Bortezomib resistance in a myeloma cell line is associated to PSMβ5 overexpression and polyploidy

doi:10.1016/j.leukres.2011.09.011

Leukemia Research

Volume 36, Issue 2, February 2012, Pages 212-218

https://doi.org/10.1016/j.leukres.2011.09.011 Get rights and content

Abstract

Bortezomib is a proteasome inhibitor important to the therapy of multiple myeloma (MM), though a number of patients show resistance to this drug. To study the cellular basis of this resistance we have generated a MM cell line displaying enhanced (5–6-fold) resistance to bortezomib by serial cultivation of RPMI 8226 cells with increasing concentrations of this drug. Bortezomib-resistant cells (8226/7B) became bigger in size than parental cells and nearly doubled the amount of DNA per cell, evolving from hypotriploidy to near-tetraploidy. 8226/7B displayed lowered Noxa accumulation and reduced caspase-3 activation in response to bortezomib. Resistant 8226/7B cells overexpressed the PSMβ5 proteasome subunit, the molecular target of bortezomib, both at the mRNA and protein level. No mutations were detected in the PSMβ5 gene. Bortezomib-resistant cells were roughly as sensitive as parental cells to other chemotherapeutic drugs, including doxorubicin, melphalan, vincristine, BMS-214662 and BMS-345541. 8226/7B cells showed partial and high cross-resistance to the proteasome inhibitors epoxomicin and MG-132, respectively. Co-treatment with the histone deacetylase inhibitor trichostatin A (TSA) potentiated bortezomib-induced apoptosis in parental RPMI 8226 cells but did not revert bortezomib resistance in 8226/7B cells. Therefore, treatment of bortezomib-refractory myeloma with drugs targeting molecular structures other than proteasome seems to be the more suitable therapeutic strategy to overcome bortezomib resistance.

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.

Section snippets

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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Bortezomib resistance in a myeloma cell line is associated to PSMβ5 overexpression and polyploidy

Abstract

Introduction

Section snippets

Materials

Characterization of bortezomib-resistant 8226/7B cells

Discussion

Conflict of interest statement

Acknowledgements

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