Advertisement

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

  • Adrian StaabEmail author
  • Markus Fleischer
  • Juergen Loeffler
  • Harun M. Said
  • Astrid Katzer
  • Christian Plathow
  • Herrmann Einsele
  • Michael Flentje
  • Dirk Vordermark
Original Article

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.

Key Words

Hypoxia inducible factor-1α Small interfering RNA Hypoxia Radiation Oxygen 

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

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.

Schlüsselwörter

Hypoxia-inducible factor-1α Small Interfering RNA Hypoxie Bestrahlung Sauerstoff 

References

  1. 1.
    Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res 2001;61:2911–6.PubMedGoogle Scholar
  2. 2.
    Bache M, Kappler M, Said HM, et al. Detection and specific targeting of hypoxic regions within solid tumors: current preclinical and clinical strategies. Curr Med Chem 2008;15:322–38.PubMedCrossRefGoogle Scholar
  3. 3.
    Balducci M, Apicella G, Manfrida S, et al. Single-arm phase II study of conformal radiation therapy and temozolomide plus fractionated stereotactic conformal boost in high-grade gliomas: final report. Strahlenther Onkol 2010;186:558–64.PubMedCrossRefGoogle Scholar
  4. 4.
    Comerford KM, Cummins EP, Taylor CT. c-Jun NH2-Terminal kinase activation contributes to hypoxia-inducible factor 1a-dependent Pglycoprotein expression in hypoxia. Cancer Res 2004;15:9057–61.CrossRefGoogle Scholar
  5. 5.
    Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;15:3387–94.Google Scholar
  6. 6.
    Dellas K, Bache M, Pigorsch SU, et al. Prognostic impact of HIF-1alpha expression in patients with definitive radiotherapy for cervical cancer. Strahlenther Onkol 2008;184:169–74.PubMedCrossRefGoogle Scholar
  7. 7.
    Epstein AC, Gleadle JM, McNeill LA, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001;107:43–54.PubMedCrossRefGoogle Scholar
  8. 8.
    Gillespie DL, Whang K, Ragel BT, et al. Silencing of hypoxia-inducible factor-1alpha by RNA interference attenuates human glioma cell growth in vivo. Clin Cancer Res 2007;13:2441–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Griffiths EA, Pritchard SA, Valentine HR, et al. Hypoxia-inducible factor-1alpha expression in the gastric carcinogenesis sequence and its prognostic role in gastric and gastro-oesophageal adenocarcinomas. Br J Cancer 2007; 96:95–103.PubMedCrossRefGoogle Scholar
  10. 10.
    Hall, EJ. Radiobiology for the radiologist. 4. ed. Philadelphia: Lippincott; 1994Google Scholar
  11. 11.
    Harris AL. Hypoxia—a key regulatory factor in tumour growth. Nature Reviews Cancer 2002;2:38–47.PubMedCrossRefGoogle Scholar
  12. 12.
    Ivan M, Kondo K, Yang H, et al. HIF alpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292:464–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72.PubMedCrossRefGoogle Scholar
  14. 14.
    Kappler, Taubert H, Holzhausen HJ, et al. Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters. Strahlenther Onkol 2008;184:393–9.PubMedCrossRefGoogle Scholar
  15. 15.
    Khan ZA, Chakrabarti S. Cellular signaling and potential new treatment targets in diabetic retinopathy. Experimental Diabetes Research 2007;2007:31867.PubMedCrossRefGoogle Scholar
  16. 16.
    Kung AL, Zabludoff SD, France DS, et al. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell 2004;6:33–43.PubMedCrossRefGoogle Scholar
  17. 17.
    Lau SK, Boutros PC, Pintilie M, et al. Three-gene prognostic classifier for early-stage non small-cell lung cancer. J Clin Oncol 2007;25:5562-9.Google Scholar
  18. 18.
    Lövey J, Kenessey I, Raso E, et al. Human recombinant erythropoietin-alpha increases the efficacy of irradiation in preclinical model. Magy Onkol 2007;51:53–61.PubMedGoogle Scholar
  19. 19.
    Malhotra R, Tyson DW, Rosevear HM, et al. Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells. BMC Cardiovascular Disorders 2008;8:9.PubMedCrossRefGoogle Scholar
  20. 20.
    Matsuyama T, Nakanishi K, Hayashi T, et al. Expression of hypoxia-inducible factor-1alpha in esophageal squamous cell carcinoma. Cancer Sci 2005;96:176–82.PubMedCrossRefGoogle Scholar
  21. 21.
    Moeller BJ, Dewhirst MW. HIF-1 and tumour radiosensitivity. Br J Cancer 2006;95:1–5.PubMedCrossRefGoogle Scholar
  22. 22.
    Semenza GL. Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov. Today 2007;12:853–9.PubMedCrossRefGoogle Scholar
  23. 23.
    Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for tumor-specific gene therapy. Gene Ther 2000;7:493–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Sorensen BS, Alsner J, Overgaard J et al. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH. Radiother Oncol 2007;83:362–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Staab A, Loeffler J, Said HM, et al. Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells. BMC Cancer 2007;7:213.PubMedCrossRefGoogle Scholar
  26. 26.
    Staab A, Loffler J, Said HM, Katzer A, et al. Modulation of glucose metabolism inhibits hypoxic accumulation of Hypoxia-inducible factor-1a (HIF-1alpha). Strahlenther Onkol 2007;183:366–73.PubMedCrossRefGoogle Scholar
  27. 27.
    Takahashi Y, Nishikawa M, Takakura Y. Inhibition of tumor cell growth in the liver by RNA interference-mediated suppression of HIF-1alpha expression in tumor cells and hepatocytes. Gene Ther 2008,15:572–82.PubMedCrossRefGoogle Scholar
  28. 28.
    Tinkl D, Grabenbauer GG, Golcher H, et al. Downstaging of pancreatic carcinoma after neoadjuvant chemoradiation. Strahlenther Onkol 2009;185:557–66.PubMedCrossRefGoogle Scholar
  29. 29.
    Trastour C, Benizri E, Ettore F, et al. HIF-1alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome. Int J Cancer 2007;120:1451–8.PubMedCrossRefGoogle Scholar
  30. 30.
    Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26:225–39.PubMedCrossRefGoogle Scholar
  31. 31.
    Vordermark D, Katzer A, Baier K, et al. M. Cell type-specific association of hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein accumulation and radiobiologic tumor hypoxia. Int J Radiat Oncol Biol Phys 2004;58:1242–50.PubMedCrossRefGoogle Scholar
  32. 32.
    Vordermark D, Shibata T, Brown JM. Green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation. Neoplasia 2001;3:527–34.PubMedCrossRefGoogle Scholar
  33. 33.
    Vordermark D, Brown JM. Endogenous markers of tumor hypoxia predictors of clinical radiation resistance? Strahlenther Onkol 2003;179:801–11.PubMedCrossRefGoogle Scholar
  34. 34.
    Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4.PubMedCrossRefGoogle Scholar
  35. 35.
    Welsh S, Williams R, Kirkpatrick L, et al. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1alpha. Mol Cancer Ther 2004;3:233–44.PubMedGoogle Scholar
  36. 36.
    Wolf M, Zehentmayr F, Niyazi M, et al. Long-term outcome of mitomycin C- and 5-FU-based primary radiochemotherapy for esophageal cancer. Strahlenther Onkol 2010;186:374–81.PubMedCrossRefGoogle Scholar
  37. 37.
    Williams KJ, Telfer BA, Xenaki D, et al. Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1. Radiother Oncol 2005;75:89–98.PubMedCrossRefGoogle Scholar
  38. 38.
    Wurstbauer K, Weise H, Deutschmann H, et al. Non-small cell lung cancer in stages I–IIIB: long-term results of definitive radiotherapy with doses. 80 Gy in standard fractionation. Strahlenther Onkol 2010;186:551–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Yang L, Kang WK. The effect of HIF-α siRNA on growth and chemosensitivity of Mia-paca cell line. Yonsei Med J 2008;49:295–300.PubMedCrossRefGoogle Scholar

Copyright information

© Urban & Vogel, Muenchen 2011

Authors and Affiliations

  • Adrian Staab
    • 1
    • 5
    • 6
    Email author
  • Markus Fleischer
    • 1
    • 2
  • Juergen Loeffler
    • 2
  • Harun M. Said
    • 1
  • Astrid Katzer
    • 1
  • Christian Plathow
    • 3
  • Herrmann Einsele
    • 2
  • Michael Flentje
    • 1
  • Dirk Vordermark
    • 1
    • 4
  1. 1.Department of Radiation OncologyUniversity of WürzburgWürzburgGermany
  2. 2.Medical Clinic IIUniversity of WürzburgWürzburgGermany
  3. 3.Department of Nuclear MedicineUniversity of FreiburgFreiburgGermany
  4. 4.Department of Radiation OncologyMartin Luther University Halle-WittenbergHalle-WittenbergGermany
  5. 5.Paul Scherrer InstituteVilligenSwitzerland
  6. 6. Center for Proton RadiotherapyPaul Scherrer InstituteVilligen PSISwitzerland

Personalised recommendations