Title

 

Authors

E. Turrini^1,2, S. Haenisch¹, S. Laechelt¹, O. Bruhn¹, P. Hrelia², I. Cascorbi<sup>1

1</sup>Institute of Experimental and Clinical Pharmacology, University of Kiel, Germany
²Dept. of Pharmacology, University of Bologna, Italy

 

Abstract

Introduction– Imatinib mesylate (IM) is the first example of targeted therapy among tyrosine kinase inhibitors and widely accepted as gold standard in the treatment of chronic myeloid leukemia (CML) (de Kogel GE et al. 2007). Despite emerging success in CML therapy, refractoriness and resistance have been observed in a significant proportion of patients. Over-expression of Bcr-Abl gene, mutation on ATP-binding site or differential expression of efflux transporters such as ABCB1 and ABCG2, as critical determinant of intracellular drug levels, may be responsible for resistance to IM (Garofalo et al. 2008). Aim – The purpose of this study was to investigate if miRNAs, as responsible of post-transcriptional gene regulation mechanisms, are altered under IM-treatment and could therefore contribute to the mechanisms of IM-resistance. Materials and methods – An in vitro model was established on chronic myeloid leukemia cells (K-562) under acute and chronic IM treatment. miRNAs profiles were analyzed using Taqman Arrays and in silico search was performed for miRNAs showing altered expression after IM treatment. For these miRNAs the results obtained from the screening were confirmed by individual TaqMan Assays. mRNA level was checked using TaqMan realtime PCR and protein expression was analyzed by Western blotting. Luciferase assay was performed to confirm the role of candidate miRNAs. Results - ABCG2 protein was 7.2-fold elevated after long-term treatment with 0.3 µM imatinib and decreased gradually at higher concentrations while ABCB1 protein was not expressed in any condition of treatment. The combined analysis of miRNA arrays and in silico search revealed two miRNAs both having ABCG2 as potential target, namely miR-212 and miR-328. They inversely correlated with ABCG2 expression after chronic treatment with IM. At IM chronic concentrations higher than 1 µM, both miRNAs were up-regulated and at the concentration of 3 µM both in a statistically significant way: miR-328 was 4.36+/-2.04 fold up-regulated (p=0.050) and miR-212, 2.36+/-0.60 fold (p=0.050). Short-term treatment induced ABCG2 protein in a dose-dependent manner as well and caused a down-regulation of miR-212 (but not miR-328) at all tested concentrations (p=0.050). Luciferase reporter assays confirmed the binding of miR-212 to the 3’-UTR region of ABCG2. Furthermore, the microRNA-mediated down-regulation of ABCG2 after IM treatment could be abolished by transfection of anti-miRNAs. Conclusions - The results of this study suggest that resistance against IM could be mediated by a miRNA dependent mechanism.

References:
de Kogel CE et al. (2007). Oncologist. 12:1390-1394
Garofalo M. et al. (2008). Curr Opin Pharmacol. 8:661-667