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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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Effect of cytostatic drug treatment on the cell cycle phases of B16 melanoma cells.Cell cycle analysis of B164A5 (A) and B16F10 (B) melanoma cells treated with 100 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET) for 72 h, respectively. The quantification of cells in G0/G1, S and G2/M were analyzed using flow cytometry by PI staining after 24 h (mean ± SD, n = 4). Significance was calculated using two-way ANOVA with a Bonferroni post-test.
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pone.0118802.g003: Effect of cytostatic drug treatment on the cell cycle phases of B16 melanoma cells.Cell cycle analysis of B164A5 (A) and B16F10 (B) melanoma cells treated with 100 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET) for 72 h, respectively. The quantification of cells in G0/G1, S and G2/M were analyzed using flow cytometry by PI staining after 24 h (mean ± SD, n = 4). Significance was calculated using two-way ANOVA with a Bonferroni post-test.

Mentions: To further differentiate the mechanisms of genistein, fingolimod and betulin, we analyzed the cell cycle phases. The concentration of the substances for this assay (100 μM genistein, 5 μM fingolimod, 5 μM betulin) were chosen based on the findings of the MTT assay compared to the apoptosis measurements described below. We assumed that the cytostatic effect is worth to get investigated at its best when the compound has relatively low apoptotic potential but high antiproliferative activity. This is true for 100 μM genistein as well as for 5 μM fingolimod and 5 μM betulin (S1 Fig.). To analyze the cell cycle phases we treated the B16 cells with the compounds for 72 h and stained them with PI (Fig. 3; S2 Fig.). High concentrations of genistein stopped the proliferation by blocking the cells during the G2/M phase (p<0.001). Fingolimod arrested the proliferation of the B16 cells by blocking the G0/G1 phase (p<0.05). Under the conditions described for both anti-melanoma compounds, the cell cycle arresting effect was more pronounced for the high metastatic cell line B16F10 compared to the low metastatic parent cell line B164A5. In contrast, betulin failed to target a specific step of the cell cycle at the concentration tested in this study.


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)

Effect of cytostatic drug treatment on the cell cycle phases of B16 melanoma cells.Cell cycle analysis of B164A5 (A) and B16F10 (B) melanoma cells treated with 100 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET) for 72 h, respectively. The quantification of cells in G0/G1, S and G2/M were analyzed using flow cytometry by PI staining after 24 h (mean ± SD, n = 4). 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.g003: Effect of cytostatic drug treatment on the cell cycle phases of B16 melanoma cells.Cell cycle analysis of B164A5 (A) and B16F10 (B) melanoma cells treated with 100 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET) for 72 h, respectively. The quantification of cells in G0/G1, S and G2/M were analyzed using flow cytometry by PI staining after 24 h (mean ± SD, n = 4). Significance was calculated using two-way ANOVA with a Bonferroni post-test.
Mentions: To further differentiate the mechanisms of genistein, fingolimod and betulin, we analyzed the cell cycle phases. The concentration of the substances for this assay (100 μM genistein, 5 μM fingolimod, 5 μM betulin) were chosen based on the findings of the MTT assay compared to the apoptosis measurements described below. We assumed that the cytostatic effect is worth to get investigated at its best when the compound has relatively low apoptotic potential but high antiproliferative activity. This is true for 100 μM genistein as well as for 5 μM fingolimod and 5 μM betulin (S1 Fig.). To analyze the cell cycle phases we treated the B16 cells with the compounds for 72 h and stained them with PI (Fig. 3; S2 Fig.). High concentrations of genistein stopped the proliferation by blocking the cells during the G2/M phase (p<0.001). Fingolimod arrested the proliferation of the B16 cells by blocking the G0/G1 phase (p<0.05). Under the conditions described for both anti-melanoma compounds, the cell cycle arresting effect was more pronounced for the high metastatic cell line B16F10 compared to the low metastatic parent cell line B164A5. In contrast, betulin failed to target a specific step of the cell cycle at the concentration tested in this study.

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