Expression and hypoxic up-regulation of neuroglobin in human glioblastoma cells

Abstract

Neuroglobin is a recently identified globin molecule that is expressed predominantly in the vertebrate brain. Neuroglobin expression increases in oxygen-deprived neurons, suggesting it protects neurons from ischemic cell death. We report that neuroglobin transcript and protein are expressed in human glioblastoma cells, and that this expression increases in hypoxia in vitro. We also show that neuroglobin is up-regulated in hypoxic microregions of glioblastoma tumor xenografts. Our finding of hypoxic up-regulation of neuroglobin in human glioblastoma cells may provide insight into how tumor cells adapt to and survive in hypoxic microenvironments.

Figure 1

Effect of hypoxia on Ngb mRNA expression in human GBM cells. (A) M010B; (B) M059J; (C) M006x: (D) M006xLo; (E) M059K; (F) U87R; (G) U87T. Ngb mRNA expression was assessed by qRT‐PCR after exposure to hypoxia (0.6% O2) for 0, 6, 24 and 48h. Data are expressed as fold increase relative to aerobic control. (n=4). *P<0.05 (ANOVA).

Figure 2

Ngb protein expression in human GBM cells. (A) M010B; (B) M059J; (C) M006x; (D) M006xLo; (E) M059K; (F) U87R; (G) U87T. Ngb expression was assessed by Western blot analyses after exposure to hypoxia (0.6% O2) for 0, 6, 24 and 48h. The integrated intensities of Ngb and α‐tubulin (control) bands were determined and expressed in arbitrary units (AU), and representative blots are shown. (n=4) *P<0.05; **P<0.01; ***P<0.001 (ANOVA).

Figure 3

Ngb expression in a M006xLo tumor xenograft. (A) Low‐power (10×) view of adjacent serial sections stained with antibodies to pimonidazole or Ngb. Regions of necrosis (N) were used as tissue landmarks. (B) High‐power views (40×) of regions indicated by the red boxes in panel A. Hypoxic cells identified by pimonidazole staining also show strong positive staining for Ngb. (C) High‐power views (40×) of regions indicated by the green boxes in panel A. Weakly positive Ngb staining was also observed throughout the tumor section, however, these cells showed no pimonidazole immunoreactivity. (D) Low power view (10×) of control tumor sections from which primary antibodies were omitted.