Melanoma is an aggressive form of skin cancer that often shows limited response to existing therapies because of frequent drug resistance and disease recurrence. Protopanaxadiol (PPD), a key bioactive metabolite obtained from Panax ginseng, has shown notable anticancer potential in various studies. However, its specific impact on melanoma cells and the underlying molecular mechanisms are not yet well understood. This study explores the anticancer activity of PPD against melanoma and clarifies the pathways involved. The anti-melanoma effects of PPD and its mechanism of action were examined using MTT and crystal violet staining assays, GO enrichment analysis, KEGG enrichment analysis, flow cytometry, Western blot analysis, and CETSA experiments. PPD markedly suppressed the viability of melanoma cells in both dose- and time-dependent ways, triggering morphological alterations typical of apoptosis. Network pharmacology analysis pointed to the MAPK signaling pathway as a key target for PPD, which was subsequently validated experimentally. Blocking JNK with the inhibitor SP600125 attenuated PPD-induced apoptosis, highlighting the essential role of JNK signaling. Moreover, PPD selectively activated MLK3 in a ROS-independent manner, without affecting ASK1 or TAK1. These findings suggest that PPD triggers the JNK pathway primarily by targeting and activating MLK3, thereby promoting apoptosis. CETSA assays confirmed direct binding between PPD and MLK3. Molecular docking combined with site-directed mutagenesis indicated that the binding site likely involves residue LEU248. These results demonstrate that PPD possesses strong anti-melanoma activity by directly engaging MLK3 and stimulating the MLK3-JNK signaling cascade, which leads to apoptotic cell death. The study offers new understanding of PPD’s molecular actions and supports its potential development as a therapeutic option for melanoma.
