Lac water extract inhibits IFN-γ signaling through JAK2-STAT1-IRF1 axis in human melanoma

Abstract

Interferon-γ (IFN-γ) is a cytokine that plays an important role in the host defense of infectious diseases and in immune surveillance during tumor development; however, it has adverse effects in the pathogenesis of autoimmune diseases and in immunosuppressive microenvironments, promoting the immunoevasion of cancer cells. In this study, we identified lac water extract (Lac) as a candidate that can suppress IFN-γ signaling amongst 112 types of natural products, using PD-L1 promoter as a readout for the IFN-γ signaling. Moreover, we determined that Lac inhibits IFN-γ-induced PD-L1 and MHC class I expression on the cell surface in melanoma cells, both of which have been identified as the downstream molecules of IFN-γ signaling. We also determined that Lac inhibited the JAK2-STAT1-IRF1 pathway. Finally, we identified laccaic acids, encompassing laccaic acid A, B, C, and E, as the active components in Lac that inhibit IFN-γ signaling. Collectively, the laccaic acids are lead compounds for a novel inhibitor that targets the JAK2-STAT1-IRF1 pathway for diseases caused by the aberrant activation of IFN-γ signaling.

Fig. 1. Herbal medicines suppress IFN-γ-induced PD-L1 promoter activity. (A) UACC257 cells were co-transfected with the reporter plasmid containing human PD-L1 promoter sequences (PD-L1 promoter) or no promoter sequences (no promoter) upstream of the firefly luciferase gene with the reference reporter plasmid containing Renilla luciferase gene. After 24 hours of IFN-γ (250 ng ml⁻¹) treatment, the cell lysates were subjected to dual-luciferase assay. The luciferase activity was normalized to that of the vehicle control in the PD-L1 promoter. Data are the means ± S.D. of three independent experiments. *p < 0.01 by two-way ANOVA followed by the Bonferroni post-hoc test compared with vehicle control and IFN-γ. (B) Using the PD-L1 reporter plasmid in UACC257 cells, the water extracts of natural products were screened. After the addition of the water extracts of natural products at 100 μg ml⁻¹, the cells were incubated with IFN-γ (250 ng ml⁻¹) for 24 hours. The luciferase activity of each natural product was normalized to that of the vehicle control. Data are the mean of two independent experiments. (C) UACC257 were treated with Lac at the indicated dose and human IFN-γ (10 ng ml⁻¹) for 24 hours. The PD-L1 expression was determined as the median fluorescence intensity (MFI) by flow cytometry analysis. Relative PD-L1 expression was normalized to the MFI in non-treated cells. Data are the means ± S.D. of three independent experiments. *p < 0.01 by one-way ANOVA followed by the Bonferroni post-hoc test compared with IFN-γ-treated cells. (D) Melanoma cells (UACC257, M14, A2058, and MeWo) and lung cancer cells (A549 and PC-14) were treated with Lac (10 μg ml⁻¹) and human IFN-γ (10 ng ml⁻¹) for 24 hours. Other conditions are similar to those in Fig. 1C.

Fig. 2. Lac inhibits IFN-γ signaling in melanoma cells. (A) M14 cells were treated with Lac (100 μg ml⁻¹) and human IFN-γ (10 ng ml⁻¹) for 24 hours. The PD-L1 or MHC class I expression was determined by flow cytometry analysis. Other conditions are similar to those in Fig. 1D. (B) PC-9 cells were treated with Lac or gefitinib, an EGFR inhibitor, for 24 hours. *p < 0.01 by one-way ANOVA followed by the Bonferroni post-hoc test compared with non-treated cells.

Fig. 3. Lac inhibits IFN-γ signaling in melanoma cells through the JAK2-STAT1-IRF1 axis. (A) M14 and A2058 cells were treated with IFN-γ (10 ng ml⁻¹) after Lac (100 μg ml⁻¹) or baricitinib (0.5 μM) for the indicated times. Whole cell lysates were subjected to western blotting. (B) M14 and A2058 cells were treated with IFN-γ (10 ng ml⁻¹) after Lac (100 μg ml⁻¹) or baricitinib (0.5 μM) for 24 hours. The PD-L1 expression was determined as the median fluorescence intensity (MFI) by flow cytometry analysis. Other conditions are similar to those in Fig. 1D.

Fig. 4. Laccaic acids might be the active components in Lac. (A) HPLC chromatogram of lac water extract or laccaic acids. Their identification was determined from HPLC-ESI-MS/MS analysis. Peaks were identified as follows: (1) laccaic acid E; (2) laccaic acid C; (3) laccaic acid B; (4) laccaic acid A. (B) The structures of laccaic acid A, B, C, and E. (C) M14 and A2058 cells were treated with the laccaic acids (5 μg ml⁻¹) and human IFN-γ (10 ng ml⁻¹) for 24 hours. PD-L1 expression on the cell surface was determined by flow cytometry analysis. Other conditions are similar to those in Fig. 1D.