Chinese holly fruit inhibits melanoma cell proliferation and immune escape by activating P53-P21 signaling and inhibiting JAK2-STAT1 signaling.

INTRODUCTION: Chinese holly (Ilex cornutaLindl. ex Paxt) fruit exhibits various biological activities, including heat-clearing and detoxifying, promoting blood circulation to remove stasis, relieving pain and detoxifying, and anti-inflammatory effects. However, its role in inhibiting tumor initiation and progression remains unclear, particularly in melanoma therapy. METHODS: In the present study, a panel of molecular biological approaches, including CCK8 assay, colony formation assay, transwell assay, cell cycle profiling, apoptosis detection, flow cytometry, IHC, Western blot, qPCR, RNAseq and metabonomics were utilized to systematically explore the regulatory effects of CHF on proliferative behaviors and immune evasion potential of melanoma cells. RESULTS AND DISCUSSION: We found that Chinese holly fruit (abbreviated as CHF) extract suppresses the proliferative activity of melanoma by activating the P53-P21 signaling pathway, which downregulates the expression of CDK2 and cyclin family members. Meanwhile, CHF promotes apoptosis by upregulating FAS and GADD45A. Metabolomic profiling revealed that differential metabolites induced by CHF extract treatment were enriched in glucose metabolism, energy metabolism, and the AMPK signaling pathway. Furthermore, CHF extract inhibits PD-L1 expression by suppressing the JAK2-STAT1 signaling pathway, thereby increasing the infiltration of CD3+ and CD8+T cells into tumor tissues and consequently suppressing melanoma immune escape. More importantly, pharmacological administration of CHF extract improves the anti-tumor efficacy of anti-PD-1/PD-L1 immunotherapy, as demonstrated by enhanced T cell infiltration and slower tumor growth in the combination treatment group compared with monotherapy with either agent. Taken together, these findings highlight a crucial role of CHF extract in anti-melanoma effects, and combination with PD-L1 antibody immunotherapy may be an attractive strategy for melanoma treatment.