ABSTRACT
Trimidox (3,4,5-trihydroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase accompanied by growth inhibition and the differentiation of mammalian cells. Here we examine the induction of apoptosis by trimidox in several human leukaemia cell lines, focusing on the release of cytochrome c and the activation of caspase proteases in the human B cell line NALM-6. Induction of apoptosis by trimidox (300 microM) was detected in NALM-6, HL-60 (premyelocytic leukaemia cells), MOLT-4 (an acute lymphoblastic leukaemia cells), Jurkat (a T-cell leukaemia cells), U937 (expressing many monocyte-like characteristics), and K562 (erythroleukaemia). NALM-6 was most affected by trimidox among leukaemia cells; therefore, we employed NALM-6 cells in the subsequent experiments. The cells showed a time-dependent increase in DNA damage after trimidox (250 microM) treatment. A significant increase in the amount of cytochrome c release was detected after treatment with trimidox. Bcl-2 and Bax protein expressions were not changed by trimidox. Caspase-3 and -9 were activated by incubation with trimidox, whereas caspase-8 was not. Furthermore, trimidox-induced apoptosis was prevented by a broad-spectrum caspase inhibitor, a caspase-3, and a caspase-9 inhibitor, but not by a caspase-8 inhibitor. Inhibition of c-Jun NH2-terminal kinase (JNK) by SP600125 appreciably protected cells from trimidox-induced apoptosis, but no effect inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580. In contrast, extracellular signal-regulated kinase (ERK) inhibitors U0126 and PD98059 strongly potentiated the apoptotic effect of trimidox. This report shows that the induction of apoptosis by trimidox occurs through a cytochrome c-dependent pathway, which sequentially activates caspase-3 and caspase-9.
Subject(s)
Apoptosis , Benzamidines/toxicity , Caspases/metabolism , Cytochromes c/metabolism , Enzyme Inhibitors/toxicity , Anthracenes/pharmacology , Butadienes/pharmacology , Caspase 3 , Caspase 9 , Cell Line, Tumor , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Leukemia, B-Cell , Nitriles/pharmacology , Oligopeptides/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Signal Transduction/drug effects , Time FactorsABSTRACT
We found that octylcaffeate, a semisynthetic caffeic acid derivative, strongly inhibited the growth of human histiolytic lymphoma U937 cells in a dose- and time-dependent manner via apoptosis. Octylcaffeate induced the fragmentation of DNA into multiples of 180 bp (an apoptotic DNA ladder) and condensation of chromatin, and increased the percentage of hypodiploid cells detected with a flow cytometer. DNA fragmentation induced by octylcaffeate was inhibited by pretreatment with Z-DEVD-FMK and Z-Asp-CH(2)D-CB, an inhibitor of caspase, clearly showing that the mode of cell death is apoptotic. These findings suggest that the cytotoxicity of octylcaffeate involves the induction of apoptosis.
