Phytomedicine : international journal of phytotherapy and phytopharmacology · 2026
Pinocembrin inhibits melanoma progression via Ets1-mediated Smad3 transcription to enhance anti-PD-1 therapy.
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Liao Yuan, Li Yu-Rou, Wang Yi-Bing, Li Jian-Chun, Tan Rui-Zhi, Wang Hong-Lian, Wang Li
Аннотация
BACKGROUND: The TGF-β/Smad3 signaling drives the malignant progression of melanoma, activates PD-L1 expression, and exerts immunosuppressive functions. Pinocembrin (PIN), a natural dihydroxyflavanone has been shown to have anti-melanoma effects, but the detailed mechanisms remain unresolved.
PURPOSE: This study aimed to elucidate the effect of PIN on the reduction of PD-L1 expression and the enhancement of anti-PD-1 therapy by inhibiting Ets1-mediated Smad3 transcription to determine the role of PIN in anti-melanoma.
METHODS: To investigate the pharmacological mechanism of PIN, we employed B16 cells for in vitro assays and established a syngeneic tumor-bearing mouse model for in vivo studies. These analyses integrated multiple approaches, including RNA-seq, bioinformatics prediction, Dual Luciferase Reporter and ChIP-qPCR, site-directed mutagenesis, CRISPR/Cas9, lentivirus-mediated stable overexpression cell lines, RT-qPCR, and Western blot. Furthermore, the proportion of immune cells within tumor tissues was determined by flow cytometry, ELISA was performed to quantify IFN-γ secretion.
RESULTS: PIN significantly inhibited melanoma growth, proliferation, migration, and PD-L1 expression. RNA-seq analysis revealed marked inhibition of the TGF-β signaling pathway, with Smad3 identified as a core downstream target. Using bioinformatics analysis, Dual Luciferase reporter assay, and ChIP-qPCR, we demonstrated that Ets1 directly bound to four specific regions within the Smad3 promoter and activated its transcriptional activity, all of which were suppressed by PIN treatment. Ets1 overexpression restored Smad3 and PD-L1 expression and reversed the tumor-suppressive effects of PIN. Moreover, Ets1 overexpression failed to restore PD-L1 expression in Smad3-deficient B16 cells. We further demonstrated that PIN exerted its anti-tumor effect primarily on tumor cells and also modulated the immune composition of the melanoma microenvironment. Notably, combining PIN with αPD-1 increased pro-tumor immune populations, reduced inhibitory subsets, and enhanced immune-mediated melanoma killing.
CONCLUSION: PIN can effectively inhibit the malignant progression of melanoma and the expression of PD-L1 through Ets1/Smad3 axis. Combined with αPD-1, PIN can further enhance its immunomodulatory function.
PURPOSE: This study aimed to elucidate the effect of PIN on the reduction of PD-L1 expression and the enhancement of anti-PD-1 therapy by inhibiting Ets1-mediated Smad3 transcription to determine the role of PIN in anti-melanoma.
METHODS: To investigate the pharmacological mechanism of PIN, we employed B16 cells for in vitro assays and established a syngeneic tumor-bearing mouse model for in vivo studies. These analyses integrated multiple approaches, including RNA-seq, bioinformatics prediction, Dual Luciferase Reporter and ChIP-qPCR, site-directed mutagenesis, CRISPR/Cas9, lentivirus-mediated stable overexpression cell lines, RT-qPCR, and Western blot. Furthermore, the proportion of immune cells within tumor tissues was determined by flow cytometry, ELISA was performed to quantify IFN-γ secretion.
RESULTS: PIN significantly inhibited melanoma growth, proliferation, migration, and PD-L1 expression. RNA-seq analysis revealed marked inhibition of the TGF-β signaling pathway, with Smad3 identified as a core downstream target. Using bioinformatics analysis, Dual Luciferase reporter assay, and ChIP-qPCR, we demonstrated that Ets1 directly bound to four specific regions within the Smad3 promoter and activated its transcriptional activity, all of which were suppressed by PIN treatment. Ets1 overexpression restored Smad3 and PD-L1 expression and reversed the tumor-suppressive effects of PIN. Moreover, Ets1 overexpression failed to restore PD-L1 expression in Smad3-deficient B16 cells. We further demonstrated that PIN exerted its anti-tumor effect primarily on tumor cells and also modulated the immune composition of the melanoma microenvironment. Notably, combining PIN with αPD-1 increased pro-tumor immune populations, reduced inhibitory subsets, and enhanced immune-mediated melanoma killing.
CONCLUSION: PIN can effectively inhibit the malignant progression of melanoma and the expression of PD-L1 through Ets1/Smad3 axis. Combined with αPD-1, PIN can further enhance its immunomodulatory function.