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Abstract
Background
Eugenol is a major component of the essential oil isolated from Eugenia caryophyllata (Myrtaceae), and has been widely used as a traditional medicine. In this study, the effects of eugenol on the cytotoxicity, induction of apoptosis and putative pathways of its actions were investigated in human promyelocytic leukemia cells (HL-60).
Methods
After applying eugenol to cultured HL-60, the changes in the mitochondrial membrane potential of the cells were monitored after double staining with propidium iodide and rhodamine 123, with 2', 7'-dicholorofluorescin diacetate was used to measure of levels of reactive oxygen species (ROS)
Results
Eugenol was shown to be a potent inducer of apoptosis; transducing the apoptotic signal via ROS generation; thereby, inducing mitochondrial permeability transition (MPT) and cytochrome c release to the cytosol. The production of ROS, mitochondrial alteration and subsequent apoptotic cell death in eugenol-treated cells were blocked by the antioxidant, N-acetylcystein (NAC).
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Fig. 1.
Effects of eugenol on the induction of apoptosis and DNA fragmentation in HL-60 cells. (A) HL-60 cells were treated with increasing concentrations of eugenol for the indicated times. The extent (%) of fragmentation was determined using DAPI as described in Materials and Methods. Data presented are the means± SD of results from three independent experiments. ◆, 20μM eugenol; ■, 40μM eugenol; ▲, 60μM eugenol; ●, 40μM eugenol in the pretreatment of 5mM NAC. (B) HL-60 cells were treated with 40μ M eugenol for the indicated time period in the presence or absence of 5mM NAC and DNA fragmentation was analyzed by agarose gel electrophoresis.
Fig. 2.
(A) Eugenol caused the cleavage of procaspase-9 and -3, the releasing of cytochrome c from mitochondria, accumulation of cytochrome c in cytosol, translocation of bax to mitochondria and inhibition of Bcl-2 in mitochondria. HL-60 cells were treated with eugenol (40μM) for indicated time points. After treatment, the cytosolic and mitochondrial fractions were separated by SDS-PAGE, transferred onto cellulose membranes, and then blotted with specific antibodies for cytochrome c, bax, bcl-2, cas pase-9 and caspase-3 and speci fic antibodies. The amount of β-actin was measured as an internal control. (B) Eugenol caused the accumulation of cytochrome c and translocation of bax to mitochondria. HL-60 cells were cultured in the presence or absence of 5mM NAC for 1h and then 40 μM eugenol was added and incubated for 8h.
Fig. 3.
Determination of mitochondrial membrane potentials (△Ψm). HL-60 cells were treated with 40 μM eugenol in the absence or presence of 5mM NAC for 1h and then △Ψm were determined using rhodamin 123 and PI by flow cytometry as described in Materials and Methods. Data are representative of three independent experiments. (A) control, (B) 40μM eugenol for 2 h, (C) 5mM NAC for 1h, (D) 5mM NAC for 1h and then 40μM eugenol was added for 2h.
Fig. 4.
Eugenol induces the generation of ROS. HL-60 cells were cultured in the presence or absence of 5mM NAC for 1h and then 40M eugenol was added and incubated for 2h. DC F fluorescence was detected by (A) flow cytometry and (B) fluorescent microscope (× 200) after 1h of eugenol treatment. Data are representative of three independent experiments. (a) con trol (b) 40 μM eugenol for 30min (c) 40μM eugenol for 1h (d) 5mM NAC for 1h and then 40μM eu genol was added for 30min.
Fig. 5.
Effects of eugenol on the level of intracellular GSH and protein thiols. Cells were treated with various concentrations of eugenol for the indicated period and then the level of intracellular GSH (A) and protein thiols (B) were determined. ●, control; ◆, 20μM; ■, 40μM; ▲, 60μM. Data are presented as mean± S.D. of results from three independent experiment.
Table 1.
Cytotoxic activity of eugenol and methyleu genol on cancer cell growth
| Cell line | IC 50∗ (μM) | ||
|---|---|---|---|
| Eugenol | Methyleugenol | Cisplatin | |
| U-937 | 39.4 | 89.3 | 22.0 |
| HL-60 | 23.7 | 76.5 | 17.7 |
| HepG2 | 118.6 | 275.9 | 54.6 |
| 3LL Lewis | 89.4 | ＞300 | 36.5 |
| SNU-C 5 | 129.4 | ＞300 | 27.4 |
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