Korean Red Ginseng Enhances Immunotherapeutic Effects of NK Cells via Eosinophils in Metastatic Liver Cancer Model

Abstract

Metastasis decreases the survival rate of patients with liver cancer. Therefore, novel anti-metastatic strategies are needed. Korean Red Ginseng (KRG) is often ingested as a functional food with an immune-boosting effect. We investigated a combination of KRG and natural killer (NK) cells as a novel immunotherapy approach. SK-Hep1 cells were injected into the tail vein of NRGA mice to establish an experimental metastasis model. KRG, NK cells, or a combination of KRG and NK cells were administered. Tumor growth was observed using an in vivo imaging system, and metastatic lesions were evaluated by histological analysis and immunohistochemistry. Bioluminescence intensity was lower in the KRG and NK cell combination group than in the other groups, indicating that the combination treatment suppressed the progression of metastasis. CD56 expression was used as a NK cell marker and hematological analysis was performed. The combination treatment also decreased the expression of matrix metalloproteinases and the area of metastatic lesions in liver and bone tissues, as well as increased the eosinophil count. Expression of cytokines-related eosinophils and NK cells was determined by Western blotting analysis. The expression of interleukin 33 (IL33) was induced by the combination of KRG and NK cells. High IL33 expression was associated with prolonged overall survival in the Kaplan-Meier plotter. Our results suggest that KRG enhances the immune activity of NK cells by IL-33 through eosinophils and suppresses metastatic liver cancer progression.

Keywords: IL33; Korean Red Ginseng; eosinophil; metastatic liver cancer; natural killer cell.

Figure 1

Anti-metastatic effect of KRG and NK cells. (A). Schedule of treatment and in vivo imaging in metastasis model (n = 3/group). (B). In vivo imaging of SK-Hep1 metastasis. The in vivo bioluminescence imaging was conducted on days 16, 20, 23, 27, and 29. Images are representative of twice replicated experiments. (C). In vivo bioluminescence intensity. Data obtained from B are expressed as the mean ± SD (n = 3/group). *, p < 0.05 (one-way ANOVA with a Dunnett’s post hoc test). (D). Hematoxylin staining of liver tissues. Dashed lines indicate metastatic lesions. Scale bar, 500 μm (Upper panel). Magnified pictures of the arrow point are shown in the lower panel. Scale bar, 100 nm. (E). Area of metastatic lesions in liver tissues. Data are expressed as the mean ± SD (two slides of liver tissue from each mouse, n = 3/group). *, p < 0.05; **, p < 0.01; ***, p < 0.005. (two-way ANOVA with a Tukey’s post hoc test).

Figure 2

Inhibition of matrix metalloproteinases and Ki67 levels by KRG and NK cells. (A). Expression of Ki67 in metastatic lesions of liver tissues. Brown dots represent Ki67-expressing cells. T, tumor site; N, normal site. Scale bar, 100 μm. (B). Quantification of Ki67-expressing cells in the tumor site. Data are expressed as the mean ± SD (randomly three points in each slide. n = 2/group). *, p < 0.05 (one-way ANOVA with a Dunnett’s post hoc test). (C). Expression of matrix metalloproteinases (MMPs) in metastatic lesions of the epiphysis. Brown dots represent MMP-expressing cells. Scale bar, 100 μm. (D). Quantification of MMPs in the metastatic lesions. Data are expressed as the mean ± SD (randomly three points in each slide. n = 2/group). *, p < 0.05 (vs control, two-way ANOVA with a Tukey’s post hoc test).

Figure 3

Detection of circulating NK cells in an experimental metastasis model. (A). CD56 levels measured via FACS analysis. (B). Ratio of CD56^dim+ cells. Data are expressed as the mean ± standard deviation (n = 3/group). **, p < 0.01; ***, p < 0.005. (two-way ANOVA with Tukey’s post hoc test) (C). CD56 positive cells in liver tissues. Brown dots represent CD56 positive cells. Scale bar, 100 μm.

Figure 4

Activation of IL33 related to eosinophils in an experimental metastasis model. (A). Ratio of white blood cell (WBC) components. Data are expressed as the mean ± standard deviation (n = 3/group). *, p < 0.05; **, p < 0.01; ***, p < 0.005. (two-way ANOVA with Tukey’s post hoc test) (B). Expression of IL33, MBP, and IFNγ levels in metastatic liver tissues. (C). Kaplan–Meier curve of liver cancer patients with high (n = 111) and low (n = 253) IL33 expression.