Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun;95(3):350-8.
doi: 10.1016/j.radonc.2010.03.024. Epub 2010 Apr 27.

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

Mark D Hurwitz  1 , Punit Kaur, Ganachari M Nagaraja, Maria A Bausero, Judith Manola, Alexzander Asea
Affiliations

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

Mark D Hurwitz et al. Radiother Oncol. 2010 Jun.

Abstract

Background and purpose: Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined.

Materials and methods: Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-alpha) using the classical sandwich ELISA technique and the relative expression of CD8(+) T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU-145 and PC-3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro.

Results: Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p=0.0002). Increases in IL-6 (3.3-fold), TNF-alpha (1.8-fold), CD8(+) CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC-3 and DU-145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC.

Conclusions: The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies.

PubMed Disclaimer

Conflict of interest statement

Disclosures

The authors have no conflict of interest.

Figures

Fig. 1
Fig. 1
Effect of gamma irradiation on circulating serum Hsp72, immune effector cells and cytokine expression. Patients were enrolled with clinically localized prostate cancer (clinical T1c–T3b, N0, M0) treated with 8 weeks of 3D conformal radiation therapy. Patients with low-risk disease defined as clinical T1–T2a, Gleason ≤6 and PSA <10 ng/ml were treated with radiation alone while patients with adverse risk factors including bulky palpable disease, Gleason ≥7, or PSA >10ng/ml typically received 8 weeks of neoadjuvant androgen suppressive therapy (AST) prior to initiation of radiation therapy (XRT) with AST continued through radiation therapy. A, Serum Hsp72 was measured at various time points pre-AST and post-XRT using the classical sandwich ELISA. Data represent the mean Hsp72 concentration pg/ml ± SD and is the sum of three independent experiments performed in quadruplicates. *, p<0.001 vs control, 0 Gy (Student’s t-test). B, CD8+ (CTL) cells or, C, CD56+ (NK) cells were analyzed by flow cytometry as described in detail in the Methods section. Data are the mean percentage of cells expressing CD8 or CD56 ± SD and are the sum of two independently performed experiments. *, p<0.05 vs respective control (Student’s t-test). D, IL-6 and, E, TNF-α in serum was measured by the classical sandwich ELISA according to the manufactures instructions (BD BioScience). Data represent the mean cytokine concentration pg/ml ± SD and is the sum of two independent experiments performed in quadruplicates. *, p<0.001 vs control (Student’s t-test).
Fig. 2
Fig. 2
Gamma irradiation stimulates the release of tumor-derived HSP into the systemic circulation. Homozygous athymic male BALB/c nude mice (8–10 week-old) were injected with PC-3 (106 cells), DU-145 (106 cells), or PBS into the right hind leg. When human xenografts reached approximately 100 mm3, mice were exposed to 0, Gy, 0.5 Gy or 5 Gy of γ-rays from a 60Cobolt source at a rate of 2 Gy/minute. Blood was drawn at various times post irradiation exposure and serum was treated with 1% Lubrol WX for 10 min at 4°C with gentle rocking, and the concentration of serum Hsp72 (top panels) or Hsp27 (bottom panels) was measured by the classical sandwich ELISA according to the manufactures instructions (StressGen). Data represent the mean serum concentration (pg/ml ± SD) and is the sum of three independent experiments performed in quadruplicates. *, p<0.001 vs control (0 Gy) as determined by the Student’s t-test.
Fig. 3
Fig. 3
Gamma irradiation-induces the release of inflammatory mediators from tumor-bearing but not from non-tumor bearing mice. A, PC-3 (106) cells (left panel) or PBS (right panel) was injected into the right hind leg of 8–10 week-old homozygous athymic male BALB/c nude mice. When human xenografts reached approximately 100 mm3, mice were exposed to 0, Gy, 0.5 Gy or 5 Gy of γ-rays from a 60Cobolt source at a rate of 2 Gy/minute. Blood was drawn at various times post irradiation exposure and serum was treated with 1% Lubrol WX for 10 min at 4°C with gentle rocking, and the concentration of serum TNF-α was measured by the classical sandwich ELISA according to the manufactures instructions (Santa Cruz). Data represent the mean serum TNF-α concentration (pg/ml ± SD) and is the sum of two independent experiments performed in quadruplicates. *, p<0.001 vs control (0 Gy) as determined by the Student’s t-test. B, PC-3 cells (106; left panel) or DU-145 cells (106; right panel) were exposed to 0 Gy, 0.5 Gy or 5 Gy and incubated for indicated times at 37°C with 5% CO2 in air. Twenty-four hours later cells were lysed and total Hsp72 expression measured by Western blot analysis. The intensity of the bands were analyzed by densitometry with a video densitometer (ChemilmagerTM 5500; Alpha Innotech, San Leandro, CA) using the AAB software (American Applied Biology). Data are representative experiment from three independently performed experiments with similar results. C, Exosomes were isolated from PC-3 (107) cells (left panel) or DU-145 (107) cells (right panel) 96 h after exposure 0.5 Gy. Isolated exosomes were analyzed by Western blot using Hsp72, Hsc73, grp94 (StressGen), tubulin, calnexin (Santa Cruz Biotechnology) specific antibodies. The intensity of the bands were analyzed by densitometry with a video densitometer (Chemilmager5500) using the AAB software (American Applied Biology). Data are a representative experiment from two independently performed experiments with similar results.
Fig. 4
Fig. 4
Low dose gamma irradiation induces the release of Hsp72 by a mechanism independent of cells death. A, PC-3 (106) cells (left panel) or DU-145 (106) cells (right panel) were exposed to 0 Gy, 0.5 Gy or 5.0 Gy and at various times post irradiation exposure cells were assayed for viability using the CytoTox 96 Non-Radioactive Cytotoxicity Assay according to the manufactures instructions (Promega), and the percentage of LDH released versus total LDH was calculated. Bars are mean percentage cell death ± SD and represent three independently performed experiments. *, p<0.001 vs control (Student’s t-test). B, PC-3 (106) cells (left panel) or DU-145 (106) cells (right panel) were exposed to 0 Gy, 0.5 Gy or 5.0 Gy. Supernatant was extracted at various times post irradiation exposure and treated with 1% Lubrol WX for 10 min at 4°C with gentle rocking, and the concentration of released Hsp72 was measured by the classical sandwich ELISA. Data represent the mean Hsp72 concentration pg/ml ± SD and is the sum of two independent experiments performed in quadruplicates. *, p<0.001 vs. control, 0 Gy (Student’s t-test).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Beckmann RP, Mizzen LE, Welch WJ. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science. 1990;248:850–854. - PubMed
    1. Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992;355:33–45. - PubMed
    1. Gyrd-Hansen M, Nylandsted J, Jaattela M. Heat shock protein 70 promotes cancer cell viability by safeguarding lysosomal integrity. Cell Cycle. 2004;3:1484–1485. - PubMed
    1. Netzer WJ, Hartl FU. Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms. Trends Biochem Sci. 1998;23:68–73. - PubMed
    1. Nylandsted J, Brand K, Jaattela M. Heat shock protein 70 is required for the survival of cancer cells. Annals of the New York Academy of Sciences. 2000;926:122–125. - PubMed

Publication types

Substances

  NODES
Note 2
twitter 2