Characterizing Oxidative Stress in Cervical Cancer Cell Lines Using a Novel Positron Emission Tomography Probe [F-18]Dihydroethidium: Implications for Radiation and Redox Directed Therapies

Abstract

Cellular redox state may play an important role in resistance of cervical cancers to radiation therapy (RT). This work examines the potential of a novel PET tracer of cellular superoxide, [F-18]DHE, to act as a predictive marker for radiation response. A combination drug strategy targeting glycolysis with 2-deoxyglucose (2-DG), and reactive oxygen species scavenging pathways with buthionine sulfoximine (BSO) and auranofin (AUR) was used to increase oxidative stress. Four cervical cancer cell lines (SiHa, ME180, C33A, and Caski) were maintained in complete media under standard conditions in 12 well plates. Treatments included the combination of 2-DG (20 mM), BSO (1 mM) and AUR (100 nM), abbreviated DBA, and RT (6 Gy). All treatments were delivered 24 hours before [F-18]DHE labeling. [F-18]DHE was added directly to the media at 20 mCi/well, and incubated for 1 hour at 37 C. Cells were then washed, lysed, and radioactivity was counted in a gamma counter. Baseline uptake of [F-18]fluorodexoyglucose (FDG) was also measured using the same labeling procedure, with the exception that cells were incubated in glucose-free media ½ hour prior to and during labeling. The uptake of [F-18]DHE was correlated against DHE fluorescence determined using flow cytometry. Clonogenic survival was determined using the colony forming assay. There are significant differences in baseline [F-18]DHE signal between the 4 cell lines (p<0.0001 by ANOVA). Baseline uptake of radiolabeled [F-18]DHE was correlated with fluorescent signal from unlabeled DHE (Pearson’s r = 0.61, p=0.2) and with [F-18]FDG uptake (r = 0.91, p = 0.04). [F-18]DHE uptake following treatment was further explored in ME180 and SiHa cells, which show the greatest contrast in baseline [F-18]DHE signal and sensitivity to RT and DBA therapy. There is a significant correlation between fold change in [F-18]DHE signal and cell survival following treatment with RT, DBA, and RT+DBA combined (r = -0.77, p = 0.01). For example there is little change in SiHa [F-18]DHE signal following radiation alone and SiHa cells are radioresistant. However, SiHa cells are very sensitive to DBA combined with RT, and there is a correspondingly large change in [F-18]DHE signal following this treatment. [F-18]DHE, a marker for cellular superoxide, shows differences in baseline uptake in the 4 cervical cell lines studied. [F-18]DHE uptake also correlates with [F-18]FDG uptake, suggesting that cells undergoing high rates of glycolysis produce larger quantities of superoxide. The change in [F-18]DHE uptake following treatment with RT, DBA, or the combination of RT+DBA correlates with clonogenic survival in vitro. [F-18]DHE may therefore be a useful predictive marker for RT and redox directed therapy. In vivo studies are ongoing.