Conceived and designed the experiments: GUG CS HW. Performed the experiments: NM-S LD DS MK. Analyzed the data: NM-S LD DS MK. Wrote the paper: HW.
The authors have declared that no competing interests exist.
Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFκB system and TNF signaling. In view of the overwhelming importance of the NFκB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells.
BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFκB pathway, but it also diminished the stronger NFκB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12.
The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.
SMAC (second mitochondria-derived activator of caspases) / DIABLO (direct inhibitor of apoptosis-binding protein with low isoelectric point) facilitates activation of apoptotic caspases by releasing these proteins from the inhibitory interaction with inhibitor of apoptosis proteins (IAPs), particularly XIAP
Inappropriate apoptosis resistance is not only of central relevance for the development of tumors per se but also a major factor that drives the evolution of therapy refractory cancer cells. No wonder, there were early on considerable efforts to design molecules that mimic the activity of SMAC to use them in cancer therapy. In fact, in a variety of
The effects of SMAC mimetics on non-transformed primary cells have been poorly addressed so far. In view of the overwhelming importance of the NFκB system for the regulation of immune cells, we analyzed here the effects of the SMAC mimetic BV6 on human primary immune cells. We found that BV6 induces no or only moderate cell death in most immune cells with exception of monocytes which proved to be quite sensitive. BV6 treatment alone showed furthermore moderate activation of the classical NFκB pathway in dendritic cells (DCs) but diminished the stronger NFκB-inducing effect of TNF and CD40L in these cells. Noteworthy, BV6 treatment was sufficient to drive maturation of monocyte-derived DCs. Taken together, the effects of SMAC mimetics on the immune system may complicate the development of tumor therapeutic concepts based on these compounds but there also arises the possibility to exploit them for the development of immune stimulatory therapies.
We synthesized the recently published bivalent SMAC mimetic BV6 and furthermore generated a monomeric and a trimeric variant derived thereof (
(A) Kym-1 cells were seeded in 96-well plates and stimulated the next day in triplicates with the indicated concentrations of moSmM, BV6 and triSmM. After additional 18 h cell viability was determined by MTT staining. (B), Kym-1 cells were incubated in triplicates overnight (16–18 h) with the indicated mixtures of z-VAD-fmk (20 µM), neutralizing TNF-specific mAb Remicade (40 µg/ml), TNF inhibitor Enbrel (50 µg/ml) and the various SMAC mimetics (10 µM) and viability was then again determined by MTT staining. (C) The indicated cell lines were treated with 10 µM BV6 for 6 hours or remained untreated. Total cell lysates were then analyzed with respect to the presence of the indicated proteins by western blotting. (D) Kym-1 and HT1080 cells were primed with 10 µM BV6 for 6 h or remained untreated and were subsequently stimulated for 0, 3, 10 min with TNF (30 ng/ml). Cells were lysed in Laemmmli sample buffer and analyzed by western blotting for the presence of the indicated molecular species. Detection of phospho-IκBα, phospho-JNK and phospho-p38 is indicative for activation of the classical NFκB pathway and the JNK and p38 MAP kinase cascades. An increase in the p52/p100 ratio further argues for enhanced signaling via the alternative NFκB pathway. Detection of tubulin served as loading control.
To evaluate the effect of BV6 on peripheral blood monocytes, the latter was freshly isolated from buffy coats by ficoll density centrifugation and anti-CD14 magnetic bead separation. Monocytes were then immediately challenged with 10 µM BV6 or remained untreated. MTT analysis and microscopic inspection of monocytes of buffy coat preparations of various independent blood donations revealed that treatment with BV6 strongly reduces the viability of monocytes (
(A) Monocytes were isolated from peripheral blood mononuclear cells by MACS separation, cultivated for 1 day in GM-CSF/IL4 supplemented medium in the presence and absence of 10 µM BV6 and finally visually inspected by microscopy. (B) Monocytes were cultivated with 10 µM BV6 for 1 day and viability was assessed using the MTT assay (triplicates, left panel). Viability of BV6 treated cells were normalized against corresponding control samples receiving no BV6 (n = 7, right panel). (C) Effect of 10 µM BV6 on monocyte viability was determined after overnight incubation by annexin-V staining. Left panel shows a representative analysis of one individual sample and the right panel summarizes the data of monocytes independently derived from 5 buffy coat samples. The mean is indicated by a horizontal line. (D) Freshly isolated monocytes (0 h) and monocytes cultivated overnight in GM-CSF/IL4 (24 h) were analyzed by western blotting with respect to the processing of the indicated caspases and PARP-1. (E and F) Freshly isolated monocytes and monocytes cultivated in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by western blotting (E) and FACS (F) with respect to the expression of the indicated proteins. The western blot data shown were representative for two – four independent experiments.
Next, we evaluated the mechanisms of BV6-induced cell death. The pan-caspase inhibitor z-VAD-fmk showed no protective effect and even resulted regularly in a further reduction of viability of BV6-treated monocytes (
(A) Freshly isolated monocytes were incubated in GM-CSF/IL4 supplemented medium for 1 h with the indicated mixtures of z-VAD-fmk (20 µM), necrostatin-1 (70 µM) and the TNF inhibitor TNFR2-Fc/Enbrel (50 µg/ml). Cells were then challenged overnight with 10 µM BV6 and cell viability was finally evaluated by help of the MTT assay. (B) Freshly isolated monocytes and monocytes cultivated overnight in the presence of the indicated mixtures of GM-CSF/IL4 and 10 µM BV6 were analyzed by FACS for cell surface expression of membrane TNF and the death receptors TNFR1, CD95 and TRAILR2. (C) Monocytes were challenged with BV6 in the presence of soluble Fc fusion proteins of TRAILR2 (5 µg/ml), CD95 (50 µg/ml) and TNFR2 (Enbrel, 20 µg/ml) or a mixture of them. After 24 h viability was determined using the MTT assay (Left panel). The functionality of the three Fc fusion proteins was controlled in cell death assays with HT1080 and recombinant 50 ng/ml TNF, 4 ng/ml Fc-CD95L and 50 ng/mlTRAIL (right panel). Data shown are representative for three independent experiments.
When monocytes were cultured in the presence of GM-CSF and IL4 they rapidly become resistant for BV6-induced cell death. Already one day treatment with these cytokines rendered monocytes almost completely resistant against BV6, although p100 processing was still efficiently triggered (
(A and B) Monocytes were cultivated in medium supplemented with GM-CSF/IL4 and were treated immediately or after 1 and 2 days with 10 µM BV6. One day after stimulation BV6 treated cells and a corresponding control sample cultivated without BV6 were analyzed by annexin-V staining (A) and western blotting (B).
(A and B) Monocyte-derived macrophages, immature and Fc-CD40L maturated monocyte-derived dendritic cells were challenged for one day with and without 10 µM BV6. Cells were then analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent DC experiments and 6 independent experiments with monocytes and macrophages). p100 processing were determined by western blotting and is shown for one representative experiment (B). (C) Monocytes cultivated overnight in GM-CSF/IL4, iDCs obtained after 7 days of cultivation with GM-CSF/IL4 and mDCs maturated with TNF or Fc-CD40L were analyzed by western blotting with respect to the expression of the indicated proteins (data shown are representative for four independent experiments).
Irrespective of their activation status, T-cells isolated from peripheral blood mononuclear cells were also found to be resistant against BV6 (
(A and B) CD4+ and CD8+ T-cells were isolated by magnetic bead separation and were stimulated for 4–7 days with PHA or remained untreated. Cells were challenged with 10 µM or remained untreated as a control and were finally analyzed by annexin-V staining for cell death induction (A; upper panel: representative analysis of one individual sample; lower panel: summary of the data of 3 independent experiments). p100 processing was determined by western blotting and are shown for one representative experiment (B).
Next, we evaluated whether BV6 interferes with cIAP1/2-dependent non-apoptotic pathways induced by TNF or the related cytokine CD40L. We thus treated iDCs overnight with BV6 and analyzed the next day TNF- and CD40L-induced phosphorylation of IκBα as an indicator of activity of the classical NFκB pathway. There was hardly detectable IκBα phosphorylation in non-stimulated iDCs, but both cytokines induced after 5 min significant IκBα phosphorylation which was after 20 min already again reduced (
(A and B) Immature monocyte-derived dendritic cells (iDCs) were generated by cultivation for 7 days with GM-CSF/IL4. To obtain mature dendritic cells (mDCs), iDCs were further treated with Fc-CD40L (200 ng/ml) for 2 days. mDCs were then cultivated in Fc-CD40L–free medium for additional 24 hours with and without 10 µM BV6 and were stimulated for the indicated times with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (A). iDCs were primed overnight with 10 µM BV6 and were then challenged for 5 and 20 min with TNF (200 ng/ml) and Fc-CD40L (200 ng/ml) (B). iDCs and mDCs were finally analyzed by western blotting to determine the presence of the indicated proteins. The results shown with mDCs are representative of three independent experiments, the results with iDCs for two experiments. (C) iDCs were treated with 10 µM BV6, 300 ng/ml TNF and 1 µg/ml of Flag-CD40L oligomerized with 1 µg of the Flag-specific mAb M2 or as a control remained untreated. After three days cells were analyzed by FACS for the cell surface expression of CD83 and CD86. (upper panel: representative analysis of one individual sample; lower panel: summary of the data of iDCs of 4 independent donors. (D) Cell culture supernatants from “C” were analyzed for the presence of IL12. IL12 production was normalized to the corresponding values of untreated cells. The average of experiments with five independent donors is shown.
A major consequence of p100 to p52 processing is the conversion of p100/RelB heterodimers residing in the cytosol towards p52/RelB heterodimeric complexes that were able to translocate into the nucleus where they stimulate transcription of target genes of the alternative NFκB pathway
SMAC mimetics have been developed with the aim to obtain drugs that allow sensitization of tumor cells for apoptosis induction by other drugs or endogenous factors by releasing apoptotic caspases from the inhibitory interaction with IAP proteins, particular XIAP
In view of the complex effects of SMAC mimetics on NFκB and TNF signaling and the overwhelming importance of the NFκB system in immune cells, we reasoned that SMAC mimetics also modulate the function of immune cells, an aspect that is certainly of relevance for the development for SMAC mimetic-based therapies. As a first step to a better understanding of the effects of SMAC mimetics on the immune system, we analyzed in this study the effects of the bivalent SMAC mimetic BV6 on various types of human immune cells. First, we evaluated the potential apoptotic effect of BV6. In most types of immune cells namely macrophages, T-cells, immature and mature dendritic cells, BV6 showed typically no significant effect on viability. In freshly isolated monocytes, however, this compound triggered pronounced cell death (
BV6 treatment regularly induced cell death only in a fraction (30–50%) of monocytes irrespective of GM-CSF/IL4 treatment (
TNF and CD40L are potent triggers of maturation and activation of DCs and utilize cIAPs for the activation of proinflammatory pathways, including the classical NFκB pathway and the p38 MAP kinase cascade. We thus analyzed the impact of BV6 on TNF/CD40L-induced signaling in DCs. Not unexpected in view of the cIAP degrading activity of SMAC mimetics, we found that BV6 priming attenuates the capability of TNF and CD40L to stimulate the activity of p38 and the classical NFκB pathway in immature (
In sum, we noticed four effects of BV6 on immune cells. Firstly, apoptosis induction in monocytes, secondly activation of the alternative NFκB pathway in all investigated immune cells, thirdly enhanced basal but reduced cytokine inducible activation of the classical NFκB pathway in dendritic cells and fourth a potent maturation-inducing activity on DCs.
The diverse functions and the complex interplay of the various cell types of the immune system make it difficult to speculate about the consequences of SMAC mimetics
Macrophages and dendritic cells have been differentiated from monocytes isolated from rests of blood buffy coats of fully anonymized donors obtained from the Institute of Clinical Transfusion Medicine and Hämotherapy of the University Hospital Würzburg and required no special written informed consent. Peripheral blood samples for isolation of PBMCs were obtained after written informed consent (study no. 15/06 approved by the ethic commission of the Medical Faculty of the University of Würzburg).
We synthesized the recently published bivalent SMAC mimetic BV6 and monomeric and trimeric variants thereof by methods modified from those used by Vucic et al
Monocytes were isolated from blood buffy coats by density centrifugation with LSM 1077 lymphocyte separation medium (PAA Laboratories, Pasching, Austria) and subsequent separation with anti-CD14 conjugated magnetic beads and a MidiMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's recommendation. Purity of monocytes (CD14+ cells) was controlled by FACS and was regularly >95%. Monocytes were resuspended in RPMI-1460 supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PenStrep) (all PAA), 10 ng/ml IL4 (ImmunoTools GmbH, Friesoythe, Germany) and 50 ng/ml GM-CSF (ImmunoTools GmbH) and immediately prepared for the various experiments. For analysis of cell viability using the MTT assay 0.5×106 cells per well of a 96-well plate were seeded in 150 µl medium. For western blot and FACS analysis, 2×106 monocytes per well were seeded in 1 ml in a 24-well plate. Monocytes were differentiated into dendritic cells, by replenishing GM-CSF and IL4 every second day and were used for experiments with immature DCs after 7 days. To obtain mature DCs, iDCs were cultivated for additional three days in the presence of 200 ng/ml Fc-CD40L
PBMCs were isolated by Ficoll-Paque density-gradient centrifugation of cells obtained after plateletpheresis of regular blood donors who gave their written informed consent. CD4+ and CD8+ T-cells were selected with CD4+ and CD8+ MicroBeads (Miltenyi Biotech, Germany) and MACS LS columns on the QuadroMACS™ Separator according to the manufacturer's protocol. Purity of CD4+ and CD8+ T-cells was controlled by FACS and was regularly >95%. CD4+ or CD8+ cells (3×106) were seeded in a 24-well plate in 1 ml RPMI 1640/10% FCS (Gibco) and were immediately analyzed with respect to BV6-induced cell death or were stimulated with 1 µg/ml phyto-hemagglutinin (Sigma) for 4–7 days to obtain activated T-cells. For analysis of BV6-induced effects, cells were washed three times with PBS to remove any remaining mitogen and were then challenged with BV6 or remained untreated as a control.
Cells (5×105) were treated as indicated, washed two times in annexin-V staining buffer and were resuspended in 50 µl annexin-V staining buffer supplemented with 1 µl annexin-V solution (ImmunoTools GmbH). After incubation on ice in the dark for 15–20 min, cells were diluted with 100 µl annexin-V staining buffer and analyzed using FACSCalibur (BD Biosciences, Heidelberg, Germany). For determination of cellular viability cells (Kym-1: 2×104 cells per well; monocytes: 50×104 cells per well) were seeded in 96-well plates. Cells were treated as indicated and cell viability was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma, Steinheim, Germany) staining.
Cells were washed once in PBS and total cell lysates were prepared by suspending cells (8×106 cells per 100 µl for monocytes, dendritic cells, macrophages and KMS cells; 1×106 cells for Kym-1, HT1080 and HT29 cells) in 4× Laemmli sample buffer (4% SDS, 0.05 M dithiothreitol, 20% glycerol, 0.1 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktails I and II (Sigma) and complete protease inhibitor cocktail (Roche Diagnostics, Munich, Germany), sonification (ten pulses) and heating for 5 min at 96°C. After clearance of total cell lysates by centrifugation (10 min, 14000 g), samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% dry milk and membranes were then incubated with the indicated primary antibodies. After removal of primary antibodies by washing in PBS containing 0.1% Tween 20, antigen-antibody complexes were detected by the help of horseradish peroxidase-conjugated secondary antibodies (Dako, Hamburg, Germany) and an ECL western blotting detection kit (Amersham Biosciences Europe, Freiburg, Germany). Primary antibodies used were specific for caspase-8 (kind gift from K. Schulze-Osthoff; University of Tübingen), caspase-3, NIK, phospho-IκBα, p38, phospho-p38, JNK, phospho-JNK (all Cell Signaling, Frankfurt, Germany), Bcl-xL (S-18) IκBα (Santa Cruz Biotechnologies Inc., Heidelberg, Germany), p100/p52 (Upstate Biotech, Schwalbach, Germany), cIAP2, PARP1 (BD Biosciences Pharmingen, Heidelberg, Germany), cIAP1 (1E1-1-10), FLIP (NF6) (Enzo Life Sciences, Lörrach, Germany) and tubulin (Dunn, Asbach, Germany).