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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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Related in: MedlinePlus

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.
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pone.0118802.g005: 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.

Mentions: To more thoroughly investigate the apoptotic potential of the three compounds, a western blot analysis was performed. Four crucial proteins involved in the process of apoptosis, namely Bax, Bcl-2, caspase 3 and PARP (poly ADP ribose polymerase) were tested (Fig. 5). Cleaved caspase 3 as well as cleaved PARP was observed in the samples incubated with 100 μM genistein in both the B164A5 and B16F10 cell lines, indicating the activation of an intrinsic or extrinsic pathway of apoptosis by proteolytic events. Neither of the three cytostatic compounds influenced the band pattern of Bcl-2 in either one of the both melanoma cell lines; however, the overall protein expression levels appeared to be slightly higher in the high metastatic B16F10 cell line compared to all samples of the B164A5 cell line. This can be also seen in the densitometric evaluation (Fig. 5C) when comparing the untreated samples of both cell lines with each other. However, the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 protein was not affected by the agents tested.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118802.g005: 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.
Mentions: To more thoroughly investigate the apoptotic potential of the three compounds, a western blot analysis was performed. Four crucial proteins involved in the process of apoptosis, namely Bax, Bcl-2, caspase 3 and PARP (poly ADP ribose polymerase) were tested (Fig. 5). Cleaved caspase 3 as well as cleaved PARP was observed in the samples incubated with 100 μM genistein in both the B164A5 and B16F10 cell lines, indicating the activation of an intrinsic or extrinsic pathway of apoptosis by proteolytic events. Neither of the three cytostatic compounds influenced the band pattern of Bcl-2 in either one of the both melanoma cell lines; however, the overall protein expression levels appeared to be slightly higher in the high metastatic B16F10 cell line compared to all samples of the B164A5 cell line. This can be also seen in the densitometric evaluation (Fig. 5C) when comparing the untreated samples of both cell lines with each other. However, the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 protein was not affected by the agents tested.

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