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Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo

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Abstract

Purpose

Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca2+ and apoptotic cell death. This study investigated the role of the Ca2+-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo.

Methods

Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects.

Results

Inhibition of the Ca2+-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.

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Acknowledgments

This work was supported by the Multiple Myeloma Research Foundation (MMRF), and the National Institutes of Health/National Cancer Institute [CA23074, CA017094]. Bortezomib (PS-341/Velcade®) was generously provided by Millennium Pharmaceuticals Inc.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Medicine, University of Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Room 4963B, Tucson, AZ, 85724, USA

    Aluvia M. Escalante, Matthew R. Karolak & Terry H. Landowski

  2. Department of Physiology, University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA

    Ryan T. McGrath, Matthew R. Karolak & Ronald M. Lynch

  3. Department of Pharmacology, University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA

    Matthew R. Karolak, Robert T. Dorr & Ronald M. Lynch

Authors
  1. Aluvia M. Escalante
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  2. Ryan T. McGrath
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  3. Matthew R. Karolak
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  4. Robert T. Dorr
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  5. Ronald M. Lynch
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  6. Terry H. Landowski
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Corresponding author

Correspondence to Terry H. Landowski.

Electronic supplementary material

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280_2013_2156_MOESM1_ESM.pdf

Supplementary material 1 CAPNS1 siRNA reduction of calpain expression and activity. 8226 myeloma cells were transduced with CAPNS1 siRNA expression constructs and selected under puromycin. (A) Cell lysates were separated by SDS-PAGE and immunoblotted for calpain small subunit 1 protein expression. (F) Myeloma cell lines 8226/siCT and 8226/siCAPNS1 were lysed, and 100 µg of total protein was incubated with the fluorescent substrate Ac-LLY-AFC in the presence of Ca2+. Enzyme activity was read by fluorescence intensity and expressed as relative fluorescence units (RGF)/µg protein. Data shown are representative of 3 independent experiments. (PDF 27 kb)

280_2013_2156_MOESM2_ESM.pdf

Supplementary material 2 (A) Quantitative analysis of acidic vesicle formation in myeloma cells. The 8226 myeloma cell line was incubated with 10 nM BZ or EBSS for 4 h, stained with monodansylcadaverine (MDC) and immobilized in Glycosan extracellular matrix on glass coverslips for fluorescent image analysis. (A) Mean number of vesicles per cell (mean + SEM. * p < 0.01, n = 40). (B) Mean vesicular-to-cytosolic ratio is the mean fluorescence intensity within the vesicles compared with cytosolic fluorescence within the same cell to normalize for differences in cell loading. (B) Relative expression of LC3II (A) and p62 (B) in 8226 myeloma cells. Pixel density of Western blot analysis (Fig 3B) was determined using Image J. Data shown are the mean of 3 independent experiments. (PDF 37 kb)

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Escalante, A.M., McGrath, R.T., Karolak, M.R. et al. Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo. Cancer Chemother Pharmacol 71, 1567–1576 (2013). https://doi.org/10.1007/s00280-013-2156-3

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  • DOI: https://doi.org/10.1007/s00280-013-2156-3

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