This study was conducted to compare the heat shock responses of cells grown in 2D and 3D culture environments as indicated by the level of heat shock protein 70 expression and the incidence of apoptosis and necrosis of prostate cancer cell lines in response to graded hyperthermia. PC3 cells were stably transduced with a dual reporter system composed of two tandem expression cassettes—a conditional heat shock protein promoter driving the expression of green fluorescent protein (HSPp-GFP) and a cytomegalovirus (CMV) promoter controlling the constitutive expression of a “beacon” red fluorescent protein (CMVp-RFP). Two-dimensional and three-dimensional cultures of PC3 prostate cancer cells were grown in 96-well plates for evaluation of their time-dependent response to supraphysiological temperature. To induce controlled hyperthermia, culture plates were placed on a flat copper surface of a circulating water manifold that maintained the specimens within ±0.1 °C of a target temperature. Hyperthermia protocols included various combinations of temperature, ranging from 37 °C to 57 °C, and exposure times of up to 2 h. The majority of protocols were focused on temperature and time permutations, where the response gradient was greatest. Post-treatment analysis by flow cytometry analysis was used to measure the incidences of apoptosis (annexin V-FITC stain), necrosis (propidium iodide (PI) stain), and HSP70 transcription (GFP expression). Cells grown in 3D compared with 2D culture showed reduced incidence of apoptosis and necrosis and a higher level of HSP70 expression in response to heat shock at the temperatures tested. Cells responded differently to hyperthermia when grown in 2D and 3D cultures. Three-dimensional culture appears to enhance survival plausibly by activating protective processes related to enhanced-HSP70 expression. These differences highlight the importance of selecting physiologically relevant 3D models in assessing cellular responses to hyperthermia in experimental settings.
Issue Section:
Research Papers
References
1.
Kennedy
, J. E.
, Ter Haar
, G. R.
, and Cranston
, D.
, 2003
, “High Intensity Focused Ultrasound: Surgery of the Future?
,” Br. J. Radiol.
, 76
, pp. 590
–599
.10.1259/bjr/171502742.
Lal
, S.
, Clare
, S. E.
, and Halas
, N. J.
, 2008
, “Nanoshell-Enabled Photothermal Cancer Therapy: Impending Clinical Impact
,” Acc. Chem. Res.
, 41
(12
), pp. 1842
–1851
.10.1021/ar800150g3.
Goldberg
, S. N.
, and Dupuy
, D. E.
, 2001
, “Image-Guided Radiofrequency Tumor Ablation: Challenges and Opportunities—Part I
,” J. Vasc. Interv. Radiol.
, 12
(9
), pp. 1021
–1032
.10.1016/S1051-0443(07)61587-54.
Dupuy
, D. E.
, and Goldberg
, S. N.
, 2001
, “Image-Guided Radiofrequency Tumor Ablation: Challenges and Opportunities—Part II
,” J. Vasc. Interv. Radiol.
, 12
(10
), pp. 1135
–1148
.10.1016/S1051-0443(07)61670-45.
Brausi
, M.
, Castagnetti
, G.
, Gavioli
, M.
, Peracchia
, G.
, de Luca
, G.
, and Olmi
, R.
, 2004
, “Radio Frequency (RF) Ablation of Renal Tumours Does Not Produce Complete Tumour Destruction: Results of a Phase II Study
,” Eur. Urol. Suppl.
, 3
(3
), pp. 14
–17
.10.1016/j.eursup.2004.02.0046.
Solbiati.
L.
, Ierace
, T.
, Goldberg
, S. N.
, Sironi
, S.
, Livraghi
, T.
, Fiocca
, R.
, Servadio
, G.
, Rizzatto
, G.
, Mueller
, P. R.
, Del Maschio
, A.
, and Gazelle
, G. S.
, 1997
, “Percutaneous US-Guided Radio-Frequency Tissue Ablation of Liver Metastases: Treatment and Follow-up in 16 Patients
,” Radiology
, 202
, pp. 195
–203
.10.1148/radiology.202.1.89882117.
Oden
, J. T.
, Diller
, K. R.
, Bajaj
, C.
, Browne
, J. C.
, Hazle
, J. D.
, Babuška
, I.
, Bass
, J.
, Biduat
, L.
, Demkowicz
, L.
, Elliott
, A.
, Feng
, Y.
, Fuentes
, D.
, Prudhomme
, S.
, Rylander
, M. N.
, Stafford
, R. J.
, and Zhang
, Y.
, 2007
, “Dynamic Data-Driven Finite Element Models for Laser Treatment of Cancer
,” Numer. Methods Partial Differ. Equation
, 23
(4
), pp. 904
–922
.10.1002/num.202518.
Fuentes
, D.
, Oden
, J. T.
, Diller
, K. R.
, Hazle
, J. D.
, Elliott
, A.
, Shetty
, A.
, and Stafford
, R. J.
, 2009
, “Computational Modeling and Real-Time Control of Patient-Specific Laser Treatment of Cancer
,” Ann. Biomed. Eng.
, 37
, pp. 763
–782
.10.1007/s10439-008-9631-89.
Lindquist
, S.
, and Craig
, E. A.
, 1988
, “The Heat-Shock Proteins
,” Annu. Rev. Genet.
, 22
, pp. 631
–677
.10.1146/annurev.ge.22.120188.00321510.
Lindquist
, S.
, 1986
, “The Heat-Shock Response
,” Annu. Rev. Biochem.
, 55
, pp. 1151
–1191
.10.1146/annurev.bi.55.070186.00544311.
Yeh
, C.
, Hsu
, S.
, Yang
, C.
, Chien
, C.
, and Wang
, N.
, 2010
, “Hypoxic Preconditioning Reinforces HIF-Alpha-Dependent HSP70 Signaling to Reduce Ischemic Renal Failure-Induced Renal Tubular Apoptosis and Autophagy
.” Life Sci.
, 86
(3–4
), pp. 115
–123
.10.1016/j.lfs.2009.11.02212.
Kurz
, A. K.
, Schliess
, F.
, and Häussinger
, D.
, 1998
, “Osmotic Regulation of the Heat Shock Response in Primary Rat Hepatocytes
,” Hepatology
, 28
(3
), pp. 774
–781
.10.1002/hep.51028032613.
Schett
, G.
, Redlich
, K.
, Xu
, Q.
, Bizan
, P.
, Gröger
, M.
, and Tohidast-Akrad
, M.
, 1998
, “Enhanced Expression of Heat Shock Protein 70 (Hsp70) and Heat Shock Factor 1 (HSF1) Activation in Rheumatoid Arthritis Synovial Tissue. Differential Regulation of Hsp70 Expression and HSF1 Activation in Synovial Fibroblasts by Proinflammatory Cytokines, Shear Stress, and Antiinflammatory Drugs
,” J. Clin. Invest.
, 102
(2
), pp. 302
–311
.10.1172/JCI246514.
Calini
, V.
, Urani
, C.
, and Camatini
, M.
, 2003
, “Overexpression of HSP70 is Induced by Ionizing Radiation in C3H 10T1/2 Cells and Protects From DNA Damage
,” Toxicol. in Vitro
, 17
(5–6
), pp. 561
–566
.10.1016/S0887-2333(03)00116-415.
Kregel
, K. C.
, 2002
, “Heat Shock Proteins: Modifying Factors in Physiological Stress Responses and Acquired Thermotolerance
,” J. Appl. Physiol.
, 92
(5
), pp. 2177
–2186
.10.1152/japplphysiol.01267.200116.
Diller
, K. R.
, 2006
, “Stress Protein Expression Kinetics
,” Annu. Rev. Biomed. Eng.
, 8
, pp. 403
–424
.10.1146/annurev.bioeng.7.060804.10044917.
Calderwood
, S. K.
, Khaleque
, M. A.
, Sawyer
, D. B.
, and Ciocca
, D. R.
, 2006
, “Heat Shock Proteins in Cancer: Chaperones of Tumorigenesis
,” Trends Biochem. Sci.
, 31
(3
), pp. 164
–172
.10.1016/j.tibs.2006.01.00618.
Khoei
, S.
, Fazeli
, G.
, Amerizadeh
, A.
, Eslimi
, D.
, and Goliaei
, B.
, 2010
, “Elimination of Enhanced Thermal Resistance of Spheroid Culture Model of Prostate Carcinoma Cell Line by Inhibitors of Hsp70 Induction
,” Yakhteh Med. J.
, 12
(1
), pp. 105
–112
. Available at: http://celljournal.org/components4.php?rQV==wHQwAkO0JXY0N3XmxHQwYDQ6QWS05WZyFGcfZGfAhjN1EDQ6QWStVGdp9lZ8BEMApDZJxWY0J3bQxWYuJXdvp2XmxHQyATNApDZJ52bpR3Yh9lZ19.
Khoei
, S.
, Goliaei
, B.
, Neshasteh-Riz
, A.
, and Deizadji
, A.
, 2004
, “The Role of Heat Shock Protein 70 in the Thermoresistance of Prostate Cancer Cell Line Spheroids
,” FEBS Lett.
, 561
(1–3
), pp. 144
–148
.10.1016/S0014-5793(04)00158-920.
Sun
, T.
, Jackson
, S.
, Haycock
, J. W.
, and Macneil
, S.
, 2006
, “Culture of Skin Cells in 3D Rather Than 2D Improves Their Ability to Survive Exposure to Cytotoxic Agents
,” J. Biotech.
, 122
(3
), pp. 372
–381
.10.1016/j.jbiotec.2005.12.02121.
Jain
, R. K.
, 2005
, “Normalization of Tumor Vasculature: An Emerging Concept in Antiangiogenic Therapy
,” Science
, 307
, pp. 58
–62
.10.1126/science.110481922.
Harris
, A. L.
, 2002
, “Hypoxia—A Key Regulatory Factor in Tumor Growth
,” Nature
, 2
, pp. 38
–47
.10.1038/nrc70423.
Santos
, I.
, Haemmerich
, D.
, Pinheiro
, C.
, and Rocha
, A.
, 2008
, “Effect of Variable Heat Transfer Coefficient on Tissue Temperature Next to a Large Vessel During Radio Frequency Tumor Ablation
,” Biomed. Eng. Online
, 7
(1
), p. 21
.10.1186/1475-925X-7-2124.
Gomer
, C. J.
, Ryter
, S. W.
, Ferrario
, A.
, Rucker
, N.
, Wong
, S.
, and Fisher
, A. M. R.
, 1996
, “Photodynamic Therapy-Mediated Oxidative Stress can Induce Expression of Heat Shock Proteins
,” Cancer Res.
, 56
(10
), pp. 2355
–2360
. Available at: http://cancerres.aacrjournals.org/content/56/10/2355.abstract25.
Luk
, J. M.
, Lam
, C.-T.
, Siu
, A. F. M.
, Lam
, B. Y.
, Ng
, I. O. L.
, and Ju
, M.-Y.
, 2006
, “Proteomic Profiling of Hepatocellular Carcinoma in Chinese Cohort Reveals Heat-Shock Proteins (Hsp27, Hsp70, GRP78) Up-Regulation and Their Associated Prognostic Values
,” Proteomics
, 6
(3
), pp. 1049
–1057
.10.1002/pmic.20050030626.
Heacock
, C. S.
, and Sutherland
, R. M.
, 1990
, “Enhanced Synthesis of Stress Proteins Caused by Hypoxia and Relation to Altered Cell Growth and Metabolism
,” Br. J. Cancer
, 62
(2
), pp. 217
–225
.10.1038/bjc.1990.26427.
Li
, G. C.
, and Shrieve
, D. C.
, 1982
, “Thermal Tolerance and Specific Protein Synthesis in Chinese Hamster Fibroblasts Exposed to Prolonged Hypoxia
,” Exp. Cell Res.
, 142
(2
), pp. 464
–468
.10.1016/0014-4827(82)90390-128.
Khoei
, S.
, Goliaei
, B.
, and Neshasteh-Riz
, A.
, 2004
, “Differential Thermo-Resistance of Multicellular Tumor Spheroids
,” Iran. J. Sci. Technol.
, 28
, pp. 107
–116
.29.
He
, X.
, and Bischof
, J. C.
, 2005
, “The Kinetics of Thermal Injury in Human Renal Carcinoma Cells
,” Ann. Biomed. Eng.
, 33
(4
), pp. 502
–510
.10.1007/s10439-005-2508-130.
Connors
, K. A.
, 1990
, Chemical Kinetics: The Study of Reaction Rates in Solution
, VCH Publishers, Inc.
, New York
.31.
Rylander
, M. N.
, Diller
, K. R.
, Wang
, S.
, and Aggarwal
, S. J.
, 2005
, “Correlation of HSP70 Expression and Cell Viability Following Thermal Stimulation of Bovine Aortic Endothelial Cells
,” ASME J. Biomech. Eng.
, 127
(5), pp. 751
–757
.10.1115/1.199366132.
Wang
, S.
, Xie
, W.
, Rylander
, M. N.
, Tucke
, P. W.
, Aggarwal
, S. J.
, and Diller
, K. R.
, 2008
, “HSP70 Kinetics Study by Continuous Observation of HSP-GFP Fusion Protein Expression on a Perfusion Heating Stage
,” Biotechnol. Bioeng.
, 99
, pp. 146
–154
.10.1002/bit.2151233.
Samali
, A.
, Holmberg
, C. I.
, Sistonen
, L.
, and Orrenius
, S.
, 1999
, “Thermotolerance and Cell Death are Distinct Cellular Responses to Stress: Dependence on Heat Shock Proteins
,” FEBS Lett.
, 461
(3
), pp. 306
–310
.10.1016/S0014-5793(99)01486-634.
Huang
, C.-T.
, Lu
, Y.-H.
, and Jen
, C.-P.
, 2010
, “Investigation on Supraphysiological Thermal Injury in Two Well-Differentiated Human Hepatoma Cell Lines, Hepg2 and Hep3B
,” J. Therm. Biol.
, 35
(8
), pp. 411
–416
.10.1016/j.jtherbio.2010.09.00135.
Marylevitch
, N. P.
, Schuschereba
, S. T.
, Mata
, J. R.
, Gilligan
, G. R.
, Lawlor
, D. F.
, and Goodwin
, C. W.
, 1998
, “Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes After Thermal Injury
,” Am. J. Pathol.
, 153
(2
), pp. 567
–577
.10.1016/S0002-9440(10)65599-X36.
Madersbacher
, S.
, Pedevilla
, M.
, Vingers
, L.
, Susani
, M.
, and Marberger
, M.
, 1995
, “Effect of High-Intensity Focused Ultrasound on Human Prostate Cancer in Vivo
,” Cancer Res.
, 55
(15
), pp. 3346
–3351
. Available at: http://cancerres.aacrjournals.org/content/55/15/3346.abstract?sid=e8f2c7a4-bcb4-4695-b408-7c40ee71395137.
Nijhuis
, E. H. A.
, Poot
, A. A.
, Feijen
, J.
, and Vermes
, I.
, 2006
, “Induction of Apoptosis by Heat and γ-Radiation in a Human Lymphoid Cell Line; Role of Mitochondrial Changes and Caspase Activation
,” Int. J. Hyperthermia
, 22
(8
), pp. 687
–698
.10.1080/0265673060104540938.
Shelton
, S. N.
, Dillard
, C. D.
, and Robertson
, J. D.
, 2010
, “Activation of Caspase-9, but not Caspase-2 or Caspase-8, is Essential for Heat-Induced Apoptosis in Jurkat Cells
,” J. Biol. Chem.
, 285
(52
), pp. 40525
–40533
.10.1074/jbc.M110.16763539.
Wilson
, L.
, and Matsudaira
, P. T.
, 1995
, Cell Death
, Vol. 46
, Academic Press
, New York
.40.
Ritossa
, F.
, 1962
, “A New Puffing Pattern Induced by Temperature Shock and DNP in Drosophila
,” Cell Mol. Life Sci.
, 18
(12
), pp. 571
–573
.10.1007/BF0217218841.
Todryk
, S.
, Melcher
, A. A.
, Hardwick
, N.
, Linardakis
, E.
, Batemen
, A.
, and Colombo
, M. P.
, 1999
, “Heat Shock Protein 70 Induced During Tumor Cell Killing Induces Th1 Cytokines and Targets Immature Dendritic Cell Precursors to Enhance Antigen Uptake
,” J. Immunol.
, 163
(3
), pp. 1398
–1408
. Available at: http://www.jimmunol.org/content/163/3/1398.abstract42.
Yoo
, J. C.
, and Mayman
, M. J.
, 2006
, “HSP70 Binds to SHP2 and Has Effects on the SHP2-Related EGFR/GAB1 Signaling Pathway
,” Biochem. Biophys. Res. Commun.
, 351
(4
), pp. 979
–985
.10.1016/j.bbrc.2006.10.15243.
Thériault
, J. R.
, Mambula
, S. S.
, Sawamura
, T.
, Stevenson
, M. A.
, and Calderwood
, S. K.
, 2005
, “Extracellular HSP70 Binding to Surface Receptors Present on Antigen Presenting Cells and Endothelial/Epithelial Cells
,” FEBS Lett.
, 579
(9
), pp. 1951
–1960
.10.1016/j.febslet.2005.02.04644.
Beere
, H. M.
, Wolf
, B. B.
, Cain
, K.
, Mosser
, D. D.
, Mahboubi
, A.
, and Kuwana
, T.
, 2000
, “Heat-Shock Protein 70 Inhibits Apoptosis by Preventing Recruitment of Procaspase-9 to the Apaf-1 Apoptosome
,” Nature Cell Biol.
, 2
(8
), pp. 469
–475
.10.1038/3501950145.
Ravagnan
, L.
, Gurbuxani
, S.
, Susin
, S. A.
, Maisse
, C.
, Daugas
, E.
, and Zamzami
, N.
, 2001
, “Heat-Shock Protein 70 Antagonizes Apoptosis-Inducing Factor
,” Nature Cell Biol.
, 3
(9
), pp. 839
–843
.10.1038/ncb0901-83946.
Kim
, Y.-M.
, de Vera
, M. E.
, Watkins
, S. C.
, and Billiar
, T. R.
, 1997
, “Nitric Oxide Protects Cultured Rat Hepatocytes From Tumor Necrosis Factor-α-Induced Apoptosis by Inducing Heat Shock Protein 70 Expression
,” J. Biol. Chem.
, 272
(2
), pp. 1402
–1411
.10.1074/jbc.272.2.140247.
Li
, C.-Y.
, Lee
, J.-S.
, Ko
, Y.-G.
, Kim
, J.-I.
, and Seo
, J.-S.
, 2000
, “Heat Shock Protein 70 Inhibits Apoptosis Downstream of Cytochrome C Release and Upstream of Caspase-3 Activation
,” J. Biol. Chem.
, 275
(33
), pp. 25665
–25671
.10.1074/jbc.M90638319948.
Komarova
, E. Y.
, Afanasyeva
, E. A.
, Bulatova
, M. M.
, Cheetham
, M. E.
, Margulis
, B. A.
, and Guzhova
, I. V.
, 2004
, “Downstream Caspases are Novel Targets for the Antiapoptotic Activity of the Molecular Chaperone HSP70
,” Cell Stress Chaperones
, 9
(3
), pp. 265
–271
.10.1379/CSC-27R1.149.
Creagh
, E. M.
, Carmody
, R. J.
, and Cotter
, T. G.
, 2000
, “Heat Shock Protein 70 Inhibits Caspace-Dependent and -Independent Apoptosis in Jurkat T Cells
,” Exp. Cell Res.
, 257
(1
), pp. 58
–66
.10.1006/excr.2000.485650.
Bretland
, A. J.
, Lawry
, J.
, and Sharrard
, R. M.
, 2001
, “A Study of Death by Anoikis in Cultured Epithelial Cells
,” Cell Prolif.
, 34
(4
), pp. 199
–210
.10.1046/j.1365-2184.2001.00198.x51.
Frisch
, S. M.
, and Screaton
, R. A.
, 2001
, “Anoikis Mechanisms
,” Curr. Opin. Cell Biol.
, 13
(5
), pp. 555
–562
.10.1016/S0955-0674(00)00251-952.
Grossmann
, J.
, 2002
, “Molecular Mechanisms of ‘Detachment-Induced Apoptosis—Anoikis’
,” Apoptosis
, 7
(3
), pp. 247
–260
.10.1023/A:101531211969353.
Kamarajan
, P.
, and Kapila
, Y. L.
, 2007
, “An Altered Fibronectin Matrix Induces Anoikis of Human Squamous Cell Carcinoma Cells by Suppressing Integrin Alpha V Levels and Phosphorylation of FAK and ERK
,” Apoptosis
, 12
, pp. 2221
–2231
.10.1007/s10495-007-0138-954.
Bunek
, J.
, Kamarajan
, P.
, and Kapila
, Y. L.
, 2011
, “Anoikis Mediators in Oral Squamous Cell Carcinoma
,” Oral Dis.
, 17
(4
), pp. 355
–361
.10.1111/j.1601-0825.2010.01763.xCopyright © 2014 by ASME
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