Fig. 4.

The role of H₂O₂, NO and peroxynitrite for bystander signaling. MKN-45 cells were pretreated with 0.05 mU/ml GOX and 1 mM nitrite for 25 min, subjected to three cycles of washing and then resuspended in fresh medium. Pretreated cells were added at increasing percentages to untreated tumor cells. A. During pretreatment with GOX/nitrite, additional assays received either 20 μM EUK-134, or 2.4 mM l-NAME or 25 μM FeTPPS. B. During coculture of GOX/nitrite-pretreated cells with untreated cells, EUK-134, or l-NAME or FeTPPS was present. The inhibitors had been added to the cells 10 min before mixing the populations. C. The experiment was performed as described under B, with the modification that the inhibitors were added 25 min after the beginning of coculture. In all assays, the percentages of apoptotic cells were determined at 4.5 h. The results show that acquisition of bystander effect-inducing potential through treatment with GOX/nitrite involves H₂O₂, NOS-derived NO and peroxynitrite. The data also show that NO and peroxynitrite play a dominant role for bystander signaling during the first 25 min, but not later. H₂O₂ plays a role at all times, as it is involved in several steps, including late HOCl signaling. Statistical analysis: A. The effects of all three inhibitors are highly significant (p < 0.001). B. The effects of all three inhibitors, as well as the difference between inhibition by EUK-134 versus inhibition by l-NAME or FeTPPS are highly significant (p < 0.001). C. Only the inhibitory effect of EUK-134 is highly significant (p < 0.001).