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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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Detection of early apoptosis in B16 melanoma cells and primary dendritic cells after treatment with cytostatic drugs.Annexin V staining of B16 melanoma cells and BMDCs treated with various concentrations of genistein (A), fingolimod (B) or betulin (C) for 24 h, respectively (mean ± SD, n = 4–7). Significance was evaluated using two-way ANOVA with a Bonferroni post-test. Early apoptotic cells (Annexin V+/ 7-AAD-) were analyzed by FACS measurement. Representative dot blots of the staining are exemplarily shown for betulin in B16F10 cells (D). FACS data generated from all three compounds were used to calculate the mean values shown in the graphs.
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pone.0118802.g006: Detection of early apoptosis in B16 melanoma cells and primary dendritic cells after treatment with cytostatic drugs.Annexin V staining of B16 melanoma cells and BMDCs treated with various concentrations of genistein (A), fingolimod (B) or betulin (C) for 24 h, respectively (mean ± SD, n = 4–7). Significance was evaluated using two-way ANOVA with a Bonferroni post-test. Early apoptotic cells (Annexin V+/ 7-AAD-) were analyzed by FACS measurement. Representative dot blots of the staining are exemplarily shown for betulin in B16F10 cells (D). FACS data generated from all three compounds were used to calculate the mean values shown in the graphs.

Mentions: Further investigations related to the pro-apoptotic effect of the three active agents were performed that analyzed the early signs of apoptosis by annexin V-FITC staining measured by fluorescence cytometry (Fig. 6). This assay was performed in parallel for the melanoma cells as well as the BMDCs to investigate whether there was a difference in the induction of apoptosis between strongly proliferating tumor cells and BMDCs. Genistein induced slightly more apoptosis in the B16 cells than in the BMDCs only at its highest concentration. In BMDCs we measured 6% whereas B164A5 cells revealed up to 15% or 23% in B16F10 cells, respectively. As expected, the immune modulator fingolimod caused a very high rate of apoptosis in both cell types and was already cytotoxic at low concentrations (see above; MTT assay and trypan blue staining (Fig. 1). Fingolimod seemed to act as a saponifying detergent at high concentrations; therefore, the apoptosis rate that was measurable by this type of assay apparently decreased again with concentrations of fingolimod that were above 15 μM because the cells have been physically destroyed already. Betulin triggered apoptosis in only up to 4% of BMDCs, even at the highest concentration applied. In contrast, betulin induced early apoptosis in 20% of the B164A5 cells starting from a concentration of 15 μM (p<0.01). As before, the B16F10 cells were not affected to the same extent. This gradually higher activity in melanoma cells appears to be promising with respect to the anti-cancerogenic potential of betulin, especially in the light of its immune stimulating activity described below.


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)

Detection of early apoptosis in B16 melanoma cells and primary dendritic cells after treatment with cytostatic drugs.Annexin V staining of B16 melanoma cells and BMDCs treated with various concentrations of genistein (A), fingolimod (B) or betulin (C) for 24 h, respectively (mean ± SD, n = 4–7). Significance was evaluated using two-way ANOVA with a Bonferroni post-test. Early apoptotic cells (Annexin V+/ 7-AAD-) were analyzed by FACS measurement. Representative dot blots of the staining are exemplarily shown for betulin in B16F10 cells (D). FACS data generated from all three compounds were used to calculate the mean values shown in the graphs.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118802.g006: Detection of early apoptosis in B16 melanoma cells and primary dendritic cells after treatment with cytostatic drugs.Annexin V staining of B16 melanoma cells and BMDCs treated with various concentrations of genistein (A), fingolimod (B) or betulin (C) for 24 h, respectively (mean ± SD, n = 4–7). Significance was evaluated using two-way ANOVA with a Bonferroni post-test. Early apoptotic cells (Annexin V+/ 7-AAD-) were analyzed by FACS measurement. Representative dot blots of the staining are exemplarily shown for betulin in B16F10 cells (D). FACS data generated from all three compounds were used to calculate the mean values shown in the graphs.
Mentions: Further investigations related to the pro-apoptotic effect of the three active agents were performed that analyzed the early signs of apoptosis by annexin V-FITC staining measured by fluorescence cytometry (Fig. 6). This assay was performed in parallel for the melanoma cells as well as the BMDCs to investigate whether there was a difference in the induction of apoptosis between strongly proliferating tumor cells and BMDCs. Genistein induced slightly more apoptosis in the B16 cells than in the BMDCs only at its highest concentration. In BMDCs we measured 6% whereas B164A5 cells revealed up to 15% or 23% in B16F10 cells, respectively. As expected, the immune modulator fingolimod caused a very high rate of apoptosis in both cell types and was already cytotoxic at low concentrations (see above; MTT assay and trypan blue staining (Fig. 1). Fingolimod seemed to act as a saponifying detergent at high concentrations; therefore, the apoptosis rate that was measurable by this type of assay apparently decreased again with concentrations of fingolimod that were above 15 μM because the cells have been physically destroyed already. Betulin triggered apoptosis in only up to 4% of BMDCs, even at the highest concentration applied. In contrast, betulin induced early apoptosis in 20% of the B164A5 cells starting from a concentration of 15 μM (p<0.01). As before, the B16F10 cells were not affected to the same extent. This gradually higher activity in melanoma cells appears to be promising with respect to the anti-cancerogenic potential of betulin, especially in the light of its immune stimulating activity described below.

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