DA-125, a novel anthracycline derivative showing high-affinity DNA binding and topoisomerase II inhibitory activities, exerts cytotoxicity via c-Jun N-terminal kinase pathway

Purpose: DA-125 [(8S,10S)-8-(3-Aminopropanoyloxyacetyl)-10-[(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl) oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacene-dione hydrochloride] is a novel anthracycline derivative with Anticancer activity. In the present study, we compared the cytotoxicity of DA-125 with that of doxorubicin in H4IIE rat hepatoma cells and investigated the mechanistic basis. Because activation of MAP kinases, in particular c-Jun N-terminal kinase (JNK), is implicated in apoptotic cell death, the signaling pathways responsible for DA-125-induced Apoptosis were studied.

Methods: Cytotoxicity and Apoptosis were measured in H4IIE cells and cells were stably transfected with a dominant-negative mutant of JNK1 (JNK1-) by MTT and TUNEL assays. Inhibition of Topoisomerase II activity was determined in vitro. Drug accumulation and DNA binding affinity were determined by fluorescence spectroscopy.

Results: The cytotoxicity of DA-125 was greater than that of doxorubicin (IC50 11.5 vs 70 microM). DA-125 induced Apoptosis with 30-fold greater potency than doxorubicin. Inhibition of Topoisomerase II by DA-125 was fourfold greater. The presence of excess beta-alanine, a DA-125 moiety, failed to alter cytotoxicity and accumulation of DA-125, indicating that the improved cytotoxicity of DA-125 did not result from the beta-alanine moiety. Greater cellular accumulation of DA-125 correlated with its high-affinity DNA binding. Although neither PD98059 nor SB203580 altered the degree of cytotoxicity induced by DA-125, JNK1 cells exhibited about a twofold greater viability than control cells. DA-125-induced Apoptosis was also decreased in JNK1- -transfected cells.

Conclusions: DA-125 potently inhibited Topoisomerase II activity and induced Apoptosis by a high rate of prooxidant production. DA-125 exhibited high-affinity DNA binding with improved cellular drug accumulation. Apoptosis induced by DA-125 involved the pathway of JNK1, but not ERK1/2 or p38 kinase.