Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine

Abstract

Licorice has been used as herbal medicine and natural sweetener. Here, we prepared Glycyrrhiza uralensis water extract (GUWE) and investigated the effect of GUWE on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We observed that GUWE dose-dependently promoted DC maturation and cytokine secretion through TLR4 signaling pathway. The capacity of DC to stimulate allogenic splenocyte proliferation was also enhanced by GUWE treatment. Compared with control group, GUWE treated DCs pulsed with human papillomavirus (HPV)-16 E6/E7 peptides significantly inhibited the tumor growth in both early and late therapeutic groups. In early therapeutic group, the frequencies of induced regulatory T cells (iTregs: CD4⁺CD25^-Fopx3⁺) and CD4⁺ and CD8⁺ T cells were significantly decreased and increased, respectively. HPV-16-specific CD8⁺ T cell responses were significantly induced and negatively correlated with iTreg frequencies and tumor weight. These results indicated the immunoregulatory activities of licorice.

Figure 1. DC maturation and cytokine production upon GUWE treatment.

DCs were induced from bone marrow of C57BL/6 mice in the presence of GM-CSF. On day 7, cells (1 × 10⁶/ml) were treated with different concentrations (4, 20 and 40 μg/ml) of GUWE for 12 h. LPS (20 ng/ml) was used as positive control. (A) After treatment, the expressions of co-stimulatory molecules and MHC II on DCs were detected by flow cytometry (upper panels). The mean fluorescence intensity (MFI) (mean ± SEM) of co-stimulatory molecules and MHC II is shown in lower panels. (B) The supernatants was collected and the production of IL-1β, IL-6, IL-12 and TNF-α was detected by ELISA. The concentrations (mean ± SEM) of cytokines are shown. Data are from 4 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated DCs.

Figure 2. The function of DCs upon GUWE treatment.

MLR was performed using C57BL/6 DCs and BALB/c splenocytes. DCs on day 7 were treated with different concentrations (4, 20 and 40 μg/ml) of GUWE or LPS for 12 h, and then treated with mitomycin C. Splenocytes were obtained from BALB/c mice. DCs and splenocytes at ratios of 1:5 and 1:10 were co-cultured for 48 h. Cell proliferation was detected by MTT assay. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05 compared to untreated DCs.

Figure 3. The effect of TLR4 inhibitor on DC maturation and cytokine production.

DCs were pretreated with or without 1 μM TAK-242 for 1 h, and then treated with 4, 20 and 40 μg/ml of GUWE or 20 ng/ml of LPS for 12 h. (A) The expressions of CD40 and CD86 were analyzed by flow cytometry. MFI of CD40 and CD86 are shown. (B) Supernatants were collected and the production of IL-12 and TNF-α was measured by ELISA. The concentrations of cytokines are shown. Data are from 3 independent experiments. p values are indicated (paired t-test).

Figure 4. The effect of GUWE on MAPK and NF-κB signaling pathways in DCs.

DCs were treated with 40 μg/ml of GUWE, then nuclear and cytoplasmic proteins were isolated at the indicated time points. The levels of protein and their phosphorylation in cytoplasm (A) or nuclei (B) were detected by Western blot. Cropped blots are shown and full-length blots are included in the Supplementary Information.

Figure 5. Tumor growth and weight after treatment with GUWE-HPV-DCs.

Tumor mice were immunized with GUWE-HPV-DCs on day 5 or 12 after injection of TC-1 cells. (A) Tumor volumes were measured. The data are shown in the left panel. The area under curve was calculated with Prism 5 and the values are shown in right panel. (B) Tumors were isolated and weighted 32 days after tumor induction. The tumor photo and weight are shown in upper and lower panels, respectively. *p < 0.05 and ***p < 0.001 (ANOVA) compared to control group.

Figure 6. The frequencies of Tregs, CD4⁺ and CD8⁺ T cells, and CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cells after treatment with GUWE-HPV-DCs.

Splenocytes were isolated 32 days after treatment with GUWE-HPV-DCs. The freshly isolated splenocytes were used to analyze the frequencies of Tregs (A) and CD4⁺ and CD8⁺ T cells (B). (C) Splenocytes were stimulated with HPV-16 E6/E7 peptides overnight. The frequency of antigen specific CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cells was analyzed by flow cytometry. *p < 0.05 and **p < 0.01 (ANOVA) compared to control group.