Juglone potentiates BRAF inhibitor­induced apoptosis in melanoma through reactive oxygen species and the p38­p53 pathway.

BRAF inhibitors are some of the most effective drugs against melanoma; however, their clinical application is largely limited by drug resistance. Juglone, isolated from walnut trees, has demonstrated anti­tumour activity. In the present study, it was investigated whether juglone could enhance the responses to a BRAF inhibitor in melanoma cells (A375R and SK­MEL­5R) with an acquired resistance. These cells were treated with juglone alone, BRAF inhibitor (PLX4032) alone, or juglone combined with PLX4032. It was demonstrated that the combination of juglone and PLX4032 had synergistic effects on BRAF inhibitor­resistant melanoma cells. Juglone potentiated PLX4032­induced cytotoxicity and mitochondrial apoptosis in both A375R and SK­MEL­5R cells, which was accompanied by a decline in mitochondrial membrane potential and a decrease in Bcl­2/Bax ratio. Moreover, juglone combined with PLX4032 markedly increased the intracellular level of reactive oxygen species (ROS) and activated p38 and p53, as compared with juglone alone or PLX4032 alone. Pre­treatment with N­acetyl­L­cysteine, a ROS scavenger, completely reversed the cytotoxicity induced by juglone combined with PLX4032. In conclusion, juglone potentiated BRAF inhibitor­induced apoptosis in resistant melanoma cells, and these effects occurred partially through ROS and the p38­p53 pathway, suggesting the potential of juglone as a sensitizer to BRAF inhibitors in the treatment of melanoma.

Icariside II overcomes BRAF inhibitor resistance in melanoma by inducing ROS production and inhibiting MITF.

Metastatic melanoma is the most aggressive skin cancer. Although BRAF inhibitor treatment has achieved great success in melanoma, resistance develops within 12 months. Icariside II (IS), a natural compound extracted from Herba Epimedii, exerts anticancer properties. In the present study, we determined by MTT, flow cytometry and western blotting, respectively that IS potentiated the PLX4032­induced downregulation of cell viability and increase in apoptosis and autophagy in BRAF inhibitor­resistant melanoma. In addition, we also revealed by flow cytometry and western blotting, respectively, that IS combined with PLX4032 increased mitochondrial and intracellular reactive oxygen species (ROS) generation and subsequently promoted depolarization of mitochondria and release of apoptotic proteins. N­acetyl cysteine (NAC) and glutathione (GSH), ROS scavengers, reversed the IS­induced enhancement of the response to PLX4032. Microphthalmia­associated transcription factor (MITF) and tyrosine­protein kinase Met (c­Met) are well­known factors that contribute to BRAF inhibitor resistance. Furthermore, c­Met is a direct transcriptional target of MITF in melanocytes and melanoma cells. It was also revealed that IS markedly inhibited MITF and c­Met expression partially by increasing ROS production in BRAF inhibitor­resistant melanoma cells.