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

Aluvia M. Escalante, Ryan T. McGrath, Matthew R. Karolak, Robert T Dorr, Ron Lynch, Terry H Landowski

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1567-1576
Number of pages10
JournalCancer Chemotherapy and Pharmacology
Volume71
Issue number6
DOIs
StatePublished - Jun 2013

Fingerprint

Calpain
Cell death
Cell Death
Cytotoxicity
HIV Protease Inhibitors
Assays
Bortezomib
In Vitro Techniques
Nelfinavir
Proteasome Inhibitors
Endoplasmic Reticulum Stress
SCID Mice
Trypan Blue
Cysteine Proteases
Annexin A5
Autophagy
Enzyme Assays
Enzyme activity
Proteasome Endopeptidase Complex
Caspases

Keywords

  • Autophagy
  • Bortezomib
  • Calpain
  • Myeloma
  • Nelfinavir

ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Pharmacology
  • Pharmacology (medical)
  • Toxicology

Cite this

Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo. / Escalante, Aluvia M.; McGrath, Ryan T.; Karolak, Matthew R.; Dorr, Robert T; Lynch, Ron; Landowski, Terry H.

In: Cancer Chemotherapy and Pharmacology, Vol. 71, No. 6, 06.2013, p. 1567-1576.

Research output: Contribution to journalArticle

@article{c5d3246d5d7045d08d72b7374dfca5da,
title = "Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo",
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.",
keywords = "Autophagy, Bortezomib, Calpain, Myeloma, Nelfinavir",
author = "Escalante, {Aluvia M.} and McGrath, {Ryan T.} and Karolak, {Matthew R.} and Dorr, {Robert T} and Ron Lynch and Landowski, {Terry H}",
year = "2013",
month = "6",
doi = "10.1007/s00280-013-2156-3",
language = "English (US)",
volume = "71",
pages = "1567--1576",
journal = "Cancer Chemotherapy and Pharmacology",
issn = "0344-5704",
publisher = "Springer Verlag",
number = "6",

}

TY - JOUR

T1 - Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo

AU - Escalante, Aluvia M.

AU - McGrath, Ryan T.

AU - Karolak, Matthew R.

AU - Dorr, Robert T

AU - Lynch, Ron

AU - Landowski, Terry H

PY - 2013/6

Y1 - 2013/6

N2 - 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.

AB - 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.

KW - Autophagy

KW - Bortezomib

KW - Calpain

KW - Myeloma

KW - Nelfinavir

UR - http://www.scopus.com/inward/record.url?scp=84878658221&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84878658221&partnerID=8YFLogxK

U2 - 10.1007/s00280-013-2156-3

DO - 10.1007/s00280-013-2156-3

M3 - Article

C2 - 23572175

AN - SCOPUS:84878658221

VL - 71

SP - 1567

EP - 1576

JO - Cancer Chemotherapy and Pharmacology

JF - Cancer Chemotherapy and Pharmacology

SN - 0344-5704

IS - 6

ER -