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(PP237) Targeting the thioredoxin system with AW464: hypoxic cytotoxicity, antiangiogenic activity and chemo-/radiosensitisation.
Mukherjee, Abhik1, Martin, Stewart*,1, 1 University of Nottingham, Dept of Clinical Oncology, Nottingham, Notts, UK
ABSTRACT- AW464 is a novel benzothiazole substituted quinol compound active against certain cancer cell lines with thioredoxin (Trx1) / thioredoxin reductase (TrxR1) signalling as a possible target. The drug has enhanced hypoxic toxicity and both direct and indirect anti-angiogenic effects (inhibition of VEGF production) at low doses (Mukherjee et al 2005). Fibroblasts and quiescent endothelial cells are relatively resistant. The current study aimed to investigate the above effects at the molecular level and to investigate the mode of cell death. Combinations of the drug with radiation or doxorubicin were also investigated, using clonogenic assays, as evidence in the literature (Karimpour S, et al 2002, Smart DDK, et al 2004) suggests that thioredoxin is involved in the cellular response to radiation. Experiments (RT-PCR; insulin/ DTNB reduction and apoptosis assays) were performed using HT29 colorectal and MCF-7 breast cancer cells, human umbilical vein endothelial cells (HUVEC) and MRCV foetal lung fibroblasts under both normoxic and hypoxic (1% O2) conditions. AW464 inhibits the insulin reducing function of Trx1 but not TrxR1, with effects being more pronounced under hypoxic conditions. There is no effect on Trx1 mRNA expression but TrxR1 mRNA may be induced early. Inhibition of VEGF mRNA is observed at low doses under hypoxic conditions but HIF1a mRNA remains unaltered, indicating a probable functional inhibition of HIF1. Untreated quiescent HUVEC show lower Trx1 activity as compared to proliferating HUVEC, but no alterations of Trx1 mRNA. Hence, in endothelial cells, targets other than Trx1 may also exist. MRCV fibroblasts have low Trx1 and TrxR1 mRNA levels and Trx1 function is also low and may explain the relative resistance of fibroblasts. Low doses of drug, under hypoxia, enhance the percentage of cells in the G2/M phase and this correlates to a greater incidence of apoptosis. Preliminary results suggest that the drug acts as both a chemo- and radiosensitiser to tumour cell lines. Inhibition of thioredoxin function may explain some but not all of the observed effects of AW464. Targeting the thioredoxin system may represent a novel way of enhancing the therapeutic efficacy of radio- or chemotherapy by preferentially targeting hypoxic cells, decreasing VEGF expression and increasing radio-/ chemosensitivity.
Key words: Thioredoxin, Angiogenesis, Hypoxia, Radiation
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