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Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells.

Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, Fleischer M, Schwab F, Baier K, Einsele H, Flentje M, Vordermark D - BMC Cancer (2007)

Bottom Line: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis.Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Würzburg, 97080 Würzburg, Germany. staab_a@klinik.uni-wuerzburg.de

ABSTRACT

Background: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells.

Methods: Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h).

Results: Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 +/- 7.2% and 39.6 +/- 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.

Conclusion: HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

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Related in: MedlinePlus

Chetomin (CHT) inhibits the hypoxic activation of hypoxia-responsive-element-(HRE-) mediated EGFP fluorescence in stably transfected HT 1080 human fibrosarcoma cells in a dose and incubation-time dependent manner. After chetomin pre-treatment for two or four hours, HT 1080 cells were cultured for 12 hours under hypoxia resulting in total chetomin treatment times of 14 or 16 h as indicated. A maximum suppression of EGFP fluorescence was achieved when cells were treated with 150 nM chetomin for 16 hours (representative FACS experiment). Higher doses had no additional effect.
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Figure 2: Chetomin (CHT) inhibits the hypoxic activation of hypoxia-responsive-element-(HRE-) mediated EGFP fluorescence in stably transfected HT 1080 human fibrosarcoma cells in a dose and incubation-time dependent manner. After chetomin pre-treatment for two or four hours, HT 1080 cells were cultured for 12 hours under hypoxia resulting in total chetomin treatment times of 14 or 16 h as indicated. A maximum suppression of EGFP fluorescence was achieved when cells were treated with 150 nM chetomin for 16 hours (representative FACS experiment). Higher doses had no additional effect.

Mentions: In HT 1080 cells stably transfected with a construct of EGFP under the control of a hypoxia-responsive 5HRE-hCMVmp promoter, the addition of chetomin markedly suppressed the hypoxic increase in EGFP fluorescence signal (Figure 1). This suppression of EGFP fluorescence signal was dose and incubation-time dependent (Figure 2). A maximum suppression of EGFP fluorescence signal was measured when chetomin was added four hours prior hypoxia treatment (0.1% O2, 12 h) and in a final concentration of 150 nM. Higher doses of chetomin led to an increasing proportion of completely EGFP-negative cells indicating substantial cytotoxicity (data not shown). Chetomin had no effect on HRE-independent EGFP fluorescence as shown by comparable fluorescence in chetomin-treated (150 nM) and control HT 1080 cells transfected with the constitutive CMV promoter (Figure 1).


Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells.

Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, Fleischer M, Schwab F, Baier K, Einsele H, Flentje M, Vordermark D - BMC Cancer (2007)

Chetomin (CHT) inhibits the hypoxic activation of hypoxia-responsive-element-(HRE-) mediated EGFP fluorescence in stably transfected HT 1080 human fibrosarcoma cells in a dose and incubation-time dependent manner. After chetomin pre-treatment for two or four hours, HT 1080 cells were cultured for 12 hours under hypoxia resulting in total chetomin treatment times of 14 or 16 h as indicated. A maximum suppression of EGFP fluorescence was achieved when cells were treated with 150 nM chetomin for 16 hours (representative FACS experiment). Higher doses had no additional effect.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2200672&req=5

Figure 2: Chetomin (CHT) inhibits the hypoxic activation of hypoxia-responsive-element-(HRE-) mediated EGFP fluorescence in stably transfected HT 1080 human fibrosarcoma cells in a dose and incubation-time dependent manner. After chetomin pre-treatment for two or four hours, HT 1080 cells were cultured for 12 hours under hypoxia resulting in total chetomin treatment times of 14 or 16 h as indicated. A maximum suppression of EGFP fluorescence was achieved when cells were treated with 150 nM chetomin for 16 hours (representative FACS experiment). Higher doses had no additional effect.
Mentions: In HT 1080 cells stably transfected with a construct of EGFP under the control of a hypoxia-responsive 5HRE-hCMVmp promoter, the addition of chetomin markedly suppressed the hypoxic increase in EGFP fluorescence signal (Figure 1). This suppression of EGFP fluorescence signal was dose and incubation-time dependent (Figure 2). A maximum suppression of EGFP fluorescence signal was measured when chetomin was added four hours prior hypoxia treatment (0.1% O2, 12 h) and in a final concentration of 150 nM. Higher doses of chetomin led to an increasing proportion of completely EGFP-negative cells indicating substantial cytotoxicity (data not shown). Chetomin had no effect on HRE-independent EGFP fluorescence as shown by comparable fluorescence in chetomin-treated (150 nM) and control HT 1080 cells transfected with the constitutive CMV promoter (Figure 1).

Bottom Line: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis.Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Oncology, University of Würzburg, 97080 Würzburg, Germany. staab_a@klinik.uni-wuerzburg.de

ABSTRACT

Background: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells.

Methods: Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h).

Results: Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 +/- 7.2% and 39.6 +/- 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.

Conclusion: HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

Show MeSH
Related in: MedlinePlus