Abstract

Proteasome inhibitors as bortezomib have been included in standard treatment regimens in several hematological malignancies, especially multiple myeloma. As resistance mechanisms depending on MDR1 expression have not been evaluated in detail for proteasome-inhibitors it was the aim of our study to investigate resistance of cells to Bortezomib and MLN-273 conferred by MDR1.

To address that question K562Dox cells have been long-term cultured in low concentrated doxorubicin to obtain MDR1 overexpression. These cells were stably transfected with a Sleeping-Beauty vector system carrying an siRNA targeting the mRNA of PgP. With that approach, PgP was reduced to 10% compared to the wild-type cell line. PgP-positive and PgP-negative cells were exposed to bortezomib and MLN-273 and proliferation measured was in a 3H-Thymidine incorporation assay. This showed a dose-dependent reduction of DNA-synthesis in cells exposed to Bortezomib, regardless of PgP-expression. In contrast, MLN-273 showed a PgP-dependency by inhibiting cells expressing PgP (K562Dox: EC 50 of 5,6 ng/ml and vector control cells K562Dox-Co: EC 50 of 29 ng/ml) less than MDR1-knock-down cells (K562Dox-siMDR1: EC50 of 0.3 ng/ml). In MDR1 positive cells this growth inhibition was reveresed by addition of verapamil, a MDR1 blocking substance (K562Dox: EC50 of 2.4 ng/ml and K562Dox-Co: EC50 of 2.8 ng/ml). This growth inhibitory effect was due to apoptosis induction, which has been shown by staining for Annexin V and propidium iodide. The direct effect of MDR1 on proteasome activity was measured by western blotting IkB╬▒, a substrate of the proteasome, which was still present in MLN-273 treated MDR1-negative cells but not in MDR1-positive cells incubated with MLN-273.

These results show that MLN-273 highly likely is a substrate for MDR1, which may gain clinical significance in treating hematological malignancies in the future.

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