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Alhadyan, Khaled (2019) Targeting thioredoxin reductase in pancreatic cancer. PhD thesis, University of Nottingham.

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Abstract

Pancreatic cancer (PC) is the fifth leading cause of cancer deaths in the United Kingdom, and responsible for 5% of all cancer deaths. Current treatment options, including surgery, chemotherapy and radiotherapy, have failed to improve five-year survival rates over the last 30 to 40 years, which remain very low, at ~5%. A number of resistance mechanisms may explain this lack of treatment success, one of these being inherent or acquired expression of redox proteins, i.e. proteins that can scavenge therapy-induced radicals, making treatment, such as radiation therapy, less efficient. Redox proteins are key members of cellular antioxidant systems, required to maintain redox homeostasis, with the thioredoxin (Trx) system being an important component. The Trx system consists of Trx, thioredoxin reductase (TrxR), and thioredoxin-interacting protein (TxNIP). Trx exists in mammalian cells in either a reduced (functional) form or an oxidised (non-functional) form. The reduced form of Trx regulates the redox state of different signalling molecules, and by regulating their redox state, can regulate cell growth, apoptosis, gene transcription, cell cycle progression and ROS levels. TrxR is the only known enzyme that can reduce (activate) Trx. Therefore, TrxR has been studied as a potential therapeutic target in different types of cancer. Indolequinone analogues (IQs) are novel bioreductive TrxR inhibitors produced at the University of Nottingham, and currently being developed as potential drugs against PC. The current thesis set out to examine the role of the Trx system, using novel TrxR inhibitors, in regulating PC cell proliferation and radiosensitivity.

The first component of the current thesis used two novel, putative TrxR inhibitors (IQ9 and IQ10) to investigate the effect that inhibiting TrxR had on cellular cytotoxicity (via proliferation and clonogenic survival assays), radiosensitivity, and cell cycle progression. This was done using three PC cell lines: PANC-1, MIA Paca-2 and BxPc-3. The study used gemcitabine (GM), an agent clinically used in the treatment of PC, and auranofin (AUR), a well-characterised TrxR inhibitor, as positive controls. Results indicate that IQs exhibited potent in vitro cytotoxicity at 24, 48 and 72 hours, with IC50 values in the low nanomolar range. In the proliferation assay, PANC-1 cells showed low sensitivity to IQs (IC50=320-920 nM), while MIA Paca-2 (IC50=90-250 nM) and BxPc-3 cells (IC50=250-470 nM) showed high and intermediate sensitivity to IQs respectively. Results also indicate IQs as less toxic than GM (IC50=40-850 nM), but more toxic than AUR (IC50=400-2,550 nM) across all PC cell lines. In the clonogenic assay, GM (IC50=20-45 nM) appeared more potent than IQs (IC50=240-900 nM) across all PC cell lines. Increased radiosensitisation was observed for PANC-1 and BxPC-3 cells treated with IQs for 48 hours at 6 Gy, with no altered radiosensitisation in MIA Paca-2 cells. Although IQs appeared to sensitise PANC-1 and BxPc-3 cells to radiation at 6 Gy, the sensitiser enhancement ratio (SER) at 1% of survival for PANC-1 (SER=1.08-1.1) and BxPc-3 (SER=1.1-1.13) cells was low. Over a period of 48 hours, IC50 of IQ9 caused a significant cell cycle arrest in PANC-1 cells in the G0/G1 phase (P=0.024), with a significant decrease in S-phase (P=0.013); no effect was observed on the cell cycle progression of BxPc-3 cells.

The second component of the current study sought to investigate the potential mechanisms of action of IQs on the Trx system. This was done by examining the IQs effects on intracellular ROS levels (using flow cytometry) on the protein expression of Trx system family members, protein expression of reductase enzymes, TrxR activity (using insulin reduction assay), and radiation sensitivity. Since IQs are classified as bioreductive agents, the second component of the study also sought to observe the effect of hypoxia and drug treatment on proliferation, clonogenic survival, and on the protein expression of Trx system members and reductase enzymes. Results suggest that PC cells treated with IQs, in combination with H2O2, showed an increase in ROS levels compared to treatment with H2O2 only, with a maximum fold increase of 2. Western blots showed reduced expression of Trx only in BxPc-3 cells treated with the IC50’s of IQs for 48 hours, with no notable changes in expression of TrxR and TxNIP. PANC-1 and MIA Paca-2 cell lines treated with IQs for 48 hours showed no changes in the expression of any of the Trx system proteins. PANC-1 and BxPc-3 cells treated with IQs showed a substantial decrease in TrxR activity, in a dose-dependent manner, with 4 hours’ treatment yielding greater inhibition than 48 hours’ treatment. Increased radiosensitisation at 6 and 8 Gy was observed in PANC-1 cells treated with IQ9 or AUR for 4 hours, but may be of little biological relevance, as only a small increase in SER values (SER≤1.13) were observed. PANC-1 and BxPc-3 hypoxic cells were sensitive to IQ9, in terms of proliferation and clonogenic survival, compared to normoxic cells, but with a small increase in hypoxia cytotoxicity ratio (HCR) (HCR=1.2-2.4). IQ9 treatment did not alter the endogenous expression of reductase enzymes such as NQO1 and CPR in PANC-1 and BxPc-3 cells, neither in normoxic or hypoxic conditions.

The third component of the study studied Trx system protein expression in patient tumour specimens, determining prognostic significance and relationships with clinicopathological criteria. Two cancer patient cohorts were assessed, using immunohistochemistry (IHC) on tissue microarrays. The first cohort comprised 85 pancreatic adenocarcinomas (PAD), while the second cohort comprised 145 cancers of the bile duct and ampulla. In the first cohort, high expression of cytoplasmic (P=0.018) and nuclear (P=0.006) Trx were significantly associated with better overall survival, with nuclear Trx expression remaining significantly associated with survival in multivariate analysis (P<0.0001). In the second cohort, low cytoplasmic TxNIP expression was also associated with the presence of perineural invasion (P=0.025). High expression of cytoplasmic TxNIP was significantly associated with better overall survival (P=0.0002), which remains significant in multivariate Cox-regression analysis (P=0.013).

In conclusion, the current study suggests that targeting the TrxR system may be a useful potential strategy for treating PC. Current findings show that IQs are effective in PC cell lines as single agents, with IC50 values in the low nanomolar range. However, IQs were less potent than GM. IQs enhance radiotherapeutic efficacy in certain PC cell lines, but with low SER values, rendering the biological relevance of IQs as radiosensitisers somewhat questionable. IQs showed high efficacy in terms of inhibiting TrxR activity and increasing ROS levels in PC cells, with no notable alteration in endogenous protein expression of TrxR. Hypoxia increased the cytotoxic effect of IQs on PC cell lines but with low HCR values, making the biological significance of IQs as potential hypoxic cytotoxins doubtful. The Trx family protein expression can be used prognostically in patients with pancreatic, bile duct and ampullary cancers, and may influence cancer cell susceptibility to anticancer agents.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Martin, Stewart Ellis, Ian
Keywords:	Thioredoxin system, Thioredoxin, Thioredoxin reductase, Thioredoxin-interacting protein, Redox proteins, Pancreatic cancer, Indolequinone, Radiotherapy, Radiosensitivity
Subjects:	W Medicine and related subjects (NLM Classification) > WI Digestive system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	56609
Depositing User:	Alhadyan, Khaled
Date Deposited:	21 Aug 2019 14:06
Last Modified:	07 May 2020 10:16
URI:	https://eprints.nottingham.ac.uk/id/eprint/56609

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