Fig. 13.

Synergistic action between five different arginase inhibitors and suboptimal concentrations of the NOD inhibitor itraconazole. 12,500 MKN-45 tumor cells or 10,000 human diploid fibroblasts Alpha-1 per assay were treated without further additions (“co”), 2 µM of inhibitors 1–4, 4 µM of inhibitor 5 in the absence or presence of 0.1 µg/ml itraconazol. Where indicated, assays received 2 mM histidine or 100 U/ml Mn-SOD before addition of the other compounds. The percentages of apoptotic cells were determined after five hours in duplicate experiments. The data shown in Fig. 13 demonstrate that all arginase inhibitors act synergistically with itraconzole in the specific apoptosis induction in tumor cells. The synergistic action depends on singlet oxygen and superoxide anions, as it was blocked by histidine as well as SOD. The combination of arginase inhibitors and itraconazol used in this experiment had no apoptosis inducing effect on normal diploid fibroblasts. The lack of reaction is due to the lack of extracellular superoxide anion generation in nontransformed cells. Statistical analysis: A: None of the individually applied compounds caused significant apoptosis induction, whereas the synergistic effects between itraconazole and the arginase inhibitors were highly significant (p<0.001). Inhibition of these synergistic effects by histidine and SOD were highly significant (p<0.001). B: There was no significant apoptosis induction in normal human diploid fibroblasts.