S Phase Cell Cycle Arrest and Apoptosis is Induced by Eugenol in G361 Human Melanoma Cells

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.

Apoptotic Effects of A Cisplatin and Eugenol Co-treatment of G361 Human Melanoma Cells

Eugenol (4-allyl-2-methoxyphenol) is a naturally occurring phenolic compound that is widely used in dentistry as a component of zinc oxide eugenol cement that is commonly applied to the mouth environment. Cisplatin is one of the most potent known anticancer agents and shows significant clinical activity against a variety of solid tumors. This study was undertaken to investigate the synergistic apoptotic effects of co-treatments with eugenol and cisplatin on human melanoma (G361) cells. To investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment, an MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis of DNA hypoploidy. Western blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed. The results indicated that a co-treatment with eugenol and cisplatin induced multiple pathways and processes associated with an apoptotic response in G361 cells including nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome activity, the increase and decrease of Bax and Bcl-2, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, and the activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD). In contrast, separate treatments of 300 microM eugenol or 3 microM cisplatin for 24 h did not induce apoptosis. Our present data thus suggest that a combination therapy of eugenol and cisplatin is a potential treatment strategy for human melanoma.

In vitro inhibition of trichosanthin-monoclonal antibody conjugate on human melanoma cells / 中国免疫学杂志

We have constructed an immunotoxin(Ng76-TCS),which was composed of a monoclonalantibody directed against human melanoma and trichosanthin(TCS)——a single chain ribosomeinactivating protein.The cultured human melanoma cells(M21)were inhibited effectively byNg 76-TCS.The cytotoxicity of Ng76-TCS to M21 cells was 2,000-fold higher than that of free TCS and Ng76 mixture.A conjugate,which was prepared with normal mice immunoglobulinand TCS(NIgG-TCS),was 160-fold less cytotoxic to M21 cells.Meanwhile Ng76-TCS was125-fold less cytotoxic to nontarget cells Hela.These results showed that the immunotoxinNg76-TCS was a potent and specific anti-human melanoma agent.