Methods of investigating drug-induced increase in the expression of the drug efflux transporter P-glycoprotein in different cell types

Epilepsy is one of the most common neurological disorders, which affects humans and animals, like dogs and cats. Drug treatment is still the most often used therapy. However, about one third of epileptic patients do not respond to distinct AEDs. The mechanisms of pharmacoresistance are poorly understood. Nonetheless, there are two major hypothesis explaining pharmacoresistance, multidrug hypothesis and drug target hypothesis (Schmidt and Loscher, 2009). According to the first hypothesis, overexpression of multidrug transporters in blood-brain barrier decreases drug uptake in the brain, which lead to insufficient drug concentration needed for effective treatment. An evidence for correlation between overexpression of multidrug transporter and pharmacoresistance came from work by Tishler et al. (Tishler et al., 1995). Group showed upregulation, one of the most examined multidrug transporters, Pgp in epileptic brain tissue. Drug target hypothesis, suggests that alterations in the structure and/or functionality of AED targets in epileptogenic brain regions lead to reduced drug effects (Loscher and Potschka, 2005a). For instance, experimental studies have shown that changes in the structure and function of inhibitory GABA(A) receptors may contribute to drug resistance in epilepsy (Bethmann et al., 2008). Presented thesis was concentrated on multidrug transporter hypothesis, especially on Pgp, as a main principal for pharmacoresistance in epilepsy. In order to overcome the pharmacoresistance in epilepsy, it is relevant to explore the mechanisms that lead to refractory epilepsy. Thus, presented thesis was meant to investigate whether commonly used AEDs might cause overexpression of multidrug transporters resulting in decrease drugs concentration in the brain. For purpose of thesis two cell lines were tested: MDCK epithelial kidney cells and GPNT rat brain endothelial cells. The reason for using those cells was mostly that they are recognized as a model of blood-brain barrier. Further, MDCK cells were used in study on drug transport, GPNT express several endothelial markers and for both cell lines cultering conditions were established. Study in cell lines began from establishing of protocols for Western blotting and uptake assay methods, which are used to investigate expression and functionality of proteins. Cells were treated with antiepileptic drugs for certain period of the time, and then protein expression and functionality were analyzed. Pgp protein in MDCK cells was recognized by anti-Pgp antibody as double bands with molecular weights of approximately 140 kDa and 160 kDa. Both bands seemed to be Pgp proteins that differed in glycosylation. As it was shown, Pgp with molecular weight around 140 kDa is core-glycosylated form of Pgp, the signal around 160 kDa in turn seems to be fully glycosylated, mature form of Pgp. In presented thesis, mature form of Pgp was investigated. Thus, for quantification of the Pgp expression, bands around 160 kDa were taken as signals representing mature proteins. In contrast to MDCK, in GPNT cells anti-Pgp antibody has recognized only single band with molecular weight around 160 kDa. Individual experiments in MDCK and GPNT cells differed in results. The observed variance could be due to particular characteristics of the used cell lines, for instance changes in Pgp expression during the culturing that could depend on passage of the cells. Furthermore, period and beginning of the treatment (differentiation of the cells) might be relevant. In general, treatment with AEDs did not influence Pgp expression in MDCK cells. In GPNT cells, Pgp expression and functionality were not affected as well. However, Pgp was functionally active in GPNT cells, which was improved by significant increase in uptake of Pgp substrates after exposure to Pgp inhibitor tariquidar. No effect on Pgp fuctionality after exposure to AEDs was observed in uptake assays with both Pgp substrates: digoxin and vinblastine. Thus, the results of presented thesis did not indicate any robust effect on Pgp expression and functionality after AEDs treatment. However, GPNT and MDCK cells might be not ideal models for studying drug-induced alterations in Pgp expression, because of high level of endogenous Pgp and other multidrug transporters. More research must be done to verify multidrug transporter hypothesis and find suitable models for Pgp expression investigation.