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Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis

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Abstract

Purpose

Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells.

Methods

Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA.

Results

Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis.

Conclusion

Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.

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Abbreviations

MM:

Multiple myeloma

MTT:

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide

PS:

Phosphatidylserine

ΔΨm :

Mitochondrial transmembrane potential

DiOC6(3):

3,3′-Dihexyloxacarbocyanine iodide

TMRE:

Tetramethylrhodamine ethyl ester

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Acknowledgments

We gratefully acknowledge Bayer for the gift of sorafenib and Dr. Atanasio Pandiella and Dolors Colomer for his advice and support. This study was supported by grants SAF2010-14920 and ISCIII-RTICC RD06/0020 from Ministerio de Ciencia e Innovación, and B16 from Gobierno de Aragón/Fondo Social Europeo.

Conflict of interest

None.

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Authors and Affiliations

  1. Departamento de Bioquimica, Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009, Zaragoza, Spain

    A. Ramírez-Labrada, N. López-Royuela, V. Jarauta, P. Galán-Malo, A. Anel, I. Marzo & J. Naval

  2. Servicio de Hematología, Hospital Clínico Universitario Lozano-Blesa, Zaragoza, Spain

    G. Azaceta & L. Palomera

  3. Immune Effector Cells Group (ICE), Aragón Health Research Institute (IIS Aragón), Edificio CIBA, Biomedical Research Centre of Aragón (CIBA), Universidad de Zaragoza, Zaragoza, Spain

    J. Pardo

  4. Aragón I+D Foundation (ARAID), Government of Aragon, Zaragoza, Spain

    J. Pardo

  5. Nanoscience Institute of Aragon (INA), Universidad de Zaragoza, Zaragoza, Spain

    J. Pardo

Authors
  1. A. Ramírez-Labrada
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  2. N. López-Royuela
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  3. V. Jarauta
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  4. P. Galán-Malo
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  5. G. Azaceta
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  6. L. Palomera
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  7. J. Pardo
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  8. A. Anel
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  9. I. Marzo
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  10. J. Naval
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Corresponding author

Correspondence to J. Naval.

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Ramírez-Labrada, A., López-Royuela, N., Jarauta, V. et al. Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis. Clin Transl Oncol 17, 121–132 (2015). https://doi.org/10.1007/s12094-014-1201-y

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  • DOI: https://doi.org/10.1007/s12094-014-1201-y

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