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Role of Intracellular Calcium in Proteasome Inhibitor-Induced Endoplasmic Reticulum Stress, Autophagy, and Cell Death

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ABSTRACT

Purpose

Proteasome inhibition induces endoplasmic reticulum (ER) stress and compensatory autophagy to relieve ER stress. Disturbance of intracellular calcium homeostasis can lead to ER stress and alter the autophagy process. It has been suggested that inhibition of the proteasome disrupts intracellular calcium homeostasis. However, it is unknown if intracellular calcium affects proteasome inhibitor-induced ER stress and autophagy.

Methods

Human colon cancer HCT116 Bax positive and negative cell lines were treated with MG132, a proteasome inhibitor. BAPTA-AM, a cell permeable free calcium chelator, was used to modulate intracellular calcium levels. Autophagy and cell death were determined by fluorescence microscopy and immunoblot analysis.

Results

MG132 increased intracellular calcium levels in HCT116 cells, which was suppressed by BAPTA-AM. MG132 suppressed proteasome activity independent of Bax and intracellular calcium levels in HCT116 cells. BAPTA-AM inhibited MG132-induced cellular vacuolization and ER stress, but not apoptosis. MG132 induced autophagy with normal autophagosome-lysosome fusion. BAPTA-AM seemed not to affect autophagosome-lysosome fusion in MG132-treated cells but further enhanced MG132-induced LC3-II levels and GFP-LC3 puncta formation, which was likely via impaired lysosome function.

Conclusions

Blocking intracellular calcium by BAPTA-AM relieved MG132-induced ER stress, but it was unable to rescue MG132-induced apoptosis, which was likely due to impaired autophagic degradation.

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Abbreviations

2-APB:

2-aminoethoxydiphenyl borate

BAF:

Bafilomycin A1

BAPTA-AM:

1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl esteris

CPP:

calcium phosphate precipitates

eIF2-alpha:

Eukaryotic Translation Initiation Factor 2-alpha

ER:

Endoplasmic Reticulum

IRE1:

Inositol-requiring Enzyme1

JNK:

c-Jun N-terminal Kinase

LC3:

microtubule-associated protein 1 light chain 3

MEF:

mouse embryonic fibroblasts

PE:

Phosphatidylethanolamine

PERK:

Protein Kinase RNA-like Endoplasmic Reticulum Kinase

PFA:

paraformaldehyde

UPR:

Unfolded Protein Response

UPS:

Ubiquitin Proteasome System

Xec:

Xestospongin C

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ACKNOWLEDGMENTS AND DISCLOSURES

Jessica A. Williams and Yifeng Hou contribute equally to this work. The research work in W.X Ding’s lab was supported in part by the NIAAA funds R01 AA020518-01 and National Center for Research Resources (5P20RR021940-07). J. A. Williams was supported by the “Training Program in Environmental Toxicology” [grant 5T32 ES007079] from the National Institute of Environmental Health Sciences. Y.F. Hou was supported by the National Natural Science Foundation of China (# 81072165) and the Shanghai Science and Technology Committee (# 09PJ1402700). The authors are indebted to Dr. Bert Vogelstein (Johns Hopkins University) and Lin Zhang (University of Pittsburgh) for the HCT116 Bax-positive and Bax-negative cell lines.

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Authors and Affiliations

  1. Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, MS 1018 3901 Rainbow Blvd., Kansas City, Kansas, 66160, USA

    Jessica A. Williams, Hong-Min Ni & Wen-Xing Ding

  2. Department of Breast Surgery, Breast Cancer Institute Cancer Hospital, Fudan University, Shanghai, China

    Yifeng Hou

Authors
  1. Jessica A. Williams
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  2. Yifeng Hou
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  4. Wen-Xing Ding
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Correspondence to Wen-Xing Ding.

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Supplemental Figure 1

MG132 increased intracellular calcium 2-fold in HCT116 cells and produced similar results in DU145 Cells. (A) HCT116 Bax (-) cells were treated with MG132 (1 μM) for 16 hours. Cells were then stained with 2.5 μM of Fluo-4-AM in calcium-free Hank’s buffer for 30 minutes followed by flow cytometry analysis. Fluorescence intensity (FI) is shown. (B) DU145 Bax (-) cells were treated with MG132 (1 μM) in the presence or absence of BAPTA-AM (10 μM) for 16 hours. Cells were then washed and stained with 2.5 μM of Fluo-4-AM in calcium-free Hank’s buffer for 30 minutes followed by flow cytometry analysis. Representative histogram data are shown. (JPEG 37 kb)

Supplemental Figure 2

BAPTA-AM inhibited MG132-induced cellular vacuolization in DU145 cells. DU145 Bax (-) cells were treated with MG132 (1 μM) in the presence or absence of BAPTA-AM (10 μM) for 12 hours followed by phase-contrast microscopy. Representative images are presented in (A). (B) Vacuolated cells were counted, and results are expressed as percent of vacuolated cells (*p<0.05 vs untreated-control; ^ p<0.05 vs MG132, One Way ANOVA). (JPEG 57 kb)

Supplemental Figure 3

The source of intracellular calcium increase was most likely not ER or extracellular calcium influx. HCT116 Bax (-) cells were treated with MG132 (1 μM) in the presence or absence of Xec (25 nM) or 2-APB (20 μM) for 12 hours followed by phase-contrast microscopy. Representative images are presented in (A). (B) Vacuolated cells were counted, and results are expressed as percent of vacuolated cells. Results are from two individual experiments. (C and D) HCT116 Bax (-) cells were treated with MG132 (1 μM) in the presence or absence of Xec (25 nM) or 2-APB (20 μM) for 16 hours. Cells were then washed and stained with 2.5 μM of Fluo-4-AM in calcium-free Hank’s buffer for 30 minutes followed by flow cytometry analysis. Representative histogram data are shown. (E) HCT116 Bax (-) cells were treated with MG132 (1 μM) in the presence or absence of varying concentrations of EGTA for 12 hours followed by phase-contrast microscopy. Representative images are shown. (PDF 254 kb)

Supplemental Figure 4

MG132 induced ER dilation in DU145 cells. DU145 Bax (-) cells were treated with MG132 (1 μM) for 16 hours, and cells were fixed with 4% PFA before immunostaining for Calnexin (green) for visualization of ER dilation and DAPI (blue) for visualization of the cell nucleus. Representative fluorescence images are shown. (JPEG 30 kb)

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Williams, J.A., Hou, Y., Ni, HM. et al. Role of Intracellular Calcium in Proteasome Inhibitor-Induced Endoplasmic Reticulum Stress, Autophagy, and Cell Death. Pharm Res 30, 2279–2289 (2013). https://doi.org/10.1007/s11095-013-1139-8

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  • DOI: https://doi.org/10.1007/s11095-013-1139-8

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