We investigated the synergistic apoptotic effects of co- treatments with Chios gum mastic (CGM) and eugenol on G361 human melanoma cells. An MTT assay was cond-ucted to investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment. The induction and augmentation of apop-tosis were confirmed by DNA electrophoresis, Hoechst stai-ning, and analyses of DNA hypoploidy. Western blot ana-lysis and immunofluorescent staining were also performed to evaluate expression and translocation of apoptosis- related proteins following CGM and eugenol co-treatment. Proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed.The results indicated that the co-treatment of CGM and eugenol induces multiple pa-thways and processes associated with an apoptotic response in G361 cells. These include nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome acti-vity, an increase of Bax and decrease of Bcl-2, a decreased DNA content, cytochrome c release 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 40 µg/ml CGM or 300 µM eugenol for 24 hours did not induce apoptosis. Our present data thus suggest that a combination therapy of CGM and eugenol is a potential treatment strategy for human melanoma.

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 μM eugenol or 3 μM 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.