Bortezomib resistance in a myeloma cell line is associated to PSMβ5 overexpression and polyploidy
Introduction
The proteasome inhibitor bortezomib (Velcade) has emerged in last years as a very useful drug for the treatment of relapsed and refractory MM, as well as in elderly patients [1], being also useful in first-line therapy [2]. The overall response rate to bortezomib in phase III trials was 43% [3], suggesting that some patients exhibit resistance to bortezomib or develop secondary resistance. In fact, it is estimated than nearly a third of MM patients never respond to treatment with bortezomib [4]. Thus, understanding the mechanisms of resistance to this proteasome inhibitor will enable a more rationale use of bortezomib as well as the design of synergistic drug combinations or treatment with alternative drugs. It was recently reported the generation of a human myelomonocytic cell line with acquired resistance to bortezomib [5]. The resistance was associated to a mutation in the psmβ5 gene (β5 subunit of proteasome) that lead to a protein with impaired binding of bortezomib and thus decreased proteasome inhibition. Later, three myeloma cell lines with acquired resistance to bortezomib have been generated [6], [7]. Similar to found in the resistant leukemia cell line, bortezomib resistance in two of the MM cell lines was also associated to a mutation in psmβ5 gene whereas increased levels of PSMβ5 protein were found in the other [6].
As a model to analyze alternative molecular therapies for MM cells displaying acquired resistance to bortezomib, we recently generated a cell line, 8226/7B, five times more resistant to bortezomib than parental RPMI 8226 myeloma cells [8]. In the present work we have characterized the cellular and molecular basis of this resistance and analyzed its sensitivity to other chemotherapeutic drugs. We also propose a putative mechanism to explain the induction of bortezomib resistance in some MM cells.
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Materials
The proteasome inhibitor bortezomib (Velcade) was kindly provided by Millenium Pharmaceuticals (Cambridge, USA). The proteasome inhibitor epoxomicin, the DNA-damaging agents doxorubicin and melphalan and the redox-sensitive dye MTT were from Sigma (Madrid, Spain). The farnesyltransferase inhibitor BMS-214662 was a gift from Bristol-Myers (Princeton, USA) and the microtubule-destabilizing agent vincristine, the IκB kinase (IKK) inhibitor BMS-345541, the proteasome inhibitor MG-132 and the
Characterization of bortezomib-resistant 8226/7B cells
8226/7B cells were generated from RPMI 8226 human myeloma cells by continuous culture for 18 months in the presence of stepwise increasing concentrations of bortezomib (from 2 to 100 nM). This cell line was chosen since it exhibited a greater IC50 relative to other MM cells [8]. Compared with parental cells, 8226/7B were less sensitive to bortezomib-induced growth inhibition, determined by the MTT-assay (IC50: 15 nM vs 75 nM, Fig. 1) and more resistant to apoptosis (LD50: 15 nM vs 85 nM), determined
Discussion
The proteasome inhibitor bortezomib is an important drug for the current therapy of MM. However, resistance to this proteasome inhibitor frequently occurs. Identifying the mechanisms of resistance is important to develop complementary or alternative treatments. To analyze this problem, we generated a cell line, 8226/7B, displaying enhanced resistance to this proteasome inhibitor by culturing RPMI 8226 cells with increasing concentrations of bortezomib for several months [8]. The 8226/7B
Conflict of interest statement
The authors have no relevant conflict of interest to disclose.
Acknowledgements
This study was supported by grants (SAF2010-14920 and ISCIII-RTICC RD06/0020) from Ministerio de Ciencia e Innovación, Spain.
Contributions. PB generated the resistant myeloma cell line and performed main experiments and analysis of apoptosis and drafted the manuscript. PG-M carried out molecular biology analysis and cDNA sequencing. IM supervised flow cytometric and protein analysis, analyzed the data and drafted the manuscript; JN designed experiments, wrote the final version of the paper and
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