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Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin.

Pfarr K, Danciu C, Arlt O, Neske C, Dehelean C, Pfeilschifter JM, Radeke HH - PLoS ONE (2015)

Bottom Line: As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining.Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression.Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system.

View Article: PubMed Central - PubMed

Affiliation: pharmazentrum frankfurt/ZAFES, Institute of General Pharmacology and Toxicology, Clinic of the Goethe University, Frankfurt/Main, Germany.

ABSTRACT
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.

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Cytotoxic effect of betulin in an antigen-specific B16OVA model system.B16OVA cell viability after treatment with prestimulated spleen cells (mean ± SD, n = 4). 1x105 B16OVA cells were treated with 3.2x106 spleen cells, isolated of OT I RAG mice or WT mice, respectively. The CD8+ OVA TCR+ effector T cell to B16OVA target cell ratio for OT I RAG cells is 7:1. The viability of the CFSE stained B16OVA cells was analyzed by FACS measurements. Significance was calculated using two-way ANOVA with a Bonferroni post-test.
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pone.0118802.g009: Cytotoxic effect of betulin in an antigen-specific B16OVA model system.B16OVA cell viability after treatment with prestimulated spleen cells (mean ± SD, n = 4). 1x105 B16OVA cells were treated with 3.2x106 spleen cells, isolated of OT I RAG mice or WT mice, respectively. The CD8+ OVA TCR+ effector T cell to B16OVA target cell ratio for OT I RAG cells is 7:1. The viability of the CFSE stained B16OVA cells was analyzed by FACS measurements. Significance was calculated using two-way ANOVA with a Bonferroni post-test.

Mentions: We measured the B16OVA killing potential of betulin treated spleen cells in a SIINFEKL-specific model system (Fig. 9). Betulin treatment alone induced cell death of B16OVA cells (p ≤ 0.01). The antigen-specific loading of DCs with SIINFEKL peptide alone led to the recognition and destruction of SIINFEKL-carrying B16OVA cells by CD8+ OVA TCR+ cytotoxic T cells (p ≤ 0.001). However, the combination of both decreased the viability of B16OVA cells in an even higher extend (p ≤ 0.001). Interestingly, the loading process of the peptides on MHC I molecules (S4A Fig.) as well the frequency of SIINFEKL-specific T cell receptors (S4B Fig.) is not influenced by betulin.


Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin.

Pfarr K, Danciu C, Arlt O, Neske C, Dehelean C, Pfeilschifter JM, Radeke HH - PLoS ONE (2015)

Cytotoxic effect of betulin in an antigen-specific B16OVA model system.B16OVA cell viability after treatment with prestimulated spleen cells (mean ± SD, n = 4). 1x105 B16OVA cells were treated with 3.2x106 spleen cells, isolated of OT I RAG mice or WT mice, respectively. The CD8+ OVA TCR+ effector T cell to B16OVA target cell ratio for OT I RAG cells is 7:1. The viability of the CFSE stained B16OVA cells was analyzed by FACS measurements. Significance was calculated using two-way ANOVA with a Bonferroni post-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355578&req=5

pone.0118802.g009: Cytotoxic effect of betulin in an antigen-specific B16OVA model system.B16OVA cell viability after treatment with prestimulated spleen cells (mean ± SD, n = 4). 1x105 B16OVA cells were treated with 3.2x106 spleen cells, isolated of OT I RAG mice or WT mice, respectively. The CD8+ OVA TCR+ effector T cell to B16OVA target cell ratio for OT I RAG cells is 7:1. The viability of the CFSE stained B16OVA cells was analyzed by FACS measurements. Significance was calculated using two-way ANOVA with a Bonferroni post-test.
Mentions: We measured the B16OVA killing potential of betulin treated spleen cells in a SIINFEKL-specific model system (Fig. 9). Betulin treatment alone induced cell death of B16OVA cells (p ≤ 0.01). The antigen-specific loading of DCs with SIINFEKL peptide alone led to the recognition and destruction of SIINFEKL-carrying B16OVA cells by CD8+ OVA TCR+ cytotoxic T cells (p ≤ 0.001). However, the combination of both decreased the viability of B16OVA cells in an even higher extend (p ≤ 0.001). Interestingly, the loading process of the peptides on MHC I molecules (S4A Fig.) as well the frequency of SIINFEKL-specific T cell receptors (S4B Fig.) is not influenced by betulin.

Bottom Line: As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining.Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression.Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system.

View Article: PubMed Central - PubMed

Affiliation: pharmazentrum frankfurt/ZAFES, Institute of General Pharmacology and Toxicology, Clinic of the Goethe University, Frankfurt/Main, Germany.

ABSTRACT
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.

Show MeSH
Related in: MedlinePlus