Interaction of hepatic uptake transporters with antineoplastic compounds and regulation of the expression of organic cation transporter 3 in renal carcinoma cells
von Venkata Marada
Datum der mündl. Prüfung:2015-01-15
Betreuer:Prof. Dr. Gerhard Burckhardt
Gutachter:Prof. Dr. Jürgen Brockmöller
Gutachter:Prof. Dr. Heidi Hahn
EnglischThe ability of a compound to exert its antineoplastic activity is determined by the amount of its accumulation inside the cell, a process largely dependent on the transporter proteins which are responsible for the passage of compounds into and out of the cell. The present study is focussed on the uptake transporter proteins; their interactions with antineoplastic compounds routinely used in cancer chemotherapy, and the regulation of expression of one such uptake transporter protein, organic cation transporter 3 (OCT3). The interactions of four such uptake transporters, that are predominantly expressed in liver namely, organic anion transporter 2 (OAT2), sodium taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3), were analysed in stably transfected human embryonic kidney cells. The transporter proteins were functionally characterized using [³H] model substrates and the uptake of this model substrate was followed in the presence of 100 μM of the antineoplastic compounds. The antineoplastic compounds which were able to inhibit the uptake of model substrate by 60% of buffer control were chosen for further analysis of interaction. No compound could inhibit the NTCP mediated estrone-3-sulfate uptake by 60% of buffer control. The affinity (Ki value) of the transporter proteins for the compounds that inhibited the uptake of model substrate by 60% of buffer control was determined by Dixon-plot analysis. OAT2 was found to strongly interact with bendamustine, irinotecan and paclitaxel with Ki values of 43.3 μM, 26.4 μM, and 10.4 μM, respectively. OATP1B1 interacted with vinblastine and paclitaxel, with Ki values of 10.2 μM and 0.84 μM, respectively. OATP1B3 interacted with chlorambucil, mitoxantrone, vinblastine, vincristine, paclitaxel, and etoposide with Ki values of 37.4 μM, 3.1 μM, 18.6 μM, 17.6 μM, 1.8 μM, and 13.5 μM, respectively. From the IC50 values generated, the possibility of these interactions to contribute to potential drug-drug interactions was calculated. Furthermore, as mentioned above, the regulation of expression of OCT3 in four renal carcinoma cells (A498, ACHN, 786-O, and LN78) with variable OCT3 expression, was analysed at the epigenetic and post-transcriptional levels. Using inhibitors for the processes of histone deacetylation and DNA methylation, the contribution of these processes was validated. It was found that they do not account for the huge difference of expression of OCT3 found between A498 and ACHN cells. In addition, the methylation status of the promoter region of OCT3 was analysed by Ion Torrent sequencing. There was no considerable difference between the methylation status of the promoter regions tested in the four renal carcinoma cell lines. The post-transcriptional regulation of OCT3 by microRNAs was also analysed. MicroRNAs that have the ability to bind to 3′ untranslated region of OCT3 were obtained from in silico prediction programs and the expression of these microRNAs was analysed by qRTPCR. Two microRNAs, hsa-mir-204 and hsa-mir-143, were selected as they showed differential expression in A498 and ACHN cells. The levels of these microRNAs were altered in these cells using small molecules called microRNA mimics and antimirs, and the expression of OCT3 was followed. However, no correlation was observed between the expression levels of these microRNAs and OCT3. In this direction, the search for potential OCT3 regulators was pursued by the next generation sequencing of genome wide microRNA analysis. From the results it is clear that a many microRNAs are differentially expressed in the four renal carcinoma cells. To make more advances in the search for microRNAs which are directly or indirectly involved in the regulation of OCT3, transcriptome analysis from the same RNA samples is being performed. This approach is best suited to dissect any factors involved in the transcriptional as well as post-transcriptional regulation of OCT3.
Keywords: Regulation of organic cation transporter 3; Next gen sequencing; microRNA analysis; epigenetic and post transcriptional mechanisms of gene regulation; Uptake transporter proteins; antineoplastic compounds; drug-transporter interactions