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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

Western blot showing OPN-expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines
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Fig2: Western blot showing OPN-expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines

Mentions: Two different head and neck cancer cell lines (tongue carcinoma cell line, CAL 27 and hypopharyngeal cancer cell line FaDu) and two glioblastoma cell lines (U251 and U87) were cultured under different oxygen conditions (1 % O2 for Cal27 and FaDU and 0.1 % O2 for U251 and U87). Furthermore, a possible effect on OPN expression caused by irradiation (2 Gy or 8 Gy) was investigated. Twenty-four hours (all cell lines) and 48 hours (U251, U87) after irradiation, lysates of the different cell lines were prepared and tested by Western blot analysis for OPN protein levels. Twenty-four hours after irradiation a clear increase of OPN protein expression was seen in all cell lines grown under hypoxic conditions compared with normoxic controls. Except for FaDu and U251 cells grown at 0.1 % O2, this effect was independent of additional irradiation (Figs. 1 and 2). Irradiation of FaDu with doses of 2 and 8 Gy apparently had a weak inhibitory effect of OPN levels under hypoxic standard culture conditions (0.1 % O2). This was also seen in U251 cells under hypoxia and 24 h post irradiation with 2 Gy. U251 and U87 cells, which survived 48 hours post irradiation under hypoxic conditions, showed a significant increase of OPN expression 48 hours after irradiation compared with cells in the normoxic control group (Fig. 2). Interestingly, in both glioblastoma cell lines a trend for a reduced osteopontin content was seen under normoxic conditions after 48 hours irrespective of irradiation (Fig. 2). Beta-actin and GAPDH were used as loading controls, respectively. The missing protein bands of ß-actin (and GAPDH, data not shown) in lysates of Cal 27 cells, grown under hypoxic conditions, can be explained by the altered expression of housekeeping genes under hypoxia in some cell lines [18, 19].Fig. 1


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)

Western blot showing OPN-expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Western blot showing OPN-expression under normoxic (N = 21 % O2) and hypoxic (H = 0.1 % O2) conditions 24 h and 48 h after irradiation with 0, 2 and 8 Gy in U251 (a) and U87 (b) glioblastoma cell lines
Mentions: Two different head and neck cancer cell lines (tongue carcinoma cell line, CAL 27 and hypopharyngeal cancer cell line FaDu) and two glioblastoma cell lines (U251 and U87) were cultured under different oxygen conditions (1 % O2 for Cal27 and FaDU and 0.1 % O2 for U251 and U87). Furthermore, a possible effect on OPN expression caused by irradiation (2 Gy or 8 Gy) was investigated. Twenty-four hours (all cell lines) and 48 hours (U251, U87) after irradiation, lysates of the different cell lines were prepared and tested by Western blot analysis for OPN protein levels. Twenty-four hours after irradiation a clear increase of OPN protein expression was seen in all cell lines grown under hypoxic conditions compared with normoxic controls. Except for FaDu and U251 cells grown at 0.1 % O2, this effect was independent of additional irradiation (Figs. 1 and 2). Irradiation of FaDu with doses of 2 and 8 Gy apparently had a weak inhibitory effect of OPN levels under hypoxic standard culture conditions (0.1 % O2). This was also seen in U251 cells under hypoxia and 24 h post irradiation with 2 Gy. U251 and U87 cells, which survived 48 hours post irradiation under hypoxic conditions, showed a significant increase of OPN expression 48 hours after irradiation compared with cells in the normoxic control group (Fig. 2). Interestingly, in both glioblastoma cell lines a trend for a reduced osteopontin content was seen under normoxic conditions after 48 hours irrespective of irradiation (Fig. 2). Beta-actin and GAPDH were used as loading controls, respectively. The missing protein bands of ß-actin (and GAPDH, data not shown) in lysates of Cal 27 cells, grown under hypoxic conditions, can be explained by the altered expression of housekeeping genes under hypoxia in some cell lines [18, 19].Fig. 1

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