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Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation

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Abstract

Purpose

Drug resistance presents a major obstacle for the treatment of some patients with chronic myeloid leukemia (CML). Pro-apoptotic ceramide mediates imatinib-induced apoptosis, and metabolism of ceramide by glucosylceramide synthase (GCS) activity, converting ceramide to glucosyl ceramide, might contribute to imatinib resistance. In this study, we investigated the role of ceramide metabolism by GCS in the regulation of imatinib-induced apoptosis in drug-sensitive and drug-resistant K562 and K562/IMA-0.2 and K562/IMA-1 human CML cells, which exhibit about 2.3- and 19-fold imatinib resistance, respectively.

Methods

Cytotoxic effects of PDMP and imatinib were determined by XTT cell proliferation assay. Expression levels of GCS were determined by RT-PCR and western blot. Intracellular ceramide levels were determined by LC–MS. Cell viability analyses was conducted by Trypan blue dye exclusion assay. Cell cycle and apoptosis analyses were examined by flow cytometry.

Results

We first showed that mRNA and protein levels of GCS are increased in drug-resistant K562/IMA as compared to sensitive K562 cells. Next, forced expression of GCS in sensitive K562 cells conferred resistance to imatinib-induced apoptosis. In reciprocal experiments, targeting GCS using its known inhibitor, PDMP, enhanced ceramide accumulation and increased cell death in response to imatinib in K562/IMA cells.

Conclusion

Our data suggest the involvement of GCS in resistance to imatinib-induced apoptosis, and that targeting GCS by PDMP increased imatinib-induced cell death in drug-sensitive and drug-resistant K562 cells via enhancing ceramide accumulation.

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Abbreviations

CML:

Chronic myeloid leukemia

IMA:

Imatinib

MDR:

Multiple drug resistance

K562/IMA-0.2 and K562/IMA-1:

K562 cells those were able to grow in the presence of 0.2 and 1 μM Imatinib

GCS:

Glucosylceramide synthase

GlcCer:

Glucosylceramide

(IC)50 :

The concentration of any chemical that inhibits growth by 50%

XTT:

2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

RT-PCR:

Reverse transcriptase-polymerase chain reaction

Pi:

Inorganic phosphate

LC/MS:

Liquid chromatography-mass spectrometry

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Acknowledgments

This work was supported by research grants from the National Institutes of Health to BO (CA-088932, DE-016572, and CA097132).

Conflict of interest

We, the authors of the manuscript, do not have any conflict of interest. We alone are responsible for the content and writing of the paper.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Izmir Institute of Technology, Faculty of Science, Gulbahce Campus, 35430, Urla, Izmir, Turkey

    Yusuf Baran

  2. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA

    Jacek Bielawski & Besim Ogretmen

  3. Department of Biology, Middle East Technical University, 06531, Ankara, Turkey

    Ufuk Gunduz

Authors
  1. Yusuf Baran
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  2. Jacek Bielawski
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  3. Ufuk Gunduz
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  4. Besim Ogretmen
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Correspondence to Yusuf Baran.

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Baran, Y., Bielawski, J., Gunduz, U. et al. Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation. J Cancer Res Clin Oncol 137, 1535–1544 (2011). https://doi.org/10.1007/s00432-011-1016-y

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  • DOI: https://doi.org/10.1007/s00432-011-1016-y

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