Abstract
BACKGROUND: Melanoma is a highly aggressive skin cancer with limited therapeutic options due to drug resistance and recurrence. Protopanaxadiol (PPD), a bioactive metabolite derived from Panax ginseng, has demonstrated promising anticancer properties. However, the effects and underlying mechanisms of PPD on melanoma remain unclear. This study aims to investigate the anticancer effects of PPD on melanoma cells and elucidate its molecular mechanisms. METHODS: To evaluate anti-melanoma activity of PPD and its mechanism MTT and crystal violet staining assays, GO enrichment analysis, KEGG enrichment analysis, flow cytometry, Western blot analysis, and CETSA experiments were carried out. RESULTS: PPD significantly inhibited the viability of melanoma cells in a dose- and time-dependent manner, inducing morphological changes characteristic of apoptosis. Network pharmacology identified the MAPK signaling pathway as a potential target of PPD, with further confirmation. Inhibition of JNK with SP600125 reversed PPD-induced apoptosis, indicating that JNK signaling plays a critical role. Additionally, PPD specifically activated MLK3, independent of ROS, but not ASK1 or TAK1, which demonstrates that PPD might activate the JNK pathway through targeting and activating MLK3, leading to apoptosis. CETSAs results demonstrated direct binding of PPD to MLK3. Molecular docking and site-directed mutagenesis further indicated the binding site might be LEU248. CONCLUSIONS: Our findings revealed that PPD exerts potent anti-melanoma effects by directly targeting MLK3 and activating the MLK3-JNK signaling pathway, leading to apoptosis. These results provide novel insights into the molecular mechanism of PPD and suggest its potential as a therapeutic agent for melanoma treatment.