A role for ceruloplasmin in the control of human glioblastoma cell responses to radiation

Abstract

Background Glioblastoma (GB) is the most common and most aggressive malignant brain tumor. In understanding its resistance to conventional treatments, iron metabolism and related pathways may represent a novel avenue. As for many cancer cells, GB cell growth is dependent on iron, which is tightly involved in red-ox reactions related to radiotherapy effectiveness. From new observations indicating an impact of RX radiations on the expression of ceruloplasmin (CP), an important regulator of iron metabolism, the aim of the present work was to study the functional effects of constitutive expression of CP within GB lines in response to beam radiation depending on the oxygen status (21% O2 versus 3% O2). Methods and results After analysis of radiation responses (Hoechst staining, LDH release, Caspase 3 activation) in U251-MG and U87-MG human GB cell lines, described as radiosensitive and radioresistant respectively, the expression of 9 iron partners (TFR1, DMT1, FTH1, FTL, MFRN1, MFRN2, FXN, FPN1, CP) were tested by RTqPCR and western blots at 3 and 8 days following 4 Gy irradiation. Among those, only CP was significantly downregulated, both at transcript and protein levels in the two lines, with however, a weaker effect in the U87-MG, observable at 3% O2. To investigate specific role of CP in GB radioresistance, U251-MG and U87-MG cells were modified genetically to obtain CP depleted and overexpressing cells, respectively. Manipulation of CP expression in GB lines demonstrated impact both on cell survival and on activation of DNA repair/damage machinery (γH2AX); specifically high levels of CP led to increased production of reactive oxygen species, as shown by elevated levels of superoxide anion, SOD1 synthesis and cellular Fe2 + . Conclusions Taken together, these in vitro results indicate for the first time that CP plays a positive role in the efficiency of radiotherapy on GB cells.

Identification of Hypoxia Prognostic Signature in Glioblastoma Multiforme Based on Bulk and Single-Cell RNA-Seq

“…CP influences oncogenesis-associated pathways, including the HIF-1 signaling pathway [50]. It is also responsive to variations in oxygen and iron concentrations and is upregulated during hypoxia [51][52][53]. Understanding the role of CP assumes particular relevance in the context of iron regulation and the radioresistant hypoxic environment.…”

“…Understanding the role of CP assumes particular relevance in the context of iron regulation and the radioresistant hypoxic environment. In a study conducted by Roy et al [51], the radiation responses of two human GBM cell lines were analyzed. They found that CP showed significant downregulation at the transcript and protein levels.…”

Ceruloplasmin is associated with the infiltration of immune cells and acts as a prognostic biomarker in patients suffering from glioma

“…Iron oxidation is another important function of CP. CP can transfer electrons to oxygen via regulating copper, which oxidizes Fe 2+ to Fe 3+ and enters the cell after binding to transferrin, thus providing sufficient iron for cell proliferation ( Forciniti et al, 2020 ; Roy et al, 2022 ). Depletion of CP caused the accumulation of ferrous iron (Fe 2+ ) in cytoplasm.…”

“…Some recent studies stated that the abnormal CP expression occurring in the brain could be attributed to a few neurological diseases ( Klomp and Gitlin, 1996 ; Qian and Ke, 2019 ; Villadsen et al, 2023 ). At present, the existing literature studies have reported that in the iron-deficient and iron-saturated cells, CP, regardless of concentration, promotes the uptake of transferrin-bound iron and ferric citrate by glioma cells ( Attieh et al, 1999 ; Roy et al, 2022 ). Moreover, high expression of CP in tumor cells, such as lung cancer ( Chang et al, 2022 ), liver cancer ( Shang et al, 2020 ), melanoma ( Liu et al, 2022a ), and breast cancer ( Chen et al, 2021 ), can inhibit ferroptosis, thus reduces tumor cell death and promotes tumor progression.…”

Human iPSC-derived neural stem cells display a radial glia-like signaturein vitroand favorable long-term safety in transplanted mice

“…GO analyses of biological processes and pathways downregulated in NSC populations vs. GSCs revealed that tumorigenic stem cells showed an increased expression of genes driving the cell cycle (including MYC and E2F TFs); oxidative phosphorylation-dependent ATP biosynthesis, typically detected in cancer vs. somatic stem cells 137 ; synthesis of selenoproteins, which play a key role in oxidative stress regulation in glioblastoma (e.g. GPX1 and GPX4 [138][139][140][141][142][143] ); and L13a-mediated silencing of ceruloplasmin, an important regulator of iron metabolism which positively correlates with the efficacy of radiotherapy on GBM cells 144 (Fig. 6C-E; Suppl.…”