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2010
Conference Paper
Titel
Comparative mechanistical investigations on the genotoxic potential of nitrovinyluracil derivatives
Titel Supplements
Abstract
Abstract
Trans-ß-nitrostyrene was recently shown to be a promising lead for the development of new cytostatic drugs. While its strong pro-apoptotic properties seems to be very promising, till now its high reactivity prevents a clinical application. To improve the therapeutic coefficient under preservation of the biologically active nitrovinylic side chain, nitrovinyluracil (NVU)-derivatives were synthesized. In the present study, the cytotoxic, pro-apoptotic, and genotoxic properties of NVU and 3 of its derivatives, allyl-NVU (A-NVU), benzyl-NVU (B-NVU), and benzhydryl-NVU (BH-NVU) were examined to assess their utilization as new anti-tumor drugs. A flowcytometric Annexin V-FITC assay and an apoptosis microarray with L-5178-Y/TK+/- mouse lymphoma cells revealed a fast pro-apoptotic effect of NVU, A-NVU, and B-NVU at a concentration of 15µM. BH-NVU, however, seemed to be too toxic to selectively induce apoptosis. By using the alkaline and neutral versions of the comet assay and a YH2AX-immunfluorescence assay with A549 lung epithelial cells, we demonstrated a profound clastogenic potential of the NVU-derivatives, comprising induction of DNA-double strand breaks (DSB). Clastogenicity increased in the order NVU < A-NVU < B-NVU < BH-NVU. This order most likely is the consequence of differences in cellular uptake, because biologic activity corresponded to hydrophobicity of the different NVU-derivatives, as determined by calculation of the log KOW values. As shown by the inability of the NVU-derivatives to induce DNA-damage in an acellular comet assay, the clastogenic effect was not of direct nature and it was significantly reduced by S9-mix. Interestingly, the NVU-derivatives preferentially mediated DNA-damage in proliferating cells. While nearly no DNA-damage was detected in resting human white blood cells, there was significant genotoxicity in phytohemagglutinine-stimulated blood. Together, these findings led us to the assumption that an inhibition of topoisomerase II (TopoII) might be involved in clastogenicity of NVU-derivatives. This hypothesis was further supported by experiments with aclarubicin (a topoisomerase inhibitor, which prevents formation of the TopoII-DNA complex), as induction of DSB by the NVU-derivatives was significantly reduced by parallel aclarubicin treatment. In conclusion, NVU-derivatives are promising candidates for tumor treatment, due to their presumably TopoII-based pro-apoptotic potential and their preference for proliferating cells. However, their high reactivity necessitates further structural modifications.