Yilmaz, Muazzez Ceren
(2025)
Targeting thioredoxin reductase in brain tumours to improve therapeutic outcome.
PhD thesis, University of Nottingham.
Abstract
Introduction
A potential strategy in cancer treatment is to increase antioxidant stress to cause tumour cell death. Radiation therapy, alongside certain chemotherapies, induces oxidative stress, leading to cell death by disrupting cellular homeostasis, either through direct or indirect DNA damage. The thioredoxin (Trx) system is one of the most important antioxidant pathways providing cellular homeostasis balance and control. Consequently, it represents a promising target for cancer therapy. GBM is an aggressive and highly malignant brain tumour. The present treatment techniques, which contain radiotherapy and chemotherapy, can extend the survival of patients by an average of 15 months. Due to the structure of the brain and the heterogeneous nature of the tumour, it is often not possible to achieve complete surgical excision of the tumour; therefore, recurrence is common.
Methods
The current study characterised a commercially available thioredoxin reductase (TrxR) inhibitor, Auranofin (AU), alongside a novel TrxR inhibitor, Indolequinone10 (IQ10), for a variety of phenotypic effects on glioblastoma cell lines. Two primary and one established glioblastoma cell lines were used in growth curves, TrxR activity assays, cell proliferation, single agent clonogenic cytotoxicity, and clonogenic radiosensitivity in drug combination studies and wound healing/haptotaxis assays, all under normoxic and hypoxic conditions. RNA-seq transcriptomic analysis was also conducted to examine potential mechanisms and the effects of AU and IQ10 on related cell signalling pathways. Differentially expressed genes from RNA-seq analysis were further analysed by reverse transcriptase-qPCR. Additionally, a bioinformatic study conducted for the specific genes altered by differential expression of thioredoxin system genes in GBM was analysed to explore potential associated biomarkers.
Results
Growth curves and clonogenic survival assays demonstrated that hypoxia reduced proliferation and plating efficiency across all cell lines except GIN38 proliferation, with Western blotting for CA9 expression confirming hypoxic conditions. TrxR activity assays revealed that IQ10 and AU effectively inhibited TrxR, with AU showing more potency in SF188 cells under hypoxia. Proliferation assays indicated that IQ10 and AU inhibited cell growth in a dose- and time-dependent manner, being more potent than TMZ by approximately 1000-fold. Clonogenic survival assays further confirmed the cytotoxicity of IQ10 and AU in brain tumour cell lines as single agents, with hypoxia slightly enhancing the effect of IQ10 while reducing the effect of AU. RNA sequencing analysis identified differentially expressed genes responding to IQ10 and AU treatment, highlighting alterations in metabolic and survival pathways. Qiagen IPA pathway analysis suggested potential inhibition of ERK/MAPK and AMPK signalling by IQ10, while AU treatment impacted insulin receptor signalling. CRISPR-Cas9-mediated TXNRD1 knockout attempts were unsuccessful despite the optimisation of transfection conditions. Clonogenic survival assays revealed that all cell lines exhibited increased radioresistance under hypoxia, with GIN28 showing the highest radiosensitivity in normoxia. Clonogenic survival assays revealed that all cell lines exhibited increased radioresistance under hypoxia, with GIN28 showing the highest radiosensitivity in normoxia. Radiation survival curves demonstrated a dose-dependent decrease in survival, with significantly higher survival fractions at 8 Gy under hypoxia across all cell lines. IQ10 significantly enhanced radiosensitivity in SF188 but had minimal effects on GIN28 and GIN38. AU slightly increased radiosensitivity in all cell lines, with a statistically significant effect in GIN38. Neither IQ10 nor AU enhanced hypoxic radiosensitivity in SF188. Additionally, wound healing assays demonstrated that neither IQ10 nor AU significantly affected GBM cell migration. The bioinformatic analysis investigated the differential gene expression and pathway enrichment of the thioredoxin system in GBM using publicly available TCGA datasets. Pathway enrichment analysis revealed significant involvement of these genes in cell cycle regulation, RNA transport, and proteasome pathways. Survival analysis using Kaplan-Meier curves indicated that high expression levels of ANAPC10 and SHFM1 were associated with decreased survival, while high RPP25 expression correlated with improved prognosis. Additionally, four genes (CES1, BANK1, IRS1, and NHLRC2) from TXNRD1 analysis were selected based on their survival significance, with BANK1 and IRS1 linked to poor survival outcomes. Western blot and qPCR analyses revealed variable TXN and TXNRD1 expression across GBM cell lines, with GIN38 and GIN28 displaying the highest levels. However, antibody validation for selected DEGs was unsuccessful due to non-specific bindings, limiting further protein-level analysis.
Conclusions
These findings provide insights into the therapeutic potential of TrxR inhibitors in GBM. IQ10 could represent a promising radiosensitiser for SF188, while AU may have a more modest effect, particularly in GIN38, but neither compound enhances radiosensitivity under hypoxia. Further investigations are needed to examine the potential role of thioredoxin system genes in GBM progression and prognosis and to assess their functional significance.
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