Pharmaco-modulation of dihydroxy coumarins towards more selective antileukemic agents

VAZQUEZ R; RIVEIRO ME; ALONSO E; VERMEULEN M; GOMEZ N; PIEHL L; SHAYO C; DAVIO C

Orlando

Congreso; 102nd Annual Meeting of the American Association for Cancer Research (AACR).; 2011

AACR

Background: We described that 7,8-dihydroxy-4-methylcoumarin (DHMC) induces apoptosis in human leukemic U-937 cells, mediated by its pro-oxidant activity and activation of JNK. We also reported that DHMC shows selective toxicity for leukemic cells over normal peripheral blood monocytes. The aim of the present work is to establish the structural requirements for DHMC to display pro-apoptotic activity performing a structure activity relationship study of DHMC derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and a-pyrones (as representative of the d-lactone ring of the coumarin nucleus). Further, in an attempt to explain its selective toxicity, the mechanism of DHMC action through the JNK pathway will be characterized. Materials and Methods: DHMC analogues, cinnamic acid derivatives and a-pyrones were tested for their ability to induce biological effects in U-937 cells. Growth inhibition was determined through 3H-thymidine incorporation. Cellular viability was tested by trypan blue exclusion assays. Pro-apoptotic activity was evaluated by flow cytometry (annexin V), determination of caspase-3 activity and Western Blot analysis of caspase-3 and cleaved PARP levels. The oxidation potential and the capacity to generate free radicals were evaluated by cyclic voltammetry and electron paramagnetic resonance, respectively. To evaluate the role of cytoplasmic p21 (Cip1/WAF1) in the mechanism of DHMC selective action, we used U-937 cells transfected with a zinc-inducible p21 deletion mutant lacking the nuclear localization signal. Results: Only DHMC derivatives bearing two adjacent aromatic hydroxyl groups (catechol moiety) showed proliferation inhibition and pro-apoptotic activity in U-937 cells. a-pyrones had no effect on cellular viability, and cinnamic acid derivatives bearing the catechol moiety exhibited proliferation inhibition but no cytotoxic activity. Although ortho-dihydroxy coumarins and ortho-dihydroxy cinnamic acid derivatives showed a similar oxidation potential, only the former could generate stable free radicals in medium-like conditions, suggesting this capacity would be critical for their pro-apoptotic activity in U-937 cells. Finally, the pro-apoptotic effect of DHMC via JNK activation was abolished in U-937 cells expressing cytoplasmic p21, indicating this protein would be involved in the resistance that normal lymphocytes and monocytes, which normally express p21 in their cytoplasm, show for DHMC. Conclusions: DHMC-derived compounds carrying a catechol moiety can only induce apoptosis in U-937 cells if the integrity of the coumarinic nucleus is preserved. Selective toxicity of DHMC for leukemic cells would involve activation of JNK in the absence of cytoplasmic p21 expression. Collectively, our results point to ortho-dihydroxy coumarins as promising prototypes for the design of more selective antileukemic drugs.