Fig. 9.

The signature of primary singlet oxygen allows for generation of secondary singlet oxygen after release of NOX1 inhibition. A. MKN-45 cells were pretreated with 0.05 mU/ml GOX and 1 mM nitrite, in the absence or presence of 100 μM AEBSF for 40 min. After three washing cycles, the cells were added at increasing percentages to untreated MKN-45 cells. B. MKN-45 cells were pretreated with GOX and nitrite for 40 min in the presence of 100 μM AEBSF. The cells were washed in three cycles and then resuspended in fresh medium. Further incubation for 25 min was performed either in the absence of inhibitors (open square), 2 mM histidine (closed diamond), 100 μM AEBSF (closed square), 25 μM FeTPPS (closed cross) or 20 mM mannitol (closed triangle). The cells were washed in three cycles and added at increasing concentrations to untreated cells. C. The experiment was performed as described under A, with the exception that pretreatment with GOX/nitrite was carried out with cells at a density of 3000  cells/100 μl instead of 12 500 cells/100 μl. The percentages of apoptotic cells were determined at 4.5 h after beginning of the coculture. The results shown under A confirm that inhibition of NOX1 allows the signature of primary singlet oxygen. The cells imprinted by primary singlet oxygen alone show a strongly reduced potential to induce bystander signaling compared to a control population that also allows secondary singlet generation. The result shown in B demonstrates that tumor cells with the specific signature by primary singlet oxygen acquire a much higher bystander effect inducing potential after the inhibition of NOX1 is abrogated and the cells are allowed to interact for 25 min. This interaction results in the generation of secondary singlet oxygen, with superoxide anions, peroxynitrite and hydroxyl radicals as intermediates. Part C shows that the signature of primary singlet oxygen is independent of cell density, whereas subsequent secondary singlet oxygen generation is dependent. Statistical analysis: A: Apoptosis induction and the differences between the curves are highly significant (p < 0.001). B:The effects of all four inhibitors is highly significant (p < 0.001) up to a percentage of pretreated cells of 3.1%. There is no significant difference between the effects of individual inhibitors. C. Apoptosis induction is highly significant (p < 0.001), but AEBSF does not cause significant inhibition.