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Influence of hypoxia and irradiation on osteopontin expression in head and neck cancer and glioblastoma cell lines.

Wohlleben G, Scherzad A, Güttler A, Vordermark D, Kuger S, Flentje M, Polat B - Radiat Oncol (2015)

Bottom Line: This effect was not seen in Cal27 or in FaDu cells.Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions.This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University hospital Wuerzburg, Josef-Schneider-Straße 11, 97080, Würzburg, Germany. Wohlleben_G@ukw.de.

ABSTRACT

Background: Tumor hypoxia is a known risk factor for reduced response to radiotherapy. The evaluation of noninvasive methods for the detection of hypoxia is therefore of interest. Osteopontin (OPN) has been discussed as an endogenous hypoxia biomarker. It is overexpressed in many cancers and is involved in tumor progression and metastasis.

Methods: To examine the influence of hypoxia and irradiation on osteopontin expression we used different cell lines (head and neck cancer (Cal27 and FaDu) and glioblastoma multiforme (U251 and U87)). Cells were treated with hypoxia for 24 h and were then irradiated with doses of 2 and 8 Gy. Osteopontin expression was analyzed on mRNA level by quantitative real-time RT-PCR (qPCR) and on protein level by western blot. Cell culture supernatants were evaluated for secreted OPN by ELISA.

Results: Hypoxia caused an increase in osteopontin protein expression in all cell lines. In Cal27 a corresponding increase in OPN mRNA expression was observed. In contrast the other cell lines showed a reduced mRNA expression under hypoxic conditions. After irradiation OPN mRNA expression raised slightly in FaDu and U87 cells while it was reduced in U251 and stable in Cal27 cells under normoxia. The combined treatment (hypoxia and irradiation) led to a slight increase of OPN mRNA after 2 Gy in U251 (24 h) and in U87 (24 and 48 h) cell lines falling back to base line after 8 Gy. This effect was not seen in Cal27 or in FaDu cells. Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions. Again the combined treatment resulted in a minor increase in OPN secretion 48 hours after irradiation with 8 Gy.

Conclusion: Osteopontin expression is strongly modulated by hypoxia and only to a minor extent by irradiation. Intracellular OPN homeostasis seems to vary considerably between cell lines. This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting.

No MeSH data available.


Related in: MedlinePlus

Quantitative real-time RT-PCR data showing OPN RNA expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation (IR) with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines. *marks statistical differences between normoxic and hypoxic conditions on the corresponding dose levels (ANOVA; p < 0.05)
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Fig4: Quantitative real-time RT-PCR data showing OPN RNA expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation (IR) with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines. *marks statistical differences between normoxic and hypoxic conditions on the corresponding dose levels (ANOVA; p < 0.05)

Mentions: In contrast there was a decrease in OPN mRNA levels in the glioblastoma cell lines when cells were cultured under standard hypoxia (see Fig. 4: 24 h post irradiation), which was more pronounced the longer the cells were in culture (see Fig. 4: 48 h post irradiation) (p-values < 0.05). Doses of 2 Gy led to a non-significant increase of OPN mRNA expression under hypoxic conditions (Fig. 4).Fig. 4


Influence of hypoxia and irradiation on osteopontin expression in head and neck cancer and glioblastoma cell lines.

Wohlleben G, Scherzad A, Güttler A, Vordermark D, Kuger S, Flentje M, Polat B - Radiat Oncol (2015)

Quantitative real-time RT-PCR data showing OPN RNA expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation (IR) with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines. *marks statistical differences between normoxic and hypoxic conditions on the corresponding dose levels (ANOVA; p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4554368&req=5

Fig4: Quantitative real-time RT-PCR data showing OPN RNA expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation (IR) with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines. *marks statistical differences between normoxic and hypoxic conditions on the corresponding dose levels (ANOVA; p < 0.05)
Mentions: In contrast there was a decrease in OPN mRNA levels in the glioblastoma cell lines when cells were cultured under standard hypoxia (see Fig. 4: 24 h post irradiation), which was more pronounced the longer the cells were in culture (see Fig. 4: 48 h post irradiation) (p-values < 0.05). Doses of 2 Gy led to a non-significant increase of OPN mRNA expression under hypoxic conditions (Fig. 4).Fig. 4

Bottom Line: This effect was not seen in Cal27 or in FaDu cells.Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions.This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University hospital Wuerzburg, Josef-Schneider-Straße 11, 97080, Würzburg, Germany. Wohlleben_G@ukw.de.

ABSTRACT

Background: Tumor hypoxia is a known risk factor for reduced response to radiotherapy. The evaluation of noninvasive methods for the detection of hypoxia is therefore of interest. Osteopontin (OPN) has been discussed as an endogenous hypoxia biomarker. It is overexpressed in many cancers and is involved in tumor progression and metastasis.

Methods: To examine the influence of hypoxia and irradiation on osteopontin expression we used different cell lines (head and neck cancer (Cal27 and FaDu) and glioblastoma multiforme (U251 and U87)). Cells were treated with hypoxia for 24 h and were then irradiated with doses of 2 and 8 Gy. Osteopontin expression was analyzed on mRNA level by quantitative real-time RT-PCR (qPCR) and on protein level by western blot. Cell culture supernatants were evaluated for secreted OPN by ELISA.

Results: Hypoxia caused an increase in osteopontin protein expression in all cell lines. In Cal27 a corresponding increase in OPN mRNA expression was observed. In contrast the other cell lines showed a reduced mRNA expression under hypoxic conditions. After irradiation OPN mRNA expression raised slightly in FaDu and U87 cells while it was reduced in U251 and stable in Cal27 cells under normoxia. The combined treatment (hypoxia and irradiation) led to a slight increase of OPN mRNA after 2 Gy in U251 (24 h) and in U87 (24 and 48 h) cell lines falling back to base line after 8 Gy. This effect was not seen in Cal27 or in FaDu cells. Secreted OPN was detected only in the two glioblastoma cell lines with reduced protein levels under hypoxic conditions. Again the combined treatment resulted in a minor increase in OPN secretion 48 hours after irradiation with 8 Gy.

Conclusion: Osteopontin expression is strongly modulated by hypoxia and only to a minor extent by irradiation. Intracellular OPN homeostasis seems to vary considerably between cell lines. This may explain the partly conflicting results concerning response prediction and prognosis in the clinical setting.

No MeSH data available.


Related in: MedlinePlus