20(S)-Ginsenoside Rh2 (GRh2) has been the subject of extensive research due to its wide range of health benefits. Nevertheless, its specific impact on melanoma is still not fully elucidated. The current work examined the anti-melanoma properties of GRh2 along with the underlying mechanisms involved. MTT assays, EdU staining, flow cytometry, cellular thermal shift assay (CETSA), confocal microscopy, molecular docking, molecular dynamics (MD) simulations, immunoblotting, and a B16F10 cell-bearing mouse model were utilized to evaluate the anti-melanoma activity and mechanism of GRh2. GRh2 inhibited cell proliferation, caused G0/G1 phase cell cycle arrest, and promoted apoptosis in melanoma cells. The compound also induced autophagy and reduced the activity of mTOR, a major negative regulator of autophagy, in these cells. Inhibition of autophagy further strengthened the anti-melanoma activity of GRh2. Molecular docking, MD simulations, and CETSA experiments indicated that GRh2 binds stably to the Src protein, an upstream kinase of STAT3. GRh2 suppressed the activation of both Src and STAT3, thereby blocking STAT3 nuclear translocation in melanoma cells. Over-activation of STAT3 diminished the cytotoxic, pro-apoptotic, and autophagy-inducing effects of GRh2. In addition, GRh2 inhibited the growth of B16F10 tumors in mice without causing noticeable toxicity. It decreased the protein levels of phospho-Src, phospho-STAT3, phospho-mTOR, and Mcl-1, while increasing cleaved-PARP and LC3B-II levels within the B16F10 tumors. GRh2 displays anti-melanoma effects mainly by suppressing Src/STAT3 signaling. This research enhances understanding of the anti-melanoma mechanism of GRh2 and suggests that consumption of GRh2 may help slow melanoma progression.
