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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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Immune response modulation of antigen specific T cells by the treatment with cytostatic drugs.The spleen cells of OT-I mice were stimulated with 1 μg/ml LPS and 13.5 μg/ml ovalbumin (OVA) as the antigen and additionally incubated with 5 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET), respectively, for 44 h (mean ± SD, n = 3). The concentration of IFN-γ (A) and IL-2 (B) in the supernatant of the cells was measured by an ELISA. Significance was calculated using two-way ANOVA with a Bonferroni post-test.
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pone.0118802.g008: Immune response modulation of antigen specific T cells by the treatment with cytostatic drugs.The spleen cells of OT-I mice were stimulated with 1 μg/ml LPS and 13.5 μg/ml ovalbumin (OVA) as the antigen and additionally incubated with 5 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET), respectively, for 44 h (mean ± SD, n = 3). The concentration of IFN-γ (A) and IL-2 (B) in the supernatant of the cells was measured by an ELISA. Significance was calculated using two-way ANOVA with a Bonferroni post-test.

Mentions: The immune stimulating potential regarding the dendritic cell activity was promising, especially of betulin. Thus, we further sought to determine whether the modified activation of the stimulated dendritic cells by these three compounds is transmitted to an altered activation of anti-cancerogenic, cytotoxic T cells. Therefore, we used spleen cells of OT I mice as a model system expressing the ovalbumin-specific transgenic T cell receptors. The T cell activity was determined by IFN-γ and IL-2 cytokine detection in the supernatant (Fig. 8). Co-cultivation of the stimulated spleen cells with genistein had no effect on the cytokine production of T cells compared to the DMSO treated cells serving as the control. Fingolimod slightly down-regulated the IL-2 protein levels in the supernatant (p<0.05). In contrast, betulin strongly elevated the IFN-γ as well as the IL-2 production of stimulated spleen cells, indicating an increased activation of CD8+ ovalbumin-specific T cells (p<0.001).


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)

Immune response modulation of antigen specific T cells by the treatment with cytostatic drugs.The spleen cells of OT-I mice were stimulated with 1 μg/ml LPS and 13.5 μg/ml ovalbumin (OVA) as the antigen and additionally incubated with 5 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET), respectively, for 44 h (mean ± SD, n = 3). The concentration of IFN-γ (A) and IL-2 (B) in the supernatant of the cells was measured by an ELISA. 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.g008: Immune response modulation of antigen specific T cells by the treatment with cytostatic drugs.The spleen cells of OT-I mice were stimulated with 1 μg/ml LPS and 13.5 μg/ml ovalbumin (OVA) as the antigen and additionally incubated with 5 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET), respectively, for 44 h (mean ± SD, n = 3). The concentration of IFN-γ (A) and IL-2 (B) in the supernatant of the cells was measured by an ELISA. Significance was calculated using two-way ANOVA with a Bonferroni post-test.
Mentions: The immune stimulating potential regarding the dendritic cell activity was promising, especially of betulin. Thus, we further sought to determine whether the modified activation of the stimulated dendritic cells by these three compounds is transmitted to an altered activation of anti-cancerogenic, cytotoxic T cells. Therefore, we used spleen cells of OT I mice as a model system expressing the ovalbumin-specific transgenic T cell receptors. The T cell activity was determined by IFN-γ and IL-2 cytokine detection in the supernatant (Fig. 8). Co-cultivation of the stimulated spleen cells with genistein had no effect on the cytokine production of T cells compared to the DMSO treated cells serving as the control. Fingolimod slightly down-regulated the IL-2 protein levels in the supernatant (p<0.05). In contrast, betulin strongly elevated the IFN-γ as well as the IL-2 production of stimulated spleen cells, indicating an increased activation of CD8+ ovalbumin-specific T cells (p<0.001).

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