RESUMO
Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (-)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0µM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC(50) of 0.4µM) and human peripheral blood mononuclear cells (PBMC, IC(50) of 1.2µM). After 24h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5µM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.
Assuntos
Apoptose/efeitos dos fármacos , Diterpenos Clerodânicos/farmacologia , Leucemia/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , DNA/genética , Fragmentação do DNA/efeitos dos fármacos , Diterpenos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Células HL-60 , Humanos , Mitocôndrias/efeitos dos fármacos , Fosfatidilserinas/metabolismoRESUMO
A crude bioactive EtOH extract of the twigs of Casearia obliqua afforded two new clerodane diterpenes, caseobliquins A (1) and B (2). Additionally, bioactivity-directed fractionation on a bioactive hexane extract of the leaves from this species led to the isolation of the known clerodane diterpenes rel-6beta-hydroxyzuelanin-2beta-benzoate and rel-2alpha-hydroxyzuelanin-6beta-benzoate (3 and 4) as a mixture and 2beta-hydroxyzuelanin-6beta-cinnamate (5). The structures of the new clerodanes 1 and 2 were established on the basis of their 1D and 2D NMR spectroscopic data, and the new compound 1 and the known substance 5 had their absolute configurations determined by circular dichroism spectroscopy. The cytotoxicity of several of the compounds isolated was evaluated against a small panel of human tumor cell lines.