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Wnt/β-catenin pathway involvement in ionizing radiation-induced invasion of U87 glioblastoma cells

Beteiligung des Wnt/β-Catenin-Signalwegs an der strahleninduzierten Invasion von U87-Glioblastomzellen

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Abstract

Background

Radiotherapy has been reported to promote the invasion of glioblastoma cells; however, the underlying mechanisms remain unclear. Here, we investigated the role of the Wnt/β-catenin pathway in radiation-induced invasion of glioblastoma cells.

Methods

U87 cells were irradiated with 3 Gy or sham irradiated in the presence or absence of the Wnt/β-catenin pathway inhibitor XAV 939. Cell invasion was determined by an xCELLigence real-time cell analyser and matrigel invasion assays. The intracellular distribution of β-catenin in U87 cells with or without irradiation was examined by immunofluorescence and Western blotting of nuclear fractions. We next investigated the effect of irradiation on Wnt/β-catenin pathway activity using TOP/FOP flash luciferase assays and quantitative polymerase chain reaction analysis of β-catenin target genes. The expression levels and activities of two target genes, matrix metalloproteinase (MMP)-2 and MMP-9, were examined further by Western blotting and zymography.

Results

U87 cell invasiveness was increased significantly by ionizing radiation. Interestingly, ionizing radiation induced nuclear translocation and accumulation of β-catenin. Moreover, we found increased β-catenin/TCF transcriptional activities, followed by up-regulation of downstream genes in the Wnt/β-catenin pathway in irradiated U87 cells. Importantly, inhibition of the Wnt/β-catenin pathway by XAV 939, which promotes degradation of β-catenin, significantly abrogated the pro-invasion effects of irradiation. Mechanistically, XAV 939 suppressed ionizing radiation-triggered up-regulation of MMP-2 and MMP-9, and inhibited the activities of these gelatinases.

Conclusion

Our data demonstrate a pivotal role of the Wnt/β-catenin pathway in ionizing radiation-induced invasion of glioblastoma cells, and suggest that targeting β-catenin is a promising therapeutic approach to overcoming glioma radioresistance.

Zusammenfassung

Hintergrund

Studien haben gezeigt, dass eine Strahlentherapie die Invasivität von Glioblastomzellen erhöht. Zwar wurden mehrere Signalwege mit diesem strahleninduzierten Eindringen in Zusammenhang gebracht, doch die genauen Mechanismen sind bisher unklar. In der vorliegenden Studie wurden die proinvasive Wirkung einer Bestrahlung auf U87-Zellen überprüft und die Beteiligung des Wnt/ß-Catenin-Signalwegs als möglicher zugrundeliegender Mechanismus diskutiert.

Methoden

U87-Zellen wurden einer Strahlung von 3 Gy oder einer Scheinbestrahlung ausgesetzt, einige Zellen waren zuvor mit dem Wnt/ß-Catenin-Inhibitor XAV 939 behandelt worden. Der Einfluss dieser verschiedenen Konditionen auf die Invasivität wurde anschließend mit dem xCELLigence Zellanalysesystems und einem Matrigel-Invasion-Assay überprüft. Die intrazelluläre ß-Catenin-Verteilung mit bzw. ohne Bestrahlung wurde anhand von Western-Blot-Analysen unterschiedlicher Kernfraktionen und Immunfluoreszenzanalysen untersucht, die Wirkung der Strahlung auf die Aktivität des Wnt/ß-Catenin-Signalwegs durch einen Luciferase-Assay. Die Expressionslevel und Aktivität von Zielgenen, wie MMP-2 und MMP-9, wurden durch eine Western-Blot-Analyse und einen Zymographie-Assay determiniert.

Ergebnisse

Die Invasivität von U87-Zellen wurde durch ionisierende Strahlung signifikant erhöht. Immunfluoreszenzanalysen zeigten eine strahleninduzierte nukleäre Translokation und eine ß-Catenin-Akkumulation. Nach Bestrahlung zeigten sich im Luciferase-Assay zudem eine erhöhte ß-Catenin/TCF-Transkriptionsaktivität, gefolgt von einer Hochregulation der Downstream-Zielgene in den Wnt/ß-Catenin-Signalwegen in bestrahlten U87-Zellen. XV 939 unterdrückte die strahleninduzierte Hochregulierung von MMP-2 und -9 und inhibierte die Aktivität dieser Gelatinasen.

Fazit

Die vorgestellten Daten zeigen die zentrale Rolle des Wnt/ß-Catenin-Signalwegs für die strahleninduzierte Erhöhung der Invasivität von Glioblastomzellen. In diesem Zusammenhang könnte die Inhibierung von ß-Catenin einen viel versprechenden Ansatz in der Behandlung strahlenresistenter Gliome darstellen.

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Acknowledgements

This research was in part supported by a grant from the National Natural Sciences Foundation of China (No. 81272780). We gratefully thank Dr. Fengjuan Fan and Dr. Anna-Lena Scherr, Centrum für Tumorerkrankungen, Heidelberg, Germany, for help with the manuscript. Authorsʼ contributions: Z.D. cultured the U87 cells and conducted RTCA and matrigel invasion assay, participated in real-time quantitative PCR and drafted the manuscript. L.Z. carried out the immunofluorescence staining and colocalization analysis. N.H. participated in the zymography and immunoblotting. M.Z. participated in the design of the study and performed the statistical analysis. X.L. conceived of the study, and participated in its design and coordination and prepared the final manuscript. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430030, Wuhan, PR China

    Zhen Dong MD

  2. Department of Histoembryology, Tongji Medical College, Huazhong University of Science and Technology, 430030, Wuhan, PR China

    Lin Zhou PhD

  3. Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430030, Wuhan, PR China

    Na Han MD & Mengxian Zhang MD

  4. Department of Oncology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, 430024, Wuhan, PR China

    Xiaojuan Lyu MD

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Z. Dong, L. Zhou, N. Han, M. Zhang, and X. Lyu state that there are no conflicts of interest. The accompanying manuscript does not include studies on humans or animals.

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Dong, Z., Zhou, L., Han, N. et al. Wnt/β-catenin pathway involvement in ionizing radiation-induced invasion of U87 glioblastoma cells. Strahlenther Onkol 191, 672–680 (2015). https://doi.org/10.1007/s00066-015-0858-7

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