism of the downregulation in Caco-2 cells still remains unclear, but GR does not seem to be involved (chapter 4.1). In a collaboration, interactions of budesonide with the PXR-target gene CYP3A4 was investigated in comparison to other glucocorticoids. Induction studies in LS180 cell line and in a hPXRtransactivation assay showed, that budesonide significantly induced intestinal CYP3A4 while other glucocorticoids did not. In vivo data in mice showed that budesonide and dexamethasone induced intestinal CYP3a11 while only dexamethasone induced liver CYP3a11. These data indicated that budesonide has the potential to induce intestinal PXR target genes but only to a small extent and therefore, the risk for interactions seems to be low (chapter 4.2). Caco-2 cells represent a good in vitro model for the small intestine but do not express functional active PXR. Therefore we aimed to establish a Caco-2 cell model with stable transfected PXR. We successfully transfected Caco-2 cells with PXR. Induction of PXR target genes P-gp and CYP3A4 was achieved after induction with the PXR-ligand rifampicin. However, the effects were not as pronounced as in LS180 cells and therefore additional investigations are indicated (chapter 4.3). In further studies, effects of curcuma longa L. extracts and single curcuminoids on transcriptional regulation of ABC-transporters, CYP enzymes and pro-inflammatory proteins were investigated. Curcumin is described as anti-inflammatory agent and is discussed as possible therapy for intestinal inflammation. Our data in tumor-necrosis-factor-α (TNF-α) induced LS180 cells indicate that curcuminoids reduce mRNA expression of different proteins involved in inflammation such as iNOS, TNF-α and COX-2. Additionally, the curcuma extract was shown to directly inhibit cyclooxygenase-2 (COX-2) activity (chapter 5.2). Curcuma extracts and curcuminoids showed no relevant effects on P-gp and CYP mRNA expression in LS180 cells indicating no interaction potential of curcuminoids and curcuma extracts on the level of transcriptional regulation (chapter 5.1). Effects of curcuma extracts and curcuminoids on inflammatory proteins and ABC-transporters or CYP expression have to be confirmed in vivo.