Molecular radiobiology
Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1α in human glioma in vitro and in vivo

https://doi.org/10.1016/j.radonc.2007.05.003Get rights and content

Abstract

Background and purpose

To identify molecular markers of tumor hypoxia and potential therapeutic targets in glioblastoma (GBM), we investigated the hypoxia-related expression of osteopontin (OPN), carbonic anhydrase 9 (CA9), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in vitro in human GBM cell lines and in vivo in human tumor samples of GBM, compared to low-grade astrocytoma (LGA).

Materials and methods

Expression of the hypoxia-induced genes OPN, CA9, EPO, VEGF and HIF-1α was analyzed in three GBM cell lines, GaMG, U373 and U251, under in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O2) and in tumor samples from two patient groups with LGA and GBM (n = 15 each), at the mRNA level (semiquantitative RT-PCR). Selected conditions and representative tumor samples were also evaluated at the protein level by Western blot.

Results

OPN and CA9 mRNA was most consistently upregulated in relation to severity and duration of in vitro hypoxia. In tumor samples, mean expression levels (LGA vs. GBM, normalized to mean expression in normal brain) were 1.71 vs. 4.57 (p < 0.001) for OPN, 1.11 vs. 3.35 (p < 0.001) for CA9, 2.79 vs. 5.28 (not significant, n.s.) for Epo, 1.13 vs. 2.0 (p = 0.007) for VEGF and 0.97 vs. 0.97 (n.s.) for HIF-1α. In tumor samples, GBM showed a particularly strong protein expression of OPN.

Conclusions

Among a panel of known hypoxia-inducible genes, OPN and CA9 emerge as most consistently induced by in vitro hypoxia in human GBM cell lines and most specifically expressed in patient GBM tumor tissue, rendering these two genes attractive targets for hypoxia-directed treatment approaches.

Section snippets

Cell and culture and hypoxia treatment

Early-passage human malignant glioma cell lines U251 and U373 from the American Type Culture Collection (ATCC, Rockville, MD) and GaMG, a cell line that was established from a patient with glioblastoma multiforme (Gade Institut of the University Bergen, Norway) [1], were grown on glass Petri dishes in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (FBS) and non-essential amino acids. Additionally, all culture media were supplemented with penicillin (100 

Regulation of hypoxia-inducible gene expression at the mRNA level in vitro in human GBM cell lines and in vivo in human GBM and LGA tumor tissue

For all genes investigated, the time course of mRNA expression in human GBM cell lines in response to hypoxic exposure in vitro at O2 concentrations of 5%, 1% and 0.1% is displayed in Fig. 1. OPN displayed a strong hypoxic induction with increasing duration and severity of hypoxia in all three cell lines (Fig. 1a). CA9 was overexpressed under hypoxia in all three lines with the highest in GaMG (3-fold) (Fig. 2a). EPO was overexpressed only at moderate hypoxia (1% O2), but not at 0.1%, and VEGF

Discussion

Due to the poor prognosis of patients with glioblastoma multiforme, intensified and individualized treatment approaches are needed to improve the results of therapy. The presence of tumor hypoxia is an adverse prognostic factor, but may represent an opportunity to specifically target hypoxic tumor cells. Needle electrode studies have shown that oxygenation in GBM is lower than in surrounding brain tissue [5] and in immunohistochemical studies, perinecrotic regions of GBM stained positive for

Conflict of interest statement

The authors declare that they have no conflict of interest related to the contents of this manuscript.

Acknowledgements

The authors thank Thomas Freiman (Department of Neurosurgery, University of Freiburg, Germany) for normal brain control tissue, Bayer Healthcare Co., for provision of the M75 monoclonal antibody and Stefanie Gerngras and Astrid Katzer for technical assistance. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (VO 871/2) to D.V. and by IZKF Würzburg (B25) to C.H. and G.H.V.

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