CONCLUSION

1. Novel antitumor quinols: the role of thioredoxinEng-Hui Chew, Jenny C. Cookson, Charles S. Matthews, Ji-Hong Zhang, Thilo Hagen, Tracey D. Bradshaw, Malcolm F.G. Stevens, Andrew D. WestwellCentre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK Novel antitumor QuinolsAW464, BW114 and AJM290 exhibit selective antiproliferative activity against colon, renal and breast carcinoma cell lines (GI50 < 0.5 μM). Growth inhibition against renal, colon and breast xenografts has also been demonstrated1, 2. Preliminary results (microarray analysis, insulin reduction assay) suggested that quinols targeta 12 kDa thiol protein thioredoxin (Trx)3. Here we report further evidence of quinols interacting with thioredoxin and the results of investigations into the induction of oxidative stress and apoptosis by quinols. • Quinols induce cellular apoptosis Figure 3: Annexin V-FITC/Propidium Iodide double staining 24hrs 12hrs Propidium Iodide • Quinols induce oxidative stress-AW464 induced ROS production in sensitive cancer cell lines (HCT116, HT29 colon cells and MCF7 breast cells). • AW464 Binding To Thioredoxin Protein Figure 1: A, B: Mass spectrum of human Trx Annexin V-FITC Reduced Trx Figure 2: A, B: MTT cell viability assays (n=4) HCT116 colon cells treated with AW464 1 µM showed an increasing proportion of cells undergoing apoptosis between 12 and 24 hours. A Figure 4: Western blot Dose-dependent PARP cleavage and caspase 3 activation was observed in HCT116 cells treated with AW464 and BW114 for 24 hours. Campothecin 1 µM, 24hrs (tve ctrl) AW464 BW114 untreated 1 3 6 0.5 1 3 (µM) BSO-induced glutathione (GSH) depletion increases cytotoxicity of quinols by 10-fold (Fig 2A). Reduced Trx + AW464 PARP Cleaved PARP B -actin Caspase 3 Cleaved Caspase 3 -actin No blockade of cytotoxicity by pan caspase inhibitor Z-VAD-FMK and caspase 3 inhibitor Z-DEVD-FMK, suggesting that quinols induced apoptosis via both caspase dependent and independent pathways. Supplementation of GSH monoethyl ester caused a 2-fold reduction in potency (Fig 2B). (A) Non-drug treated thioredoxin: mass peak 11,606 (B) AW464 treated thioredoxin: mass peaks 12,336, 12,586 and 12,821 corresponding to thioredoxin plus 3, 4 and 5 AW464 molecules respectively. Subsequent MS/MS analysis of trypsin digested peptides showed additional mass of AW464 only at cysteine residues CONCLUSION We conclude that thioredoxin emerges as a molecular target of novel antitumor quinols. We aslo report that quinols induce oxidative stress and cellular apoptosis. Reference: 1. Wells et al J. Med. Chem. 2003, 46, 532. 2. Berry et al J. Med.Chem. 2005, 48, 639. 3. Bradshaw et al Cancer Res. 2005, 65 (in press).