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Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

Hypoxia-inducible-Factor-1α-Small-Interfering-RNA inhibiert die hypoxische Akkumulation von HIF-1 α und erhöht die Strahlensensitivität von HT-1080-Fibrosarkomzellen in vitro

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Background:

Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells.

Material and Methods:

HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER´) was calculated as the ratio of the doses to achieve the same survival at 0.1% O2 as at ambient oxygen tensions. OER´ was obtained at cell survival levels of 50%, 37%, and 10%.

Results:

HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro.

Conclusion:

Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro.

Hintergrund und Ziel:

Hypoxia-inducible Factor-1 (HIF-1) wurde als potentielles therapeutisches Target identifiziert. Ziel der Arbeit war es, zu untersuchen, ob die selektive HIF-1-Inhibition mittels Small Interfering RNA (siRNA) gegen HIF-1α die Strahlensensibilität von hypoxischen HT-1080-Zellen beeinflussen kann.

Material und Methodik:

Die HIF-1α-Expression in humanen HT-1080-Fibrosarkomzellen wurde mittels RNA-Interferenz nach Transfektion der Zellen mit siRNA unter hypoxischen Bedingungen (0,1%, O2, 12 h), bzw. Normoxie (20% O2) in vitro inhibiert. Die HIF-1α-Genexpression wurde mit quantitativer Realtime-Polymerasekettenreaktion (qRT-PCR), das HIF-1α-Protein mittels Western Blot quantifiziert. Das klonogene Überleben wurde nach Bestrahlung unter Hypoxie und Normoxie bestimmt und daraus eine Oxygen Enhancement Ratio (OER´) bei den Überlebensniveaus 50%, 37% and 10% berechnet.

Resultate:

HIF-1α-siRNA erhöht die Strahlensensibilität unter hypoxischen Bedingen, verglichen mit HT-1080-Zellen, die mit Kontroll-siRNA behandelt wurden. Die OER` war bei allen Überlebensniveaus reduziert.

Schlussfolgerung:

Eine selektive Inhibition der HIF-1-Aktivierung durch HIF-1α-siRNA wirkt synergistisch mit einer Bestrahlung und erhöht die Strahlensensitivität hypoxischer Tumorzellen in vitro.

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Authors and Affiliations

  1. Department of Radiation Oncology, University of Würzburg, Würzburg, Germany

    Adrian Staab MD, Markus Fleischer, Harun M. Said, Astrid Katzer, Michael Flentje & Dirk Vordermark

  2. Medical Clinic II, University of Würzburg, Würzburg, Germany

    Markus Fleischer, Juergen Loeffler & Herrmann Einsele

  3. Department of Nuclear Medicine, University of Freiburg, Freiburg, Germany

    Christian Plathow

  4. Department of Radiation Oncology, Martin Luther University Halle-Wittenberg, Halle-Wittenberg, Germany

    Dirk Vordermark

  5. Paul Scherrer Institute, Villigen, Switzerland

    Adrian Staab MD

  6. Center for Proton Radiotherapy, Paul Scherrer Institute, 5232, Villigen PSI, Switzerland

    Adrian Staab MD

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  1. Adrian Staab MD
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  2. Markus Fleischer
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Correspondence to Adrian Staab MD.

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Staab, A., Fleischer, M., Loeffler, J. et al. Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro. Strahlenther Onkol 187, 252–259 (2011). https://doi.org/10.1007/s00066-011-2167-0

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  • DOI: https://doi.org/10.1007/s00066-011-2167-0

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