Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin.

Kathrin Pfarr,Corina Danciu,Olga Arlt,Josef M Pfeilschifter,Cristina Dehelean,Heinfried H Radeke,Christina Neske

doi:10.1371/journal.pone.0118802

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.

Highlights

For decades the incidence of melanoma has been increasing, especially in the fair-skinned population
This study will investigate the influence of several drugs on the bone marrow (BM) derived dendritic cells (DCs) of C57BL/6 mice, which were stimulated with lipopolysaccharide (LPS) as a ligand for TLR4/2
We investigated the immune modulating function of the betulin related compounds but the derivatives failed to influence the activation of Bone marrow-derived dendritic cells (BMDCs) as measured by IL-12p70 release

Summary

Introduction

For decades the incidence of melanoma has been increasing, especially in the fair-skinned population. TLR4 and TLR2 have recently been shown to bind endogenous DAMPs, which are dispatched by stressed or dying cells These two TLRs appear to be critical for the orchestration of potentially therapeutic anti-cancer immune responses [6]. This study will investigate the influence of several drugs on the bone marrow (BM) derived DCs of C57BL/6 mice, which were stimulated with lipopolysaccharide (LPS) as a ligand for TLR4/2 This stimulation leads to the expression of IL-12 through NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells), IRF-3 (interferon regulatory factor 3) or AP-1 (activator protein1) as well as to the activation of STAT4 (signal transducer and activator of transcription 4) [7]. IL-12 is responsible for the Th1- and CTL-driven anti-cancer immune response, which may augment an increase of CTLs in combination with oxaliplatin [3]

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Discussion

Conclusion