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Additive Effects of 5-Aza-2’-deoxycytidine and Irradiation on Clonogenic Survival of Human Medulloblastoma Cell Lines

Additive Effekte von 5-Aza-2’-deoxycytidin und Bestrahlung auf das klonogene Überleben von humanen Medulloblastomzelllinien

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Background and Purpose:

In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2’-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.

Material and Methods:

Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 μM, 6 days) or high-dose (3/5 μM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.

Results:

5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of α-, β-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.

Conclusion:

5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.

Hintergrund und Ziel:

In den letzten Jahren wurden epigenetische Modulatoren in die Tumortherapie eingeführt. In dieser Arbeit untersuchten die Autoren den Antitumoreffekt der 5-Aza-2’-deoxycytidin-(5-aza-dC-)induzierten Demethylierung in Kombination mit Bestrahlung auf humane Medulloblastom-(MB-)Zellen, welche die häufigsten malignen Hirntumoren im Kindesalter bilden.

Material und Methodik:

Drei MB-Zell-Linien wurden mit 5-aza-dC in Niedrigdosis (0,1 μM, 6 Tage) oder Hochdosis (3/5 μM, 3 Tage) behandelt und mit 2, 4, 6 oder 8 Gy Einzeldosis bestrahlt. Die Untersuchung des Methylierungsstatus und der mRNA-Expression von drei Kandidatengenen erfolgte durch methylierungsspezifische PCR (Polymerase-Kettenreaktion) und quantitative Real-Time-RT-PCR. Lebendzellraten und Mortalität wurden durch Trypanblau-Ausschlusstest und die Proliferationsrate im BrdU-Assay bestimmt. Das Langzeitüberleben wurde durch einen klonogenen Assay ermittelt.

Ergebnisse:

Die 5-aza-dC-Behandlung führte zur partiellen Promotordemethylierung und zu einem Anstieg der Expression hypermethylierter Kandidatengene. Eine signifikante Verminderung der Lebendzellzahl und Proliferation bei Zunahme der Mortalität wurde sowohl in 5-aza-dC-behandelten als auch in bestrahlten MB-Zellen beobachtet. Bei kombinierter Behandlung summierten sich die Effekte der Einzelbehandlungen. Während die Inkubation mit 5-aza-dC in Hochdosis hinsichtlich der Demethylierung effektiver war als in Niedrigdosis, ergaben sich im klonogenen Assay keine Unterschiede zwischen beiden Behandlungsschemata, was darauf hinweist, dass die 5-aza-dC-induzierte Demethylierung nicht relevant für die Verminderung des Zellüberlebens ist. Mit 5-aza-dC vorbehandelte Zellen zeigten im untersuchten Bestrahlungsdosisbereich eine signifikant niedrigere „plating efficiency“ als unbehandelte Zellen. Die Überlebenskurven bestrahlter MB-Zellen wiesen jedoch keine signifikanten Unterschiede der α/β-Werte und der Überlebensfraktion nach 2 Gy in 5-aza-dC-behandelten gegenüber unbehandelten Zellen auf.

Schlussfolgerung:

5-aza-dC erhöhte die Strahlensensitivität von MB-Zellen nicht, reduzierte jedoch die Klonogenität signifikant gegenüber alleiniger Bestrahlung, was weitere Untersuchungen zur potentiellen klinischen Anwendung von 5-aza-dC bei MB, möglicherweise in Kombination mit anderen Chemotherapeutika, rechtfertigt.

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Authors and Affiliations

  1. Department of Radiotherapy and Radiooncology, Universitätsklinikum Leipzig AöR, Leipzig, Germany

    Ina Patties, Jutta Jahns, Guido Hildebrandt, Rolf-Dieter Kortmann &  Annegret Glasow

  2. Department of Radiotherapy, University of Rostock, Rostock, Germany

    Guido Hildebrandt

  3. Klinik und Poliklinik für Strahlentherapie und Radioonkologie, Universitätsklinikum Leipzig AöR, Stephanstraße 9a, 04103, Leipzig, Germany

    Annegret Glasow

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  1. Ina Patties
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  2. Jutta Jahns
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  5. Annegret Glasow
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Correspondence to Annegret Glasow.

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Patties, I., Jahns, J., Hildebrandt, G. et al. Additive Effects of 5-Aza-2’-deoxycytidine and Irradiation on Clonogenic Survival of Human Medulloblastoma Cell Lines. Strahlenther Onkol 185, 331–338 (2009). https://doi.org/10.1007/s00066-009-1956-1

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  • DOI: https://doi.org/10.1007/s00066-009-1956-1

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