Limits...
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.

Show MeSH

Related in: MedlinePlus

Time- and dose-dependent effects of cytostatic drug treatment on B16 cell proliferation.The decreasing intensity of the CFSE dye as a degree for the proliferation rate of the cells is shown (mean ± SD, n = 3). B164A5 (A, C) and B16F10 (B, D) melanoma cells were stained with CFSE and treated for 4 days with different concentrations of genistein (A, B) or betulin (C, D), respectively. Subsequent flow cytometry analysis of this cytosolic dye dilution as an indicator of frequency and number of cell divisions was determined.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4355578&req=5

pone.0118802.g002: Time- and dose-dependent effects of cytostatic drug treatment on B16 cell proliferation.The decreasing intensity of the CFSE dye as a degree for the proliferation rate of the cells is shown (mean ± SD, n = 3). B164A5 (A, C) and B16F10 (B, D) melanoma cells were stained with CFSE and treated for 4 days with different concentrations of genistein (A, B) or betulin (C, D), respectively. Subsequent flow cytometry analysis of this cytosolic dye dilution as an indicator of frequency and number of cell divisions was determined.

Mentions: To confirm these results, we measured the cytotoxic effect of the compounds by exclusion dye staining with trypan blue after 24 h (Fig. 1C, D). In line with the MTT data, the cell viability of the genistein-treated samples remained relatively high. Even at the highest concentration of genistein tested, over 70% of the cells were still alive (p<0.001). However, fingolimod displayed a similar high toxicity in the trypan blue staining as in the MTT assay. After stimulation of the cells with 50 μM fingolimod, over 90% of the cells were stained blue (p<0.001). In contrast with the MTT assay, the B16 cells treated with 150 μM betulin demonstrated a trypan blue exclusion capacity of 60% (p<0.01), whereas in the MTT test these cells had shown almost no mitochondrial activity starting from a concentration as low as 15 μM. As will be discussed, this finding may indicate an arrest in proliferation without the actual killing of the cells. To gather more information about these differences, a four day study was performed to characterize the cell division rate of the treated melanoma cells in more detail using the fluorescent dye CFSE. Because treatment of the cells with fingolimod led to a very efficient and fast cell death, the CFSE assay was performed for genistein and betulin treated cells only (Fig. 2). After 24 h, the proliferation of the B164A5 cells was already decreased when treated with 30 μM genistein (p<0.05). In contrast, 50 μM betulin was necessary for the same effect within the same time frame. Additionally, a low concentration of 5 μM genistein inhibited the proliferation rate of the B164A5 cells after 48 h, whereas 5 μM betulin needed 72 h to decrease the cell division rate (p<0.05). These two results again indicated the delayed antiproliferative effect of betulin. Over a longer period of time and in higher concentrations starting from 100 μM, betulin seemed to be a stronger antiproliferative agent than genistein. In the B16F10 cells, however, the antiproliferative response of betulin was weaker.


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)

Time- and dose-dependent effects of cytostatic drug treatment on B16 cell proliferation.The decreasing intensity of the CFSE dye as a degree for the proliferation rate of the cells is shown (mean ± SD, n = 3). B164A5 (A, C) and B16F10 (B, D) melanoma cells were stained with CFSE and treated for 4 days with different concentrations of genistein (A, B) or betulin (C, D), respectively. Subsequent flow cytometry analysis of this cytosolic dye dilution as an indicator of frequency and number of cell divisions was determined.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118802.g002: Time- and dose-dependent effects of cytostatic drug treatment on B16 cell proliferation.The decreasing intensity of the CFSE dye as a degree for the proliferation rate of the cells is shown (mean ± SD, n = 3). B164A5 (A, C) and B16F10 (B, D) melanoma cells were stained with CFSE and treated for 4 days with different concentrations of genistein (A, B) or betulin (C, D), respectively. Subsequent flow cytometry analysis of this cytosolic dye dilution as an indicator of frequency and number of cell divisions was determined.
Mentions: To confirm these results, we measured the cytotoxic effect of the compounds by exclusion dye staining with trypan blue after 24 h (Fig. 1C, D). In line with the MTT data, the cell viability of the genistein-treated samples remained relatively high. Even at the highest concentration of genistein tested, over 70% of the cells were still alive (p<0.001). However, fingolimod displayed a similar high toxicity in the trypan blue staining as in the MTT assay. After stimulation of the cells with 50 μM fingolimod, over 90% of the cells were stained blue (p<0.001). In contrast with the MTT assay, the B16 cells treated with 150 μM betulin demonstrated a trypan blue exclusion capacity of 60% (p<0.01), whereas in the MTT test these cells had shown almost no mitochondrial activity starting from a concentration as low as 15 μM. As will be discussed, this finding may indicate an arrest in proliferation without the actual killing of the cells. To gather more information about these differences, a four day study was performed to characterize the cell division rate of the treated melanoma cells in more detail using the fluorescent dye CFSE. Because treatment of the cells with fingolimod led to a very efficient and fast cell death, the CFSE assay was performed for genistein and betulin treated cells only (Fig. 2). After 24 h, the proliferation of the B164A5 cells was already decreased when treated with 30 μM genistein (p<0.05). In contrast, 50 μM betulin was necessary for the same effect within the same time frame. Additionally, a low concentration of 5 μM genistein inhibited the proliferation rate of the B164A5 cells after 48 h, whereas 5 μM betulin needed 72 h to decrease the cell division rate (p<0.05). These two results again indicated the delayed antiproliferative effect of betulin. Over a longer period of time and in higher concentrations starting from 100 μM, betulin seemed to be a stronger antiproliferative agent than genistein. In the B16F10 cells, however, the antiproliferative response of betulin was weaker.

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