Research Papers

Thermally Induced Apoptosis, Necrosis, and Heat Shock Protein Expression in Three-Dimensional Culture

[+] Author and Article Information
Alfred S. Song

Department of Biomedical Engineering,
The University of Texas,
1 University Station, BME4.202A,
Austin, TX 78712;
Baylor College of Medicine,
1 Baylor Plaza, MS BCM 368,
Houston, TX 77030
e-mail: alfred.song@bcm.edu

Amer M. Najjar

Department of Cancer Systems Imaging,
The University of Texas MD
Anderson Cancer Center,
Unit 059, 1515 Holcombe Boulevard,
Houston, TX 77030
e-mail: amer.najjar@mdanderson.org

Kenneth R. Diller

Department of Biomedical Engineering,
The University of Texas,
1 University Station, BME4.202A,
Austin, TX 78712
e-mail: kdiller@mail.utexas.edu

1Corresponding author.

Manuscript received January 14, 2014; final manuscript received March 17, 2014; accepted manuscript posted March 24, 2014; published online May 12, 2014. Assoc. Editor: Ram Devireddy.

J Biomech Eng 136(7), 071006 (May 12, 2014) (10 pages) Paper No: BIO-14-1020; doi: 10.1115/1.4027272 History: Received January 14, 2014; Revised March 17, 2014; Accepted March 24, 2014

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.

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Grahic Jump Location
Fig. 1

Normalized WST-1 absorbance of PC3 cells for long durations of heating at various temperatures at approximately 12 h later. Each point is normalized to an average value of WST1 absorbance for cells heated at 37 °C, as evidenced by the uniform shade in the 37 °C row along the bottom of the figure (n = 3). Note that the time scale is not linear.

Grahic Jump Location
Fig. 2

Apoptosis and necrosis of PC3 cells heated at various temperatures for 10 min. (a) Unheated control, (b) 44 °C, (c) 48 °C, (d) 52 °C, and (e) 60 °C. Cells were analyzed by flow cytometry using annexin-V FITC and PI staining 24 h after heat shock.

Grahic Jump Location
Fig. 3

Apoptosis and necrosis of PC3 cells heated at 44 °C for various durations. (a) Unheated control, (b) 10 min, (c) 30 min, (d) 60 min, and (e) 120 min. Cells were analyzed by flow cytometry using annexin-V FITC and PI staining 24 h after heat shock.

Grahic Jump Location
Fig. 4

Flow cytometry scatter plots of PC3 cells heated at 45 °C for various durations. (a) Unheated control, (b) 30 min, (c) 60 min, (d) 120 min, (e) 180 min. Cells were analyzed by flow cytometry using annexin-V FITC and PI staining 24 h after heat shock.

Grahic Jump Location
Fig. 5

Apoptosis and necrosis of PC3 cells heated in the presence of caspase inhibitors. (a)–(c) Pan-caspase inhibition (Z-VAD-FMK) with thermal dose of 45 °C for 120 min. (d)–(f) Caspase 3 inhibition (Ac-DEVD-CHO) with thermal dose of 45 °C for 180 min. (a) Unheated control for Z-VAD-FMK, (b) heated without Z-VAD-FMK, (c) heated with Z-VAD-FMK, (d) unheated control for Ac-DEVD-CHO, and (e) heated without Ac-DEVD-CHO, and (f) heated with Ac-DEVD-CHO. A delay in the progression to late apoptosis or necrosis is observed in the presence of inhibitor Z-VAD-FMK. No difference is detected in the presence of caspase 3 inhibitor. Cells were analyzed by flow cytometry using annexin-V FITC and PI staining 24 h after heat shock.

Grahic Jump Location
Fig. 6

Caspase 3 activity of PC3 cells heated at various temperatures and durations assessed by Ac-DEVD-AMC fluorometric assay. Cells heated for 3 or 5 min (blue markers) and 10 or 22 min (red markers) at 37 °C (+), 40 °C (○), 44 °C (□), 50 °C (♦), 55 °C (*), 56 °C (▾), 57 °C (Δ), or caspase extract positive control (◁). Cells were processed and analyzed at 6, 12, and 24 h after heat shock. Assay duration is approximately 3 h. Y axis units are arbitrary. No caspase 3 activity from PC3 cells was detected.

Grahic Jump Location
Fig. 7

Caspase 3 activity as assessed by Ac-DEVD-AMC fluorometric assay in PC3 Cells. Cells were heated at 45 °C at the indicated durations. Negative and positive controls were water and apoptotic Jurkat cell extract, respectively. Cells were processed and analyzed 24 h after heat shock. No significant caspase 3 activity was detected (n = 3).

Grahic Jump Location
Fig. 8

Apoptosis and necrosis of 2D and 3D cultured PC3 cells after heating. Cells were heated at the indicated temperatures for 120 min and observed 24 h after heat shock. Samples heated at 45 °C demonstrate a significant difference (n = 3, t-test p < 0.05) between 2D and 3D cultures. Populations have been normalized to respective 37 °C control groups.

Grahic Jump Location
Fig. 9

HSP70 induction in 2D and 3D cultures of PC3 cells at 44 °C and 45 °C for 120 min. The difference in HSP70 induction between the 2D and 3D culture was found to be statistically significant for heating at 45 °C for 120 min (n = 3, t-test p < 0.001).




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