Abstract
Gliomas are the most common primary brain cancer diagnosed in adults. Gliomas arise from glial cells within the central nervous system, and vary in severity based on a World Health Organisation scale. The 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are a super-family of enzymes that govern numerous metabolic pathways in gliomas. The 2-OGDD enzymes utilise dioxygen and 2-oxoglutate as substrates, and ascorbate and iron as co-factors. When the availability of any of these cofactors and substrates is limiting, enzyme activity is reduced. We hypothesised that iron was required for 2-OGDD activity in gliomas.
A total of 59 human glioma samples were obtained from the He Taonga Tapu Cancer Society Tissue Bank (H19/163) and Dunedin School of Medicine (MEC/08/02/016). The elemental iron content of the glioma samples was measured using inductively coupled quadrupole plasma mass spectrometry (ICP-Q-MS). Ferrous iron (Fe2+) was quantified by the ferrozine assay. Western blotting was used to determine the levels of the iron-related proteins transferrin receptor, ferroportin, and heme oxygenase 1. Ferritin protein was measured using enzyme-linked immunosorbent assay (ELISA). Optimisation steps using mouse brain were undertaken prior to the use of clinical glioma samples. 2-OGDD activity measures, including hypoxic response and DNA methylation, were previously determined within the Mackenzie Cancer Research Group.
Elemental iron content in ~10 mg glioma samples varied over 140-fold (8.2 mg/kg to 1500 mg/kg), with a median of 57 mg/kg. Ferrous iron ranged from undetectable to 5133 mg/kg, with a median of 684 mg/kg. Elemental iron measured by ICP-Q-MS correlated weakly with ferrous iron measured by ferrozine assay (r=0.330, p=0.016). Western blots of glioma sample lysates showed varying presence of ferroportin and heme oxygenase 1, however, transferrin receptor was not detected. Ferritin levels were high with 32 samples above 5 mg/ml. There were no associations between elemental iron and any of the iron-related proteins, nor with clinicopathological data including tumour grade and location in the brain. Counter to our hypothesis, there was no association between elemental or ferrous iron and 2-OGDD activity. There was a significant relationship between increased ferrous iron in glioblastoma tumours and longer patient survival (p=0.013). To our knowledge, this is the first study to have investigated the relationship between iron and 2-OGDD enzymes in brain cancer samples. The lack of association between iron pathway proteins and elemental iron may be indicative of dysregulation. The lack of relationship between iron and 2-OGDD activity suggests that iron levels in glioma may not be a limiting factor for enzyme activity. Ferrous iron content of glioblastoma tissue may be predictive of patient survival.