Arbutin overcomes tumor immune tolerance by inhibiting tumor programmed cell death-ligand 1 expression

Abstract

Arbutin, predominantly derived from the bearberry plant, exhibits promising immunomodulatory properties. Given its ability to influence the programmed cell death-ligand 1/ programmed cell death-1 (PD-L1/PD-1) pathway, it is emerging as a potential alternative treatment for cancer. A reduced expression of PD-L1, as seen after arbutin treatment, can bolster immune responses critical step in effective tumor immunotherapy. However, the molecular mechanism by which arbutin inhibits PD-L1 is still incompletely known. The expression of PD-L1 was decreased after tumor cells were treated with arbutin. Arbutin can downregulate the expression of PD-L1 on the cell surface via the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The findings suggest the protective role of arbutin and provide novel insights into immunotherapy, which involves inhibiting the AKT/mTOR signaling pathway. Arbutin might serve as a potential therapeutic agent alone or in combination with other treatments.

Keywords: Arbutin; programmed cell death protein ligand-1; tumor immune tolerance.

Figure 1

Effect of Arbutin on cell viability in B16F10 and LL2 cell. The B16F10 and LL2 cells (5 × 10⁵ cells/well) were placed into 96-well plates and incubated at 37℃ for 24 h. The B16F10 (A) and LL2 (B) cells were treated with indicated concentrations of arbutin or cisplatin (2 µg/ml) for 6 h. Cell viability was evaluated by WST-1 cell proliferation assay. (mean ± SD, n=5). The B16F10 and LL2 cells (5 × 10⁴ cells/well) were placed into 96-well plates and incubated at 37℃ for 24 h. The B16F10 and LL2 cells were treated with indicated concentrations of arbutin for 6 h. (C) Then the cells were fixed and stained for BrdU (green) and nuclei were counterstained with Hoechst 33342 (blue). (D) The cells were counted under a fluorescence microscope (mean ± SD, n=3; ***, p < 0.001).

Figure 2

Arbutin inhibited PD-L1 expression in B16F10 and LL2 cells. Arbutin reduced PD-L1 expression in B16F10 and LL2 cells. (A) B16F10 and LL2 cells (5 × 10⁵ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 h. Then treatment with Salmonella (5 × 10⁷ cells/well) for 1.5h or CoCl₂ (200 μM) for 6 h, the expression of PD-L1 was measured by Western blotting. B16F10 (B) and LL2 (C) cells (5 × 10⁵ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 h. Then treatment with arbutin (0-1.56 μM) for 6 h, the expression of PD-L1 was measured by Western blotting. Arbutin reduced PD-L1 expression on the surface of cells. After treatment with arbutin (1.56 μM) for 6 h, the expression of PD-L1 on the surface of B16F10 (C) and LL2 (D) cells was measured by flow cytometry. (mean ± SD, n=5; *, p < 0.05).

Figure 3

Arbutin-mediated PD-L1 protein expression. B16F10 (A) and LL2 (B) cells (5 × 10⁵ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 h. Then treatment with arbutin (0-1.56 μM) for 6 h, the expression of PD-L1 and phosphorylation-AKT/mTOR were measured by Western blotting. Quantification histograms are presented beneath each Western blotting plot. Data are expressed as the mean ± SD of three-time repeated determinations. Each experiment was repeated three times with similar results.

Figure 4

Arbutin reduces PD-L1 expression through the AKT/mTOR pathway. B16F10 (A) and LL2 (B) cells (5 × 10⁵ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 h. Transfect the cells with control or constitutively active AKT plasmid (5ug) at 37°C for 6 h, then treat with arbutin (1.56 μM) for 6 h. Analyze the expression levels of the AKT/mTOR proteins and PD-L1 in the cells using Western blotting. Quantification histograms are presented beneath each Western blotting plot. Data are expressed as the mean ± SD of three-time repeated determinations. Each experiment was repeated three times with similar results.

Figure 5

Arbutin affected apoptosis in immune cells. B16F10 and LL2 cells (5 × 10⁵ cells/well) were placed into 6-well plates, incubated at 37 °C for 24 h, and then treated with arbutin (1.56 μM) for 6 h. WEHI-3 (A) and EL4 (B) cells were co-cultured with arbutin-treated B16F10 and LL2 cells before harvesting. The protein expression was analyzed by Western blotting. (C) EL4 cells were co-cultured with arbutin-treated B16F10 and LL2 cells or treated with cisplatin (2 µg/ml) before harvesting. The protein expression was analyzed by Western blotting. (D) The EL4 cell number were measured by staining with trypan blue. (n = 6, data are mean± SD; * p < 0.05; **, p < 0.01; *** , p < 0.001). Each experiment was repeated three times with similar results.

Figure 6

Arbutin inhibits tumor growth and PD-L1 expression in vivo. B16F10 and LL2 tumor cells were subcutaneously injected into C57BL/6 mice on day 0. Tumor growth was allowed for seven days, and arbutin (50 mg/kg) was administered via intraperitoneal injection for seven consecutive days from day 8. Tumor volumes were measured every three days. On Day 15, tumors were extracted using a lysis buffer. The supernatant was collected, and PD-L1 expression levels were analyzed using Western blotting for (A) B16F10 (n=3) and (B) LL2 (n=3) tumors. Tumor volumes were compared between the control group and the arbutin-treated group for (C) B16F10 (n=10) and (D) LL2 (n=9) tumors. (mean ± SEM; *, p < 0.05).