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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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Dose-dependent effects of cytostatic drug treatment on B16 cell proliferation and viability.Proliferation was determined by an MTT assay (mean ± SD, n = 4) for genistein (GEN, square), fingolimod (FIN, triangle) and betulin (BET, circle) in the B16 melanoma cell lines. B164A5 (A) and B16F10 (B) cells were treated at concentrations ranging from 1 to 150 μM for 24 h and stained with the MTT reagent for 4 h. Additionally, the percentage of living cells was tested by trypan blue exclusion (mean ± D, n = 5) for the B164A5 (C) and B16F10 (D) melanoma cell lines. The cells were treated as described above and stained with trypan blue after 24 h. Significant differences compared to the untreated control cells were calculated by two-way ANOVA with a Bonferroni post-test.
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pone.0118802.g001: Dose-dependent effects of cytostatic drug treatment on B16 cell proliferation and viability.Proliferation was determined by an MTT assay (mean ± SD, n = 4) for genistein (GEN, square), fingolimod (FIN, triangle) and betulin (BET, circle) in the B16 melanoma cell lines. B164A5 (A) and B16F10 (B) cells were treated at concentrations ranging from 1 to 150 μM for 24 h and stained with the MTT reagent for 4 h. Additionally, the percentage of living cells was tested by trypan blue exclusion (mean ± D, n = 5) for the B164A5 (C) and B16F10 (D) melanoma cell lines. The cells were treated as described above and stained with trypan blue after 24 h. Significant differences compared to the untreated control cells were calculated by two-way ANOVA with a Bonferroni post-test.

Mentions: The first approach to elucidate the cytotoxic mechanism of these three compounds was carried out by an MTT proliferation assay. Genistein, fingolimod, and betulin all dose-dependently reduced the activity of the mitochondrial oxidoreductase of the B16 cells (Fig. 1A, B). Both of the murine melanoma cell lines, B164A5 and B16F10, were affected in nearly the same range. An increasing concentration of genistein decreased the B16 cell viability to a lower extent than the other two substances. At the highest tested concentration of genistein, the cell viability was still 30% (p<0.001). In contrast, a concentration of only 15 μM of fingolimod or betulin was sufficient to suppress the proliferation rate of both melanoma cell lines to less than 20% (p<0.001). These differences were also reflected by the IC50 values calculated (Table 1).


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)

Dose-dependent effects of cytostatic drug treatment on B16 cell proliferation and viability.Proliferation was determined by an MTT assay (mean ± SD, n = 4) for genistein (GEN, square), fingolimod (FIN, triangle) and betulin (BET, circle) in the B16 melanoma cell lines. B164A5 (A) and B16F10 (B) cells were treated at concentrations ranging from 1 to 150 μM for 24 h and stained with the MTT reagent for 4 h. Additionally, the percentage of living cells was tested by trypan blue exclusion (mean ± D, n = 5) for the B164A5 (C) and B16F10 (D) melanoma cell lines. The cells were treated as described above and stained with trypan blue after 24 h. Significant differences compared to the untreated control cells were calculated by 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.g001: Dose-dependent effects of cytostatic drug treatment on B16 cell proliferation and viability.Proliferation was determined by an MTT assay (mean ± SD, n = 4) for genistein (GEN, square), fingolimod (FIN, triangle) and betulin (BET, circle) in the B16 melanoma cell lines. B164A5 (A) and B16F10 (B) cells were treated at concentrations ranging from 1 to 150 μM for 24 h and stained with the MTT reagent for 4 h. Additionally, the percentage of living cells was tested by trypan blue exclusion (mean ± D, n = 5) for the B164A5 (C) and B16F10 (D) melanoma cell lines. The cells were treated as described above and stained with trypan blue after 24 h. Significant differences compared to the untreated control cells were calculated by two-way ANOVA with a Bonferroni post-test.
Mentions: The first approach to elucidate the cytotoxic mechanism of these three compounds was carried out by an MTT proliferation assay. Genistein, fingolimod, and betulin all dose-dependently reduced the activity of the mitochondrial oxidoreductase of the B16 cells (Fig. 1A, B). Both of the murine melanoma cell lines, B164A5 and B16F10, were affected in nearly the same range. An increasing concentration of genistein decreased the B16 cell viability to a lower extent than the other two substances. At the highest tested concentration of genistein, the cell viability was still 30% (p<0.001). In contrast, a concentration of only 15 μM of fingolimod or betulin was sufficient to suppress the proliferation rate of both melanoma cell lines to less than 20% (p<0.001). These differences were also reflected by the IC50 values calculated (Table 1).

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