Abdullah, Nurul Akmaryanti
(2019)
The role of redox proteins in the radiotherapeutic response of breast cancer.
PhD thesis, University of Nottingham.
Abstract
Radiotherapy is an effective treatment for many types of cancer including breast cancer. Ionising radiation induces cytotoxicity mainly via the generation of reactive oxygen species (ROS). The overexpression of redox proteins in various tumours, including breast cancer, has been shown to cause multidrug and radioresistance. Targeting redox proteins represents an innovative approach to enhancing the radiosensitivity of tumour cells. The present study aimed to assess how inhibitors of glutathione (GSH) and thioredoxin (Trx) systems can alter the radiosensitivity of breast cancer cells.
Several agents were used including Piperlongumine (PL) (targeting the GSH system), PL analogues, Indolequinone IQ9 (a novel thioredoxin reductase (TrxR) inhibitor) and metformin (a type-2 diabetes drug which inhibits the Trx system). Basal-like and luminal breast cancer cell lines were used as in vitro breast cancer models. The cytotoxic effect of drugs was assessed using growth curves and clonogenic survival assays. Clonogenic radiosensitisation was measured by treating cells with drugs and subsequently exposing them to various irradiation doses of (0-8 Gy) x-rays. The sensitiser enhancement ratio (SER) was calculated to assess the radiosensitisation effect of the drugs when combined with irradiation. Intracellular ROS levels were assessed using flow cytometry and the protein expression of Trx, thioredoxin-interacting protein (Txnip) and TrxR was assessed using Western blotting. TrxR activity was evaluated using insulin reduction assays.
First, the radiosensitisation effect of PL and its analogues (LH91 and LH92) was studied in MDA-MB-231 and T47D cells. In terms of cell proliferation and clonogenic survival, MDA-MB-231 cells were more sensitive to PL, LH91 and LH92 than T47D cells (IC50 values of 3-13µM and 9-15µM, respectively). PL slightly increased the radiosensitivity of MDA-MB-231 (SER 1.12) but not that of T47D. LH91 and LH92 did not alter the breast cancer cell radioresponse. No changes in ROS levels were observed following treatment with PL, LH91 and LH92, either alone or with subsequent exposure to hydrogen peroxide (H2O2). Such results may explain the lack of a radiosensitisation effect of PL analogues in breast cancer cells.
The effect of the TrxR inhibitor IQ9 in breast cancer cell lines was also investigated with respect to TrxR activity, proliferation, clonogenic survival, radioresponse and Trx system expression in MDA-MB-231, MDA-MB-468 and MDA-MB-436, T47D and MCF-7 cell lines. TrxR activity was assessed at two time points following IQ9 treatment (4 and 48 hours). IQ9 inhibited TrxR activity more efficiently in basal-like (IC50 272-439nM) than luminal (IC50 619-640nM) breast cancer cells after 4-hours of treatment. Additionally, inhibition of TrxR activity was more effective after 4-hours than 48-hours of IQ9 treatment. Cell proliferation and clonogenic survival assays revealed that IQ9 exhibited a potent anticancer effect against breast cancer cells with IC50 in the low nanomolar range (174-2072nM). Radiosensitivity of all basal-like breast cancer cells was enhanced following 4-hours of IQ9 treatment (SER 1.20-1.43), whereas a minimal or no increase in radiosensitivity was observed in MCF-7 (SER 1.08) and T47D (SER 1.00). In contrast, prolonged exposure to IQ9 (48 hours) before irradiation did not alter radiosensitivity. IQ9 treatment increased Trx expression only in MDA-MB-231 and no changes in Txnip expression were observed. IQ9 upregulated TrxR expression in luminal but not in basal-like breast cancer cells. Such results may indicate that luminal breast cancer cells increase TrxR expression to compensate for the inhibition of TrxR activity and thus reduce IQ9 radiosensitisation effects. The dual inhibition of the GSH and Trx systems using L-buthionine-S, R-sulfoximine (BSO) and IQ9 to improve the radiosensitivity of breast cancer cells was also investigated. The results show that the combined treatment of BSO and IQ9 without irradiation substantially decreased colony formation of MDA-MB-231 cells compared to the control and single agent alone. However, such effect was not observed when combined with irradiation.
The final aim of the study was to evaluate whether modulating the Trx system using metformin can radiosensitise breast cancer cells and to explore whether the effect is phenotype specific. Metformin decreased breast cancer cell growth and colony formation with IC50 ranging from 3-20mM. Treatment with metformin substantially radiosensitised MCF-7 and MDA-MB-468, but not T47D cells with SER values of 1.45 and 1.30, respectively, suggesting that metformin radiosensitises breast cancer cells in a cell-specific manner. The effect of metformin may be partly explained by increasing ROS levels with subsequent exposure to H2O2. Metformin completely attenuated Txnip expression in MCF-7 and had no effect on that of T47D. MDA-MB-468 did not express Txnip. Taken together, the data indicate that the increase in radioresponse by metformin may be subject to the increase in ROS level and the loss in function of Txnip.
In summary, this study highlights IQ9 and metformin as potential radiosensitisers in breast cancer via modulating Trx system function. In addition, this study provides preliminary support for the therapeutic effect of LH91, LH92, IQ9 and metformin as potential anticancer agents for the treatment of breast cancer.
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