Eugenol is an essential oil found in cloves and cinnamon that is used widely in perfumes. However, the significant anesthetic and sedative effects of this compound have led to its use also in dental procedures. Recently, it was reported that eugenol induces apoptosis in several cancer cell types but the mechanism underlying this effect has remained unknown. In our current study, we examined whether the cytotoxic effects of eugenol upon human melanoma G361 cells are associated with cell cycle arrest and apoptosis using a range of methods including an XTT assay, Hoechst staining, immunocytochemistry, western blotting and flow cytometry. Eugenol treatment was found to decrease the viability of the G361 cells in both a time- and dose-dependent manner. The induction of apoptosis in eugenol-treated G361 cells was confirmed by the appearance of nuclear condensation, the release of both cytochrome c and AIF into the cytosol, the cleavage of PARP and DFF45, and the downregulation of procaspase-3 and -9. With regard to cell cycle arrest, a time-dependent decrease in cyclin A, cyclin D3, cyclin E, cdk2, cdk4, and cdc2 expression was observed in the cells after eugenol treatment. Flow cytometry using a FACScan further demonstrated that eugenol induces a cell cycle arrest at S phase. Our results thus suggest that the inhibition of G361 cell proliferation by eugenol is the result of an apoptotic response and an S phase arrest that is linked to the decreased expression of key cell cycle-related molecules.

Eugenol (4-allyl-2-methoxyphenol) is a phenol derivative and generally used in dental treatment. A few investigator reported that eugen이-induced C)πoto잉city by apopto디c pathway, but it is not yet well understood In the present study, to investigate the eugenol-induced cytoto잉city by apoptosis, we have examined the apoptotic molecules and pathway in primary human gingival fibroblast (HGF) and human salivary gland cells (HSG). To identify apoptotic cell death, 3-(4,5-dimethylthiazol-2-yl)-2 ,5-diphenyl tetrazolium bromide (MTT) reduction assay with or without N-acetylcysteine (NAC), and the morphological study by propidium iodide (pI) staining were screened. And to investigate the apoptotic pathway, reverse transcriptase-polymerase chain reaction (RT-PCR) for apoptotic molecules and caspase aαivity assay were performed. Both M1T reduction assay and an addition of NAC showed that eugenol act as a pro-oxidant led to cell death. With the morphological study, both cells showed apoptotic change by nuclear fragmentation and/or chromatin condensations. With the apoptotic machinery study, the Bax and Bcl-2 mRNA expression were not detected in HGF. But, for HSG, the increased expression of Bax with decreased of Bcl-2 was observed. And the expression of Apaf-l was not detected or nα significantly increased in HGF and HSG, respectively. With measure of caspase activity, there was no change of caspase activities in HGF. But, for HSG, there was decrease of caspase 9 activity and increased caspase 3 activity. We suggest eugenol-treated HGF underwent apoptosis independent of Bcl family and caspase. However, for eugenol-πeated HSG, apoptosis occurred via Bcl famiIy and caspase pathway.