Immune Netw.  2016 Feb;16(1):75-84. 10.4110/in.2016.16.1.75.

Immunogenic Cell Death Induced by Ginsenoside Rg3: Significance in Dendritic Cell-based Anti-tumor Immunotherapy

• Son KJ
• Choi KR
• Lee SJ
• Lee H

• KMID: 2154813
• DOI: http://doi.org/10.4110/in.2016.16.1.75

Abstract

Cancer is one of the leading causes of morbidity and mortality worldwide; therefore there is a need to discover new therapeutic modules with improved efficacy and safety. Immune-(cell) therapy is a promising therapeutic strategy for the treatment of intractable cancers. The effectiveness of certain chemotherapeutics in inducing immunogenic tumor cell death thus promoting cancer eradication has been reported. Ginsenoside Rg3 is a ginseng saponin that has antitumor and immunomodulatory activity. In this study, we treated tumor cells with Rg3 to verify the significance of inducing immunogenic tumor cell death in antitumor therapy, especially in DC-based immunotherapy. Rg3 killed the both immunogenic (B16F10 melanoma cells) and non-immunogenic (LLC: Lewis Lung Carcinoma cells) tumor cells by inducing apoptosis. Surface expression of immunogenic death markers including calreticulin and heat shock proteins and the transcription of relevant genes were increased in the Rg3-dying tumor. Increased calreticulin expression was directly related to the uptake of dying tumor cells by dendritic cells (DCs): the proportion of CRT+ CD11c+ cells was increased in the Rg3-treated group. Interestingly, tumor cells dying by immunogenic cell death secreted IFN-gamma, an effector molecule for antitumor activity in T cells. Along with the Rg3-induced suppression of pro-angiogenic (TNF-alpha) and immunosuppressive cytokine (TGF-beta) secretion, IFN-gamma production from the Rg3-treated tumor cells may also indicate Rg3 as an effective anticancer immunotherapeutic strategy. The data clearly suggests that Rg3-induced immunogenic tumor cell death due its cytotoxic effect and its ability to induce DC function. This indicates that Rg3 may be an effective immunotherapeutic strategy.

Keyword

Ginsenoside Rg3; DC; Immunogenic cell death; CRT; HSPs

MeSH Terms

Animals
Apoptosis
Calreticulin
Carcinoma, Lewis Lung
Cell Death*
Dendritic Cells
Heat-Shock Proteins
Immunotherapy*
Melanoma
Mortality
Panax
Saponins
T-Lymphocytes
Calreticulin
Heat-Shock Proteins
Saponins

Figure

• 

  Figure 1 The effect of ginsenoside Rg3 treatment on the viability of LLC and B16F10 cells. The LLC (A) and B16F10 (B) cells (2×103 cells/well) were seeded in 96-well plates and treated with different Rg3 concentrations (10~500 µg/ml) and Doxorubicin (0.01 µg/ml) for 48 h. Each value is the mean±SE of three experiments. Each experiment was done in triplicate. Significant effects compared to control are indicated with asterisks (*p<0.05 or **p<0.01).

• 

  Figure 2 Apoptotic cell death was observed in LLC (A) and B16F10 (B) cell lines. Cells were treated for 18 h with the indicated Rg3 (100, 300, 500 µg/ml) and Doxorubicin (0.01 µg/ml) concentrations. The percentage of apoptotic cells were determined by measuring Annexin V/PI expression using flow cytometry. Each experiment was done in triplicate. Significant effects versus control are indicated with asterisks (*p<0.05, **p<0.01); # indicates the significant changes (#p<0.05) compared to Doxorubicin (0.01µg/ml).

• 

  Figure 3 Rg3 induced the expression of CRT and HSPs on LLC (A) and B16F10 (B) cell lines. Representative histograms of one of the experiments and mean±SE of three experiments showing the expression of CRT, HSP60, HSP70, and HSP90 after 18 h of Rg3 treatment on tumor cells are shown. Each experiment was done in triplicate. Significant effects versus control are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001); #,##indicates the significant changes (#p<0.05 or ##p<0.01) compared to Doxorubicin (0.01 µg/ml).

• 

  Figure 4 (A) The effect of ginsenoside Rg3 (100, 300, 500 µg/ml) on cytokine secretion from LLC cells. Each value is the mean±SE of three experiments. Each experiment was done in triplicate. Significant effects versus control are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001); #indicate the significant changes compared to Doxorubicin (0.01 µg/ml). (B) The effect of ginsenoside Rg3 (100, 300, 500 µg/ml) on cytokine secretion from B16F10 cells. Each value is the mean±SE of three experiments. Each experiment was done in triplicate. Significant effects versus control are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001); #indicates the significant changes compared to Doxorubicin (0.01 µg/ml).

• 

  Figure 5 (A) LLC was incubated overnight with or without doxorubicin or Rg3 at 37℃. LLCs were then stained with CRT-FITC. DCs were labeled with CD11c-PerCP5.5 then co-cultured with CRT-FITC labeled LLC for 6 h. FITC and PerCP5.5 double stained cells were considered as DCs taken tumor cells. Each experiment was done in triplicate. (B) B16F10 was incubated overnight with or without doxorubicin or Rg3 at 37℃. B16F10s were then stained with CRT-FITC. DCs were labeled with CD11c-PerCP5.5 then co-cultured with CRT-FITC labeled B16F10 for 6 h. FITC and PerCP5.5 double stained cells were considered as DCs taken tumor cells. Each experiment was done in triplicate. Significant effects versus control are indicated with asterisks (*p<0.05); #indicates the significant changes (#p<0.05) compared to Doxorubicin (0.01µg/ml).

• 

  Figure 6 (A) Clustering of gene expression in LLC after treatment with Doxorubicin or Rg3. 11 genes were selected based on the fold change in expression (log2). Then, these genes were divided into control. The graph produced showed clusters using the Genplex software and the expression levels of genes are shown as graphs. (B) Clustering of gene expression in B16F10 after treatment with Doxorubicin or Rg3. 11 genes were selected based on the fold change in expression (log2). Then, these genes were divided into control. The graph produced showed clusters using the Genplex software and the expression levels of genes are shown as graphs.

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