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Pharmaceutical Research

, Volume 30, Issue 9, pp 2279–2289 | Cite as

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

  • Jessica A. Williams
  • Yifeng Hou
  • Hong-Min Ni
  • Wen-Xing Ding
Research Paper

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.

KEY WORDS

autophagy cell death ER stress intracellular calcium proteasome inhibitor 

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

Notes

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.

Supplementary material

11095_2013_1139_MOESM1_ESM.jpg (38 kb)
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)
11095_2013_1139_MOESM2_ESM.jpg (58 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)
11095_2013_1139_MOESM3_ESM.pdf (255 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)
11095_2013_1139_MOESM4_ESM.jpg (31 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)

REFERENCES

  1. 1.
    Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–79.PubMedCrossRefGoogle Scholar
  2. 2.
    Mitchell BS. The proteasome—an emerging therapeutic target in cancer. N Engl J Med. 2003;348(26):2597–8.PubMedCrossRefGoogle Scholar
  3. 3.
    Adams J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell. 2004;5(5):417–21.PubMedCrossRefGoogle Scholar
  4. 4.
    Chauhan D, Hideshima T, Anderson KC. Proteasome inhibition in multiple myeloma: therapeutic implication. Annu Rev Pharmacol Toxicol. 2005;45:465–76.PubMedCrossRefGoogle Scholar
  5. 5.
    Ding WX, Ni HM, Yin XM. Absence of Bax switched MG132-induced apoptosis to non-apoptotic cell death that could be suppressed by transcriptional or translational inhibition. Apoptosis: Int J Programmed Cell Death. 2007;12(12):2233–44.CrossRefGoogle Scholar
  6. 6.
    Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6(4):463–77.PubMedCrossRefGoogle Scholar
  7. 7.
    Lum JJ, DeBerardinis RJ, Thompson CB. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol. 2005;6(6):439–48.PubMedCrossRefGoogle Scholar
  8. 8.
    Kamada Y, Sekito T, Ohsumi Y. Autophagy in yeast: a TOR-mediated response to nutrient starvation. Curr Top Microbiol Immunol. 2004;279:73–84.PubMedCrossRefGoogle Scholar
  9. 9.
    Ding WX, Ni HM, Gao W, Yoshimori T, Stolz DB, Ron D, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol. 2007;171(2):513–24.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhu K, Dunner Jr K, McConkey DJ. Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells. Oncogene. 2010;29(3):451–62.PubMedCrossRefGoogle Scholar
  11. 11.
    Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 2006;441(7095):880–4.PubMedCrossRefGoogle Scholar
  12. 12.
    Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell. 2009;33(4):517–27.Google Scholar
  13. 13.
    Ding WX, Ni HM, Gao W, Chen X, Kang JH, Stolz DB, et al. Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy. Mol Cancer Ther. 2009;8(7):2036–45.PubMedCrossRefGoogle Scholar
  14. 14.
    Avivar-Valderas A, Salas E, Bobrovnikova-Marjon E, Diehl JA, Nagi C, Debnath J, et al. PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment. Mol Cell Biol. 2011;31(17):3616–29.PubMedCrossRefGoogle Scholar
  15. 15.
    Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011;17(4):654–66.PubMedCrossRefGoogle Scholar
  16. 16.
    Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990;87(7):2466–70.PubMedCrossRefGoogle Scholar
  17. 17.
    Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007;25(2):193–205.PubMedCrossRefGoogle Scholar
  18. 18.
    Gao W, Ding WX, Stolz DB, Yin XM. Induction of macroautophagy by exogenously introduced calcium. Autophagy. 2008;4(6):754–61.PubMedGoogle Scholar
  19. 19.
    Sarkar S, Korolchuk V, Renna M, Winslow A, Rubinsztein DC. Methodological considerations for assessing autophagy modulators: a study with calcium phosphate precipitates. Autophagy. 2009;5(3):307–13.PubMedCrossRefGoogle Scholar
  20. 20.
    Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, et al. Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 2008;4(5):295–305.PubMedCrossRefGoogle Scholar
  21. 21.
    Itakura E, Kishi-Itakura C, Mizushima N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell. 2012;151(6):1256–69.PubMedCrossRefGoogle Scholar
  22. 22.
    Jager S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P, et al. Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci. 2004;117(Pt 20):4837–48.PubMedCrossRefGoogle Scholar
  23. 23.
    Rusten TE, Stenmark H. How do ESCRT proteins control autophagy? J Cell Sci. 2009;122(Pt 13):2179–83.PubMedCrossRefGoogle Scholar
  24. 24.
    Eskelinen EL, Illert AL, Tanaka Y, Schwarzmann G, Blanz J, Von Figura K, et al. Role of LAMP-2 in lysosome biogenesis and autophagy. Mol Biol Cell. 2002;13(9):3355–68.PubMedCrossRefGoogle Scholar
  25. 25.
    Pryor PR, Mullock BM, Bright NA, Gray SR, Luzio JP. The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. J Cell Biol. 2000;149(5):1053–62.PubMedCrossRefGoogle Scholar
  26. 26.
    Ganley IG, Wong PM, Gammoh N, Jiang X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell. 2011;42(6):731–43.PubMedCrossRefGoogle Scholar
  27. 27.
    Li X, Yang D, Li L, Peng C, Chen S, Le W. Proteasome inhibitor lactacystin disturbs the intracellular calcium homeostasis of dopamine neurons in ventral mesencephalic cultures. Neurochem Int. 2007;50(7–8):959–65.PubMedCrossRefGoogle Scholar
  28. 28.
    Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science. 2000;290(5493):989–92.PubMedCrossRefGoogle Scholar
  29. 29.
    Ding WX, Guo F, Ni HM, Bockus A, Manley S, Stolz DB, et al. Parkin and mitofusins reciprocally regulate mitophagy and mitochondrial spheroid formation. J Biol Chem. 2012;287(50):42379–88.PubMedCrossRefGoogle Scholar
  30. 30.
    Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23(1):33–42.PubMedCrossRefGoogle Scholar
  31. 31.
    Mikoshiba K. IP3 receptor/Ca2+ channel: from discovery to new signaling concepts. J Neurochem. 2007;102(5):1426–46.PubMedCrossRefGoogle Scholar
  32. 32.
    Peppiatt CM, Collins TJ, Mackenzie L, Conway SJ, Holmes AB, Bootman MD, et al. 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium. 2003;34(1):97–108.PubMedCrossRefGoogle Scholar
  33. 33.
    Ni HM, Bockus A, Wozniak AL, Jones K, Weinman S, Yin XM, et al. Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy. 2011;7(2):188–204.PubMedCrossRefGoogle Scholar
  34. 34.
    Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8(4):445–544.PubMedCrossRefGoogle Scholar
  35. 35.
    Ni HM, Williams JA, Yang H, Shi YH, Fan J, Ding WX. Targeting autophagy for the treatment of liver diseases. Pharmacol Res. 2012;66(6):463–74.PubMedCrossRefGoogle Scholar
  36. 36.
    Rossi D, Barone V, Giacomello E, Cusimano V, Sorrentino V. The sarcoplasmic reticulum: an organized patchwork of specialized domains. Traffic. 2008;9(7):1044–9.PubMedCrossRefGoogle Scholar
  37. 37.
    Burdakov D, Petersen OH, Verkhratsky A. Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell Calcium. 2005;38(3–4):303–10.PubMedCrossRefGoogle Scholar
  38. 38.
    Michalak M, Robert Parker JM, Opas M. Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium. 2002;32(5–6):269–78.PubMedCrossRefGoogle Scholar
  39. 39.
    Ding WX, Ni HM, Gao W, Hou YF, Melan MA, Chen X, et al. Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem. 2007;282(7):4702–10.PubMedCrossRefGoogle Scholar
  40. 40.
    Starkov AA. The molecular identity of the mitochondrial Ca2+ sequestration system. FEBS J. 2010;277(18):3652–63.PubMedCrossRefGoogle Scholar
  41. 41.
    Gordon PB, Holen I, Fosse M, Rotnes JS, Seglen PO. Dependence of hepatocytic autophagy on intracellularly sequestered calcium. J Biol Chem. 1993;268(35):26107–12.PubMedGoogle Scholar
  42. 42.
    Lloyd-Evans E, Platt FM. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases. Cell Calcium. 2011;50(2):200–5.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jessica A. Williams
    • 1
  • Yifeng Hou
    • 2
  • Hong-Min Ni
    • 1
  • Wen-Xing Ding
    • 1
  1. 1.Department of Pharmacology, Toxicology and TherapeuticsThe University of Kansas Medical CenterKansas CityUSA
  2. 2.Department of Breast Surgery, Breast Cancer Institute Cancer HospitalFudan UniversityShanghaiChina

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