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DNA fragmentation and caspase-independent programmed cell death by modulated electrohyperthermia

DNA-Fragmentierung und caspaseunabhängiger programmierter Zelltod durch modulierte Elektrohyperthermie

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Abstract

Background and purpose

The electric field and the concomitant heat (electrohyperthermia) can synergistically induce cell death in tumor tissue, due to elevated glycolysis, ion concentration, and permittivity in malignant compared with nonmalignant tissues. Here we studied the mechanism and time course of tumor destruction caused by electrohyperthermia.

Material and methods

Bilateral implants of HT29 colorectal cancer in the femoral regions of Balb/c (nu/nu) mice were treated with a single 30-min shot of modulated, 13.56-MHz, radiofrequency-generated electrohyperthermia (mEHT). Tumors at 0, 1, 4, 8, 14, 24, 48, and 72 h posttreatment were studied for morphology, DNA fragmentation, and cell death response-related protein expression using tissue microarrays, immunohistochemistry, Western immunoblots, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.

Results

Modulated EHT treatment induced significant tumor destruction in HT29 xenografts with a peak of a sevenfold increase compared with the untreated controls. The significant treatment-related elevation of DNA fragmentation—detected with TUNEL assay—and apoptotic bodies between 24 and 72 h posttreatment was proof of a programmed cell death response. This was associated with significant mitochondrial accumulation of bax and mitochondrial-to-cytoplasmic release of cytochrome c proteins between 8 and 14 h. Cleaved caspase-3 levels were low and mainly localized to inflammatory cells. The substantial cytoplasmic-to-nuclear translocation of apoptosis-inducing factor (AIF) and its 57-kDa activated fragment detected between 14 and 24 h after treatment indicated AIF as an effector for DNA fragmentation.

Conclusion

Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Zusammenfassung

Hintergrund und Ziel

Die gleichzeitige Einwirkung elektrischer Felder und Hitze (Elektrohyperthermie) kann durch eine im Vergleich zu gesunden Zellen erhöhte Glykolyse, Ionenkonzentration und elektrische Leitfähigkeit in Tumorgewebe synergistisch zum Zelltod führen. Diese Arbeit untersucht den Mechanismus und den Zeitverlauf der durch Elektrohyperthermie induzierten Tumorzerstörung.

Material und Methoden

Bilaterale Implantate von HT29-Kolorektalkrebszellen im Oberschenkelbereich von Balb/c-(nu/nu-)-Mäusen wurden 30 min lang einer durch eine modulierte Radiofrequenz von 13,56 MHz erzeugten Elektrohyperthermie (mEHT) ausgesetzt. Jeweils 0, 1, 4, 8, 14, 24, 48 und 72 h nach Behandlung wurden die Tumore hinsichtlich Morphologie, DNA-Fragmentierung und Zelltodantwort auf Proteinexpressionsebene untersucht, wobei Gewebe-Mikroarrays, immunhistochemische Methoden, Western-Blots und TUNEL-Assays ("Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling") eingesetzt wurden.

Ergebnisse

Die mEHT-Behandlung führte in HT29-Xenografts zu einer signifikanten Zerstörung von Tumorgewebe vom bis zum 7-Fachen der unbehandelten Kontrollen. Die signifikante behandlungsinduzierte DNA-Fragmentierung (Detektion durch TUNEL-Assay) und Bildung von Apoptosekörpern 24–72 h nach Behandlung ließ erkennen, dass es zum programmierten Zelltod gekommen war. Daneben wurde nach 8–14 h eine signifikante Akkumulation von Bax in den Mitochondrien und eine Freisetzung von mitochondrialem Cytochrom c in das Zellplasma beobachtet. Die Konzentration an gespaltener Caspase-3 war niedrig und im Wesentlichen auf Entzündungszellen beschränkt. Die 14–24 h nach Behandlung festgestellte umfangreiche Translokation von zytoplasmatischem apoptoseinduzierendem Faktor (AIF) und seinem aktiven 57-kDa-Fragment in den Kern deutete auf AIF als Auslöser für die DNA-Fragmentierung hin.

Schlussfolgerung

Die mEHT-Behandlung kann in kolorektalen HT29-Adenokarzinom-Xenografts Tumorgewebe durch Induktion des programmierten Zelltods zerstören, wobei hauptsächlich ein caspaseunabhängiger Pfad beschritten wird.

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Acknowledgments

The authors are grateful to Edit Parsch, Renata Papp, Marcell A. Szasz, Agoston Ghidan, Diana Brauswetter, and Sandor Kiss for their technical assistance. This work was supported by grants KMOP-1.1.4-07/1-2008-0083, KMOP 1.1.1-08/1-2008-0059, and TÉT-10-1-2011-0914 in Hungary.

Compliance with ethical guidelines

Conflict of interest

N. Meggyeshazi, G. Andocs, L. Balogh, P. Balla, G. Kiszner, I. Teleki, A. Jeney, and T. Krenacs state that there are no conflicts of interest. All national guidelines on the care and use of laboratory animals have been followed and the necessary approval was obtained from the relevant authorities.

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Authors and Affiliations

  1. 1st Department of Pathology and Experimental Cancer Research MTA-SE Tumor Progression Research Group, Semmelweis University, Ulloi ut 26, 1085, Budapest, Hungary

    N. Meggyeshazi, P. Balla, G. Kiszner, I. Teleki, A. Jeney & T. Krenacs PhD

  2. Department of Veterinary Clinical Medicine, Faculty of Veterinary Science, Tottori University, Tottori, Japan

    G. Andocs

  3. Frederic Joliot Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary

    L. Balogh

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  1. N. Meggyeshazi
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  2. G. Andocs
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Correspondence to T. Krenacs PhD.

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Meggyeshazi, N., Andocs, G., Balogh, L. et al. DNA fragmentation and caspase-independent programmed cell death by modulated electrohyperthermia. Strahlenther Onkol 190, 815–822 (2014). https://doi.org/10.1007/s00066-014-0617-1

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  • DOI: https://doi.org/10.1007/s00066-014-0617-1

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