Costunolide, isolated from the stem bark of Magnolia sieboldii, is a sesquiterpene lactone that exhibits various biological and immunological actions. We investigated the induction mechanism of apoptosis by costunolide in a human B cell leukemia NALM-6 cell culture system. Costunolide (10 m mM)-induced apoptosis time-dependently increased, estimated by nuclear damage observation and flow cytometric analysis. Costunolide did not change Fas-associated factor 1 (FAF1), but the phosphorylation of Fas-associated death domain (FADD) at serine 194 increased from early treatment. The activation of caspase-8 and -9 and degradation of poly-(ADP-ribose) polymerase (PARP) was time-dependently detected by incubation with costunolide. Pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors significantly blocked costunolide-induced apoptosis, but caspase-9 inhibitor failed to block apoptosis. Telomerase activity was significantly suppressed after treatment with costunolide, and human telomerase reverse transcriptase (hTERT), a critical determinant of the enzyme activity of telomerase, decreased the expression of both mRNA and protein levels by costunolide. Costunolide-induced repression of telomerase was prevented by pretreatment of cells with caspase-3, -8 and broad spectrum caspase inhibitors, but caspase-9 inhibitor was no effect. These data suggest that one of the costunolide-induced apoptotic mechanisms is that the receptor-mediated pathway precedes the mitochondria-dependent pathway, caused by the inhibition of telomerase activity via suppression of hTERT in NALM-6 cells.

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Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H 2 O 2 ), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 m mM, 117.5 m mM, or 41.8 m mM, respectively. Induction of cytotoxicity by H 2 O 2 (500 m mM) or tBuOOH (100 m mM) was concentration-dependently attenuated by incubation with Trimidox (10-150 m mM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.

Abstract. We examined the effect of trimidox (3,4,5-trihydroxybenzamidoxime) on the production of nitric oxide (NO) by lipopolysaccharide (LPS) in mouse RAW 264.7 macrophages. Trimidox (50 -300 µM) concentration-dependently inhibited NO production by LPS (0.01, 0.1, or 1 µg / ml) after incubation for 24 h. LPS-induced expression of inducible NO synthase (iNOS) and degradation of IκBα were prevented by trimidox. The protective effect against NO production by LPS was not only observed in prior incubation but also later incubation with trimidox until iNOS was activated by LPS. These results suggest that trimidox has a predominantly protective effect against LPS-induced production of NO via iNOS expression.

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