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The EP4 receptor and PKA-dependent modification of CRE binding proteins participate in intrinsic mdr1b gene activation in primary rat hepatocyte cultures

: Ziemann, C.; Rüdell, G.; Riecke, A.; Steinfelder, H.J.; Oetjen, E.; Hirsch-Ernst, K.I.; Kahl, G.F.

Naunyn-Schmiedebergs archives of pharmacology 369 (2004), Supplement 1, pp.R122
ISSN: 0028-1298
ISSN: 1432-1912
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (Spring Meeting) <45, 2004, Mainz>
Journal Article, Conference Paper
Fraunhofer ITEM ()

Natural protein phosphatase inhibitors have been characterised in various reports as cytotoxic agents, which due to apoptosis induction display anti-tumour activity. We have recently shown that synthetic nitrostyrene derivatives are also inhibitors of ser/thr phosphatases with a pronounced pro-apoptotic effect in different cell types. Thus nitrostyrenes might represent a new class of cytostatic compounds. Their pro-apoptotic potential resembled that of natural phosphatase inhibitors, e.g. cantharidic acid, but in comparison with cantharidic acid, apoptosis induction by nitrostyrene progressed much faster. Therefore, we were interested in additional mechanisms, participating in apoptosis by nitrostyrene derivatives. In the present study, we could demonstrate that nitrostyrene and its pro-apoptotic triaminopyrimidine adduct (each 10-25µM), but not the non-pro-apoptotic diaminopyridine adduct (10-25µM), displayed concentration-dependent rapid DNA damage in HL60-leukaemia cells. As assessed by the alkaline comet assay, this effect became already evident after 30 min of incubation. In contrast, the natural phosphatase inhibitor cantharidic acid did not induce DNA damage within the first 3 hours of incubation. We further investigated induction of apoptosis and DNA damage by nitrostyrene derivatives in comparison with that of doxorubicin (DOX) in two HL60 sublines, one, equivalent to the parental line (HL60-neo) and the other exhibiting over-expression of the anti-apoptotic protein Bcl-xL (HL60-Bcl-xL). HL60-neo cells displayed DNA damage and apoptosis induction (as assessed by DNA-fragmentation and caspase 3/7 activation) by pro-apoptotic nitrostyrene derivatives as well as by DOX (lµM), whereas HL60-Bcl-xL cells exhibited nearly no responsiveness towards DOX. Unresponsive-ness of HL60-Bcl-xL cells was not due to over-expression of the multidrug transporter MDR1, as there was no evidence for enhanced MDR1 mRNA expression. Interestingly, the pro-apoptotic effect of nitrostyrene derivatives as well as the early DNA damage was retained in the HL60-Bcl-xL subline. In conclusion, we could demonstrate a correlation between early DNA damage and the fast and prominent pro-apoptotic effect of nitrostyrene derivatives, which even persists in apoptosis compromised and thus chemotherapy resistant leukaemia cells.