Fig 1.
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.
Table 1.
IC50 values of genistein, fingolimod and betulin in B16 melanoma cell lines.
Fig 2.
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.
Fig 3.
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.
Fig 4.
DAPI staining of B16 melanoma cells after treatment with cytostatic drugs.
Representative images of DAPI-stained B164A5 (A–C) and B16F10 (D–F) cells after treatment with DMSO (A, D), 100 μM genistein (GEN: B, E) or 5 μM betulin (BET: C, F) for 48 h, respectively. Arrows indicate nucleus fragmentation. Apoptotic cells per field of three independently repeated experiments are counted and shown in bar graphs for the B164A5 (G) and B16F10 (H) cell line.
Fig 5.
Analysis of proapoptotic and antiapoptotic molecules in B16 melanoma cells after treatment with cytostatic drugs.
Representative Western blot results (A) as well as densitometric analysis of performed Western blots for Bax (B), Bcl-2 (C), cleaved caspase 3 (D) as well as cleaved PARP (E) expression in B16 melanoma cells after treatment with genistein, betulin or fingolimod for 72 h, respectively (mean ± SD, n = 3). The signal strength given by the adjusted volume of the bands of the respective proteins was calculated in comparison to the respective β-actin band of each sample. Significance was determined by an unpaired t-test.
Fig 6.
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.
Fig 7.
Immune response modulation of primary dendritic cells by the treatment with cytostatic drugs.
Effects of 5 μM genistein (GEN), 5 μM fingolimod (FIN) or 5 μM betulin (BET), respectively, on the activation of LPS stimulated bone marrow derived dendritic cells (BMDCs). A) Released IL-12p70 was measured by an ELISA in the supernatant of LPS stimulated BMDCs for the indicated time points (mean ± SD, n = 3). B) Determination of secreted IL-12p70 protein levels 20 h after stimulation of BMDCs as well as the mRNA levels of both IL-12 subunits after an incubation period of 6 h (mean ± SD, n = 8–12). Quantitative assessment of mRNA levels was correlated to the expression of GAPDH mRNA in each sample. Significance was calculated by an unpaired t-test. C) IL-12p70 protein was detected in the supernatant of the stimulated BMDCs incubated with BET, betulinic acid (BA) and its derivatives, B-10 and NVX-270 (mean ± SD, n = 3). D) Secretion of other cytokines, such as IL-23, IL-6 and IL-10, was investigated by an ELISA of the supernatant of BET treated BMDCs (mean ± SD, n = 11). E and F) IL-12p70 protein was detected in the supernatant of LPS stimulated BMDCs incubated with BET as well as 200 ng/ml Rapamycin or 20 μM Piceatannol, respectively (mean ± SD, n = 3). For the calculations of significances, two-way ANOVA with a Bonferroni post-test was used.
Fig 8.
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.
Fig 9.
Cytotoxic effect of betulin in an antigen-specific B16OVA model system.
B16OVA cell viability after treatment with prestimulated spleen cells (mean ± SD, n = 4). 1x105 B16OVA cells were treated with 3.2x106 spleen cells, isolated of OT I RAG mice or WT mice, respectively. The CD8+ OVA TCR+ effector T cell to B16OVA target cell ratio for OT I RAG cells is 7:1. The viability of the CFSE stained B16OVA cells was analyzed by FACS measurements. Significance was calculated using two-way ANOVA with a Bonferroni post-test.