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Combination of Ionising Irradiation and Hyperthermia Activates Programmed Apoptotic and Necrotic Cell Death Pathways in Human Colorectal Carcinoma Cells

Kombination von ionisierender Strahlung und Hyperthermie aktiviert programmierte apoptotische und nekrotische Zelltodessignalwege in humanen kolorektalen Tumorzellen

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Abstract

Purpose:

The malignancy of tumor cells can be attenuated by interfering with cell death pathways. Since hyperthermia (HT) is a very potent radiosensitizer, the influence of HT (41.5 °C for 1 hour) alone and in combination with ionising irradiation (X-ray; 5 Gy or 10 Gy) on the form of cell death as well as on the expression of proteins known to be major components in tumor cells’ apoptotic and necrotic pathways were examined in colorectal tumor cells.

Material and Methods:

The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluorescence- based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/propidium iodide staining.

Results:

Combining X-ray with HT led to similar activation of caspase-3/7 and p53 expression in comparison to irradiation only while the amount of the pro-apoptotic proteins PUMA and Bax was increased in HCT15 and SW480 cells. HT alone or combinations with X-ray further resulted in a temporarily increased level of the anti-apoptotic protein Bcl-2. Irradiation plus HT further led to an up-regulation of IRF-5. The levels of RIP-1, a marker for programmed necrosis, increased in tumor cells which were treated with HT and/or X-ray. Combining 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased colony formation.

Conclusion:

The combined treatment of colorectal tumor cells with X-ray and HT activates distinct tumor cell pathways and fosters the early appearance of a necrotic tumor cell phenotype.

Zusammenfassung

Hintergrund:

Die Malignität von Tumorzellen kann durch Eingriffe in Zelltodeswege abgemindert werden. Da Hyperthermie (HT) strahlensensibilisierend wirkt haben wir in kolorektalen Tumorzelllinien den Einfluss von HT (41.5 °C für 1 Stunde) alleine oder in Kombination mit ionisierender Strahlung (X-ray, 5 oder 10 Gy) auf Tumorzelltodesformen und auf die Expression von Proteinen, welche Hauptbestandteile von apoptotischen und nekrotischen Tumorzellsignalwegen sind, untersucht.

Material und Methodik:

Die Expression der Proteine wurde mit Western-Blot-Technik und die relative Aktivität von Caspasen 3/7 mit Fluoreszenz basierendem Ansatz bestimmt. Die Koloniebildungskapazität wurde mit klonogenem Assay und Zelltod mittels AnnexinVFITC/ Propidiumjodid Färbung ermittelt.

Ergebnisse:

Kombinationen von X-ray mit HT führten zu vergleichbaren Aktivierung von Caspase 3/7 und p53-Expression im Vergleich zur alleinigen Bestrahlung, wohingegen die Menge der pro-apoptotischen Proteine PUMA und Bax in HCT15- und SW480-Zellen zunahm. Alleinige HT Behandlung oder Kombinationen mit X-ray resultierten in einem vorübergehend erhöhten Level an anti-apoptotischem Protein Bcl-2. Bestrahlungen plus HT führten weiterhin zu einer Hochregulation an IRF-5. Die Mengen an RIP-1, welches ein Marker für programmierte Nekrose darstellt, waren nach Behandlung der Tumorzellen mit HT und/oder X-ray erhöht. Die Kombination von Bestrahlung mit 5Gy mit HT im Vergleich zur alleinigen Bestrahlung resultierte in einer signifikant erhöhten Anzahl an nekrotischen Tumorzellen und einer signifikant erniedrigten Koloniebildung.

Schlussfolgerung:

Kombinationsbehandlungen von kolorektalen Tumorzellen mit X-ray und HT aktivieren distinkte Tumorzelltodessignalwege und fördern das rasche Auftreten eines nekrotischen Tumorzellphänotyps.

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Authors and Affiliations

  1. Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany

    Frederick Mantel, Benjamin Frey, Stefan Haslinger, Petra Schildkopf, Renate Sieber, Oliver J. Ott, Barbara Lödermann, Rolf Sauer, Rainer Fietkau &  Udo S. Gaipl PhD

  2. Department of Radiotherapy and Oncology, University of Frankfurt, Frankfurt, Germany

    Franz Rödel

  3. Department of Radiation Oncology, Radiation Immunobiology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Universitätsstr. 27, 91054, Erlangen, Germany

    Udo S. Gaipl PhD

Authors
  1. Frederick Mantel
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  2. Benjamin Frey
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  3. Stefan Haslinger
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  4. Petra Schildkopf
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  6. Oliver J. Ott
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  7. Barbara Lödermann
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  8. Franz Rödel
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  9. Rolf Sauer
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  11. Udo S. Gaipl PhD
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Correspondence to Udo S. Gaipl PhD.

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* F. Mantel and B. Frey contributed equally to this work

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Mantel, F., Frey, B., Haslinger, S. et al. Combination of Ionising Irradiation and Hyperthermia Activates Programmed Apoptotic and Necrotic Cell Death Pathways in Human Colorectal Carcinoma Cells. Strahlenther Onkol 186, 587–599 (2010). https://doi.org/10.1007/s00066-010-2154-x

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  • DOI: https://doi.org/10.1007/s00066-010-2154-x

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