Modulation of function of MDR-associated ABC transporters by chemotherapeutic drugs

The major cause of chemotherapy failure is acquired resistance of cancer cells not only to the prime drug but also to other unrelated ones. There are three main types of ABC transporters in mammals that have been found to cause multidrug resistance (MDR) of cancer cells when over-expressed - MDR1, MRP1, and BCRP (reviewed in Li et al., 2007). Recently it has been reported that expression of ABC transporters could be regulated by mitogen activated protein kinases (MAPKs), namely p38 and ERK subtype (Katayama et al. 2007), and also PI3K/Akt pathway (Liang et al. 2009). Those kinases were found to be able to modulate the activity of some transcription factors that bind into ABC transporters gene promoters, i.e. NFkappaB, API, or p53 (Scotto et al. 2003). We aim to connect effects of selected chemotherapeutics on function of ABC transporters with activity of particular kinase and its targets.

First, we selected chemotherapeutic drugs for their specificity using WST-1 cytotoxicity assay and cell lines over-expressing studied transporters (MDR1, MRP1 and BCRP). In concentrations we further used Roscovitin is a substrate of MDR1 protein, Camptothecin and Actinomycin D are substrates of MRP1 protein, Valinomycin of BCRP protein, and Doxorubicin and Geldanamycin are general substrates of all the studied ABC transporters. We evaluated their effect on function and expression of ABC transporters in A549 cells using "Dye Exclusion Assays" by means of flow cytometry (JC1 was used as a fluorescent substrate of MDR1, Calcein AM for MRP1 and Bodipy-prazosin for BCRP). Results were correlated with Immunocytochemistry and qRT-PCR. The selected cytotoxic drugs were shown to activate studied kinases to the different degrees. One of the most effective activators of stress kinases (JNK, p38 MAPK) was Roscovitin, which was also able to modulate function and expression of the studied ABC transporters. Further we used pharmacologic inhibitors of these kinases to assess how they affect the up-regulated function of ABC transporters. Those inhibitors either potentiated the effect of the chemotherapeutic drug or had not any effects. In the next step we aim to assess the role of the involved transcription factors.

Project is supported by GACR 301/08/0717, AVOZ50040507, and AVOZ50040702 of Academy of Science.