Yao, Anqi
(2020)
Targeting thioredoxin system to improve radiotherapeutic response of brain cancers.
PhD thesis, University of Nottingham.
Abstract
Radiotherapy is often prescribed for brain cancer, particularly high-grade malignant gliomas. Efficacy is, however, often limited by radioresistance. The thioredoxin (Trx) system, a key antioxidant pathway in defence against oxidative stress, represents a potentially attractive target to overcome such resistance. Trx system alterations contribute to altered treatment response in a number of tumour types but information is lacking in brain tumours. The aim of the current study is to explore the potentially prognostic role of Trx system proteins in different types brain tumours; and investigate the chemotherapeutic and radiosensitising effects of a novel TrxR inhibitor IQ10 on brain cancer cells and the mechanisms underlying its action.
Expression of Trx system proteins was evaluated in four different brain tumour types by immunohistochemistry. A panel of five different brain cancer cell lines were used in this study: SNB19 (adult glioblastoma), SF188 and KNS42 (paediatric glioblastoma), and DAOY and UW228-3 (medulloblastoma); and a normal cell type, MRC5 lung fibroblasts, was used for comparing drug cytotoxicity. Professor Chris Moody’s group (School of Chemistry, University of Nottingham) developed a series of Indolequinone (IQ) compounds that potently inhibited TrxR activity in pancreatic cancer cells. One of the most potent IQs, IQ10, was assessed for its inhibition of TrxR activity and Trx family expression in brain cancer cells using the insulin reduction assay and Western blotting respectively. The cellular cytotoxicity of IQ10 was evaluated using growth curves, resazurin reduction and clonogenic survival assays under both normoxic and hypoxic (1% O2) conditions and in 2D vs. 3D spheroid cultures. Validation of TrxR as an important target for IQ10 in brain cancer cells was carried out using an siRNA approach. IQ10’s role in regulating radioresponse of brain cancer cells was examined via clonogenic assays. In addition, intracellular ROS levels were determined by H2DCFDA flow cytometry and γH2AX foci by immunofluorescence staining. Expression of genes associated with epithelial-mesenchymal transition was examined by RT2 ProfilerTM PCR Array.
The retrospective immunohistochemical study demonstrated that Trx system proteins were expressed to variable levels across a variety of brain tumour types, with high expression of cytoplasmic TrxR consistently correlating with a worse prognosis in all tumour types, suggesting that it is potentially a very important therapeutic target in brain cancers generally. In vitro results showed that IQ10 treatment significantly inhibited TrxR activity in a dose-dependent manner but did not affect the expression levels of Trx system proteins in brain cancer cells. IQ10 exhibited potent single agent anti-proliferative and cytotoxic effects against brain cancer cells under both normoxic and hypoxic conditions in 2D and 3D systems, with IC50s in the submicromolar/low micromolar range. IQ10 was up to ~1000 fold more potent than temozolomide, the agent used clinically to treat brain tumours and used as a comparator in the current study. IQ10 seemed to preferentially kill brain cancer cells but spare normal fibroblasts MRC5. More importantly, TrxR was found to be a promising radiobiological target in that inhibition of TrxR by IQ10 substantially sensitised both 2D and 3D cultured brain cancer cells to radiation (sensitiser enhancement ratio’s ranging between 1.2 - 1.7), with such radiosensitising effect being due, in part, to functional inhibition of TrxR, which made cells less able to deal with oxidative stress, leading to oxidative DNA damage. In addition to its cytotoxic and radiosensitising effects, IQ10 showed potential to inhibit invasion/migration as treatment significantly downregulated EMT associated gene expression in brain cancer cells.
Collectively, although further confirmation is required both in vitro and in vivo, the current study highlights TrxR as a potential therapeutic target in brain cancers and suggests that IQ10 could be a promising agent, either single or combined with radiation, to improve patient outcome.
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