Trimidox Induces Apoptosis via Cytochrome c Release in NALM‐6 Human B Cell Leukaemia Cells

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 mM) 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 mM) 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, trimidoxinduced 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 NH 2 -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.

Inhibitory Effect of Trimidox on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 264.7 Macrophages

“…There was no effect until incubation with trimidox at 200 µM and slightly decreased cell survival at 300 µM (78.4 ± 5.6% of control), but it has no significant effect. This concentration had been shown to produce cell damage in several types of leukemia cells in our previous report (4). Incubation of the cells with a combination of trimidox (300 µM) and LPS (1 µg / ml) for 24 h had no significant effect on cell viability, as estimated by MTT assay (data not shown).…”

“…Trimidox (3,4,5-trihydroxybenzamidoxime) was shown to be one of the most potent in this series of compounds (3). Our lab has reported that trimidox induced apoptosis in human leukemia cell lines (4).…”

“…The expression of iNOS, IκBα, proteins was detected by Western blotting, modified by our previous method (4). Briefly, cells were harvested and extracted by lysis buffer.…”

Trimidox-Induced Apoptosis Is Mediated Through Induction of p53 in NALM-6 Cells

“…Trimidox (3,4,5-trihydroxybenzamidoxime) was shown to be one of the most potent in this series of compounds (5). Our lab has previously reported that trimidox induced apoptosis in a number of typical human leukemia cell lines, and the most susceptible cell was NALM-6, a B-cell line of acute lymphoblasyic leukemia, which expresses wild-type p53 (6). The aim of this study was to determine the contribution of p53 to trimidox-induced apoptosis.…”

“…Apoptotic cells were estimated by nuclear morphological observation and DNA fragmentation analysis using our previous methods (6,10).…”