Impact of Organic Anion Transporter 3 (OAT3) on Bendamustine Uptake of Lymphoma Cells.
Authors: Hagos Yohannes , Wulf Gerald G. , Shnitsar Vladimir , Hundertmark Philip , Gupta Shvangi , Burckhardt Gerhard 
Published: October 2019

Abstract
One main problem of tumor therapy is the resistance of malignant cells to cytostatics due to high expression of efflux transporters. Whereas the role of these efflux transporters for tumor cell resistance is well established, little is known about uptake transporters, which may increase the sensibility of tumor cells for cytostatics. In the present study we addressed the interaction of cytostatics established for the treatment of lymphoma, namely melphalan, chlorambucil or bendamustine with human Organic Anion Transporter (OATs), which belong to the Solute carrier (SLC) gene family. We selected these cytostatics, because they show structural similarity to p-aminohyppurate (PAH), the model substrate of OATs. OATs are mainly expressed in the kidney, where they are responsible for the excretion of endogenous and exogenous organic anions like urate or various drugs e.g. diuretics. Initially, we examined the cis-inhibitory effect of melphalan, chlorambucil and bendamustine on OAT1-mediated [3H]PAH uptake as well as OAT3- and OAT4- mediated [3H]estrone sulfate uptake in HEK293 cells, which were stably transfected with these transporters. Melphalan did not show any significant inhibitory effect on all tested OATs. 100 μM chlorambucil reduced OAT1-, OAT3- and OAT4-mediated uptake of PAH or estrone sulfate down to 14.6 ± 0.17%, 16.3 ± 4.0% and 66.0 ± 1.4%, respectively. 100 μM bendamustine inhibited only OAT3-mediated estrone sulfate uptake up to 91.9 ± 0.5% compared to control cells. OAT1- or OAT4- facilitated transport of PAH and estrone sulfate remained unchanged by bendamustine, suggesting that bendamustine interacts exclusively with OAT3. To determine the affinity of OAT3 for bendamustine and chlorambucil, we performed concentration dependent inhibition of OAT3-mediated estrone sulfate uptake and calculated the Ki values for both cytostatics. Dixon-Plot evaluation confirmed a competitive inhibition of OAT3 by bendamustine as well as chlorambucil. The results demonstrated higher affinity of OAT3 for bendamustine with a Ki value of 2.7 μM than for chlorambucil, showing a Ki value of 38.2 μM. To elucidate the expression of OATs in lymphoma cell lines, we performed RT-PCR experiments. Our data demonstrate high expression of OAT3 in all cell lines compared to lymphocytes isolated from a normal person. No expression of OAT1 and OAT4 was observed any lymphoma cell lines. The expression of OAT3 in B-cell lymphoma cell lines Karpas, Raji, SudHL4 and T-cell lymphoma cell lines L428, Jurkat and Hut78 was quantified by real time PCR. The highest expression of OAT3 was observed in the order Jurkat>Hut78>SudHL4>L428>Raji>Karpas. The expression of OAT3 was confirmed by real time PCR in four patients with chronic lymphocytic leukamia. OAT3- dependent cytostatic effects of bendamustine was examined by [3H] thymidine incorporation. 30 min incubation of OAT3-expressing HEK293 cells with 10, 50 or 100 μM bendamustine significantly reduced the proliferation of transfected versus non-transfected cells. We conclude that the molecular background for the cytostatic efficiency of bendamustine in lymphoma cells is due to 1) the expression of OAT3 in lymphoma cells and 2) a the high affinity of OAT3 for bendamustine.