Background
Glioblastoma (GBM) is a WHO-categorized grade IV brain-tumor with a median post-diagnosis survival rate of 14.6 months in patients. Reports on the association of iron-overload with cancer risks, emphasizes the significance of strict iron-regulation in the brain. In this study, free and bound iron levels in GBM cells have been determined. Moreover, the GBM cells have exposed to bovine lactoferrin (bLf), a natural iron-binding protein, to establish its anti-cancer properties.
Methods
Intracellular free iron levels were preliminarily measured in lysates of human GBM cells, CCF-STTG1 and T98G, and C6 rat glioma cells, as compared to healthy mice brain cells. Immunocytochemistry was performed to determine the receptor-expression for the major iron-transporter protein, transferrin, on GBM cells basally and post-exposure to iron-free bLf (apo-bLf), a member of the transferrin family. Expression of ferritin, an iron-regulatory biomarker, was determined on GBM cells basally and post-treatment with ferric ammonium citrate (FAC), a drug prescribed to anaemic patients. Furthermore, GBM cells were treated with apo-bLf, to determine its effects in terms of cytotoxicity, targeting cell proliferation and iron-chelation abilities.
Results
Increased free iron in GBM cell lysates (CCF-STTG1, T98G, and C6), as compared to healthy mice brain cells, was observed. This was supported by a basal ferritin expression in CCF-STTG1 and T98G, which visually overexpressed on FAC treatment for 24h. Basal surface expression of lactoferrin/ transferrin receptors (CD71, LRP1, and Intelectin1/2), on CCF-STTG1 and T98G, was observed, which visibly overexpressed, on apo-bLf exposure. Moreover, apo-bLf induced a time and dose-dependent cytotoxicity in these cells, grown as monolayers and tumor-spheroids. Interestingly, apo-bLf was also able to significantly reduce the intracellular iron content in CCF-STTG1.
Conclusion
As opposed to the established fact of iron, being an essential element in human physiology, it may paradoxically act as a pro-cancer factor in GBM, when in excess. Our results demonstrate an exciting potential to investigate the iron-regulatory pathways in GBM, as potential targets for bLf anti-cancer therapy.