Background
Ewing sarcoma is the second most common form of bone sarcoma in children and adolescents [
1], deriving from a mesenchymal stem cell or neuronal crest cell [
2,
3]. Its pathogenesis results from a balanced translocation of the
EWS gene creating fusion proteins which code for chimeric transcription factors promoting cell growth [
4,
5]. Although 5-year survival in Ewing sarcoma patients is about 70%, the outcome for patients with metastatic disease or relapse drops to about 10–20% [
1]. Resistance to the cytotoxic drugs used in conventional chemotherapy often occurs in persisting, recurrent or relapsed tumours, that may be avoided by specifically targeting pathogenetic mechanisms in Ewing sarcoma cells to kill cancer clones before resistance can be developed [
6,
7]. Effective agents can also naturally occur in plant extracts, although their direct mechanisms of action may not be immediately clear.
The hemiparasite,
Viscum album L. (European mistletoe), contains a large variety of different immunomodulatory and cytotoxic substances that can be highly effective against cancer cells. Active agents are primarily viscotoxins and mistletoe lectins I-III [
8‐
10], but also include triterpenes and flavonoids [
11‐
15]. Standardised aqueous mistletoe extracts are commercially available and popular in complementary cancer medicine. However, they contain only the hydrophilic mistletoe lectins and viscotoxins. Mistletoe lectins and also triterpene acids, such as betulinic acid or oleanolic acid and its derivatives, have been shown to inhibit cell growth and induce apoptosis in melanoma, breast cancer and leukaemia cells [
16‐
18]. Despite the broad ranging anti-tumour effects of
Viscum album L., there is little known about the signalling pathways affected during mistletoe-mediated apoptosis. Betulinic acid as well as oleanolic acid and its derivatives have been reported to activate stress-mediated MAPKs in gastric cancer, osteosarcoma, pancreatic cancer, breast adenocarcinoma, glioma and melanoma cells [
19‐
23]. In leukaemia cells, mistletoe lectins were shown to activate MAPK8 [
16,
24], and Korean mistletoe lectin was shown to activate TLR4 in dendritic cells [
25]. But also AKT signalling has been implicated during mistletoe lectin or oleanolic acid treatment of gastric cancer, hepatocarcinoma, epidermoid cancer, colon carcinoma, ovarian cancer, prostate cancer, osteosarcoma and trophoblast cells, and oleanolic acid and its derivatives have been demonstrated to induce MTOR and NFKB1 signalling in prostate cancer, colon cancer and osteosarcoma cells [
23,
26‐
34]. We have also previously demonstrated the therapeutic effect of recombining hydrophilic and hydrophobic mistletoe constituents in the viscumTT extract for Ewing sarcoma (Twardziok et al., 2016, manuscript accepted 07/2016) and acute leukaemia cells in vitro and in vivo cancer models [
35,
36]. In Ewing sarcoma the mechanism leading to apoptosis involves the activation of caspases and the downregulation of the anti-apoptotic MCL1 and the IAP family members BIRC5 and XIAP. The aim of the present study was to analyse the impact of viscumTT and the single extracts on the transcriptome and proteome of Ewing sarcoma cells and to further illuminate the involved signalling pathways.
Methods
Viscum and TT extracts were prepared from
Viscum album L. harvested from apple trees (
malus) as previously described [
36] by the Birken AG (Niefern-Oeschelbronn, Germany), who kindly provided the lyophilized viscum and TT extracts for this study. Intact mistletoe lectin I (ML-I) was analysed by ELISA in viscum extract [
37]. Oleanolic and betulinic acid were quantified, as a measure of extract activity, using GC-FID [
18]. Lyophilized viscum extract was reconstituted in PBS (Gibco® Life Technologies, Darmstadt, Germany) to a final concentration of 2 μg/mL intact ML-I and <1 μg/mL viscotoxins. Lyophilized TT extract (containing cyclodextrins) was reconstituted in phosphate-buffered saline to a final concentration of 4000 μg/mL oleanolic and 350 μg/mL betulinic acid.
Cell culture
Human Ewing sarcoma cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The TC-71 and MHH-ES-1 cell lines were maintained in Iscove’s Modified Dulbecco’s Medium (Gibco Life Technologies) and RPMI 1640 base medium both supplemented with L-glutamine (Gibco Life Technologies), respectively. Base media were supplemented with 10% heat-inactivated FBS (Biochrom, Berlin, Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (Biochrom). For assays, TC-71 cells were seeded into 6-well (2 × 10
5) or 12-well (1 × 10
5) microtiter plates or 25 cm
2 (5 × 10
5) or 75 cm
2 (1.5 × 10
6) cell culture flasks depending on experimental set-up. MHH-ES-1 cells were seeded into 6-well (4 × 10
5) or 12-well (2 × 10
5) microtiter plates. Cells were cultured 24 h to allow cell attachment, and treated 24 h with
Viscum album L. extracts added to culture media. Viscum, TT and viscumTT concentrations were assessed by dose-effect-curves of apoptosis measurements as previously described [
38].
RNA isolation
TC-71 cells were incubated with increasing concentrations of the extracts for 24 h. RNA was isolated using the NucleoSpin® RNA Kit according to the manufacturer’s protocol (Macherey-Nagel, Düren, Germany) in five independent experiments. Purity and concentration was determined by OD260/280 on the NanoDrop™ 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
TC-71 cells were treated once with ~IC
50 extract concentrations (viscum 2 ng/mL ML-I, TT 50 μg/mL oleanolic acid, viscumTT 1 ng/mL ML-I + 10 μg/mL oleanolic acid) for 24 h. After total RNA isolation from treated and control cells, Illumina TruSeq RNA sample preparation including a polyA selection step via oligo-dT beads was used to isolate mRNA and generate cDNA libraries. Samples were sequenced by paired-end mRNA sequencing on an Illumina HiSeq 2500 (50 bp reads,
n = 1). Reads were mapped uniquely to human genome hg19 using CLC genomics software. Normalisation and identification of differentially expressed genes was performed using DE-Seq software [
39] (Bioconductor open source software) to calculate fold-change relative to untreated control cells, false discovery rate (FDR) [
40] and
p value using the negative binomial distribution, with
p ≤ 0.05 considered as significant. The heat map and Venn diagram illustrating differential gene expression as log
2-fold change relative to untreated control cells was performed by using the R software package (
http://www.r-project.org/). Gene enrichment and functional annotation analysis was performed with the top differentially expressed genes (
p ≤ 0.01) using DAVID Bioinformatics Resources 6.7 NIAID/NIH (
http://david.abcc.ncifcrf.gov/).
cDNA synthesis and qPCR validation
Using 2 μg RNA from treated or control TC-71 cells, cDNA was synthesised using the High Capacity RNA-to-cDNA Kit according to the manufacturer’s protocol (Applied Biosystems, Waltham, MA, USA). To confirm mRNA sequencing results, gene expression was measured by real-time PCR on the StepOnePlus™ System in 96-well fast plates under standard conditions (10 min, 95 °C; 15 s, 95 °C and 60 s, 60 °C, 40×) using Power SYBR Green Master Mix including ROX as a passive reference (Applied Biosystems). The quantitative PCR reactions were 20 μl total volume containing 5 ng cDNA and 500 nM primers, which were predesigned and purchased from Integrated DNA Technologies (IDT, Leuven, Belgium):
DDIT3: Hs.PT.58.14610020;
JUN: Hs.PT.58.25094714.g;
MAP2K6: Hs.PT.58.3312889;
GAPDH: Hs.PT.39a.22214836. Primer efficiency was determined to be 90–100%. Expression was normalised using
GAPDH expression. The relative expression of genes was calculated by ∆∆CT method: ΔΔCT = (CT(target,untreated) – CT(ref,untreated)) − (CT(target,treated) – CT(ref,treated)); fold-change =2^
(−∆∆CT) [
41].
Proteomic profiling and bioinformatics analysis
TC-71 cells were grown in 25 cm
2 cell culture flasks in triplicate and treated with viscum, TT or viscumTT extracts at ~ IC
50 concentrations (viscum 2 ng/mL ML-I, TT 50 μg/mL oleanolic acid, viscumTT 1 ng/mL ML-I + 10 μg/mL oleanolic acid) for 24 h. Cells were harvested and lysed under denaturing conditions in a buffer containing 6 M guanidinium chloride, 10 mM tris(2-carboxyethyl)phosphine, 40 mM chloroacetamide and 100 mM Tris pH 8.5. Lysates were sonicated and boiled at 95 °C for 5 min. Lysates were diluted 1:10 in 10% acetonitrile in 25 mM Tris, pH 8.5, and 2% of each total lysate volume was digested with 1 μg trypsin at 37 °C overnight. Peptides were acidified by adding formic acid to a final concentration of 1%, desalted using C18 StageTips (Thermo Scientific, Waltham, MA, USA) and lyophilised. Pellets were dissolved in 5% acetonitrile and 2% formic acid and one quarter of the digest was injected for nanoflow reverse-phase liquid chromatography (Dionex Ultimate 3000, Thermo Scientific) coupled online to a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (nanoLC-MS/MS). Briefly, the LC separation was performed using a PicoFrit analytical column (75 μm ID × 40 cm long, 15 μm Tip ID (New Objectives, Woburn, MA) packed in-house with 2.1 μm C18 resin (Reprosil-AQ Pur, Dr. Maisch, Ammerbuch-Entringen, Germany) under 50 C controlled temperature. Peptides were eluted using a nonlinear gradient from 2 to 40% solvent B in solvent A over 180 min at 266 nL/min flow rate. Solvent A was 0.1% formic acid and solvent B was 79.9% acetonitrile, 20% H
2O, 0.1% formic acid). Nanoelectrospray was generated by applying 3 kV. A cycle of one full Fourier transformation scan mass spectrum (300–1700 m/z, resolution of 35,000 at m/z 200) was followed by 12 data-dependent MS/MS scans with normalised collision energy of 25 eV. A dynamic exclusion window of 30 s was used to avoid repeated sequencing of the same peptides. MS data were analysed by MaxQuant (v1.5.0.0) [
42]. The algorithm MaxLFQ [
43], which is integrated into MaxQuant, was used for label free quantification (LFQ). Peptides were searched against the human proteome UniProtKB database released in 11/2014 with 88,717 entries, released in 11/2014, using an FDR of ≤0.01 for proteins and peptides with a minimum length of seven amino acids. A maximum of two missed cleavages in the tryptic digest was allowed. Cysteine carbamidomethylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. Principal component analysis (PCA, Fig.
3b) and a heat map of the replicates (Fig.
3a) were carried out using the Perseus software (v1.5.1.6). Gene set enrichment analysis (GSEA, v2.1.0) [
44] was applied to assess a priori defined protein sets with statistically significant expression differences between treated and control cells. We used GSEA standard settings, except that minimum size exclusion was set to five and KEGG v2.1.0 as well as reactome v5.0 was used as the gene set database. The String software tool (v10) was used to visualise protein-protein interaction networks of proteins up- or downregulated at least 2-fold [
45]. Protein nodes that were not integrated into the protein-protein interaction network were removed.
Western blotting
TC-71 and MHH-ES-1 cells were incubated with viscum, TT or viscumTT in increasing concentrations for 24 h. The cells were washed twice with phosphate-buffered saline and incubated in Lysis Buffer 17 (R&D systems, Minneapolis, MN) containing protease inhibitors (complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics GmbH) to obtain cell lysates. Protein concentration was determined using Bradford solution (Bio-Rad, Munich, Germany). TC-71 and MHH-ES-1 cell lysates (30 μg protein/lane) were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad system) and blocked with 5% non-fat milk in 50 mM Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight at 4 °C in TBST containing 5% BSA and primary antibody, washed thrice in TBST and incubated 1 h with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse, Bio-Rad) then visualized by ECL (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). Primary antibodies were directed against p-MAPK14 (Thr180/Tyr182, #9211 Cell Signaling Technology, Danvers, MA, USA), LC3B (#2775 Cell Signaling Technology), EIF2AK3 (#3192, Cell Signaling Technology), p-MAPK8 (sc-6254, Santa Cruz biotechnology, Dallas, TX, USA), HSPA5 (#G8918, Sigma-Aldrich) and ß-actin conjugated directly to peroxidase (#A3854, Sigma-Aldrich).
Inhibitor treatment and apoptosis measurement
To measure the impact of TLR4 signalling, MAPK14 and MAPK8 activation or oxidative stress on apoptotic induction, TC-71 cells were pre-incubated with specific inhibitors for 1 h followed by an incubation with ~ IC50 extract concentrations for 24 h. Specific inhibitor treatment included 5–50 μM SB203580 (Cell Signaling Technology), 1 μM – 25 μM SP600125 (Sigma-Aldrich), 1–10 mM N-acetylcysteine (NAC, Sigma-Aldrich), 0.1–10 μg mL−1 LPS-RS (InvivoGen, San Diego, CA, USA). DMSO (Sigma-Aldrich) or PBS was added to extracts as solvent control, depending on the inhibitor diluent. After treatment, cells were washed twice with PBS, resuspended in 100 μl binding buffer and stained with APC-conjugated annexin V (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s protocol, then counterstained with 1 μl 1 mg/mL propidium iodide (Sigma Aldrich). Cells were analysed by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany), and the data were evaluated using FlowJo Software (TreeStar, Ashland, OR, USA).
Statistical analyses
All qPCR experiments were performed in triplicate and experiments were repeated four times. Results are presented as mean values ± standard error of the mean (SEM). Inhibitor treatment and measurement of apoptosis was repeated thrice independently. The results are also presented as mean values ± SEM. Western blots were performed in three independent experiments. mRNA sequencing was performed in one experiment, while proteomic profiling was performed in triplicate. The cut-off for significantly deregulated pathways was set to p ≤ 0.01 for mRNAseq and p ≤ 0.05 and FDR ≤ 0.25 for proteomic profiling. Principal component analysis was performed to compare differences within the triplicates used for proteomic profiling. Protein-protein interaction networks of proteins up- or downregulated were regarded as significant by at least 2-fold change with a confidence level of 0.7 [45].
Discussion & conclusions
ViscumTT reconstitutes the aqueous lectins and viscotoxins as well as the hydrophobic (with cyclodextrins solubilised) triterpene acids of mistletoe into a defined total extract, combining all components predicted to have cytotoxic properties. ViscumTT extract treatment significantly altered both the transcriptome and proteome of TC-71 cells affecting cellular stress response pathways related to cell death. Upregulation of cellular stress associated proteins was also confirmed in MHH-ES-1 cells. While viscum treatment alone displayed similar effects on the transcriptome and proteome of TC-71 cells, the TT extract alone showed different and less alterations. On the one hand, it is possible that viscumTT and TT treatment displayed different significantly deregulated genes and proteins due to the very different olanolic acid concentrations used in TT and the synergistically apoptosis-inducing viscumTT extract to reach IC50 (TT 50 μg/mL oleanolic acid, viscumTT 10 μg/mL oleanolic acid), while the difference between ML-1 concentration in viscum (2 ng/mL ML-1) and viscumTT (1 ng/mL ML-1) extract treatment were more alike. On the other hand, it is conceivable that TT-activated pathways are different from those activated by viscum or viscumTT. We have previously demonstrated that viscum and even more, viscumTT effectively induced apoptosis in Ewing sarcoma cells in vitro and ex vivo accompanied by a loss of mitochondria membrane potential and activation of CASP-8, −9 and −3, while TT alone showed only moderate apoptosis induction without a significant loss of mitochondria membrane potential and activation of CASP-8, −9 and −3. However, TT potentiates the effect of viscum resulting in a synergistic apoptosis-induction by viscumTT (Twardziok et al. 2016, manuscript accepted).
We show here that viscum, TT and total mistletoe viscumTT extract affect different transcriptomic changes in Ewing sarcoma TC-71 cells. Both, viscumTT and viscum, upregulated
JUN and other genes involved in cell death, MAPK signalling and oxidative stress. TT upregulated genes involved in TLR signalling and cell death as well as genes involved in inflammatory response. Although our results are preliminary due to a limited validity of one single mRNA-sequencing experiment for each extract treatment, others reported similar findings after mistletoe extract treatment. For instance, Yang et al. reported that recombinant mistletoe lectin differentially regulated several genes in the MAPK signalling cascade in hepatocellular carcinoma cells [
26]. Furthermore, a similar transcriptomic profile involving apoptosis and MAPK signalling pathway was shown in breast cancer cells using DNA microarray chips after treatment with an commercial aqueous mistletoe extract [
48]. Interestingly, they showed that aqueous mistletoe extracts from oak and apple tree hosts more strongly upregulate immune defence and stress response in breast cancer cells, whereas aqueous extracts from the host tree white fir affect cell-cell adhesion and cytoskeleton pathways. In line with this, our extracts from the apple tree as host evoked cellular stress and immune defence responses.
We further show that viscumTT, viscum and TT affect different proteomic changes in TC-71 cells. Treatment with the extracts resulted in the downregulation of proteins involved in translation and transcription/spliceosome and an upregulation of proteins involved in aminoacyl-tRNA biosynthesis and the proteasome. ViscumTT and viscum contain mistletoe lectins, which are classified as ribosome-inactivating proteins type-II provoking a breakdown of translation [
49‐
51] leading to proteasomal degradation. Ribosome-inactivating proteins type-II have n-glycosidase activity removing single adenines from rRNA and they were originally thought to act exclusively on ribosomes, but there is evidence growing that they are also able to inactivate non-ribosomal nucleic acid substrates [
52,
53]. Therefore, it is conceivable that they also affect the spliceosome, which consists of snRNAs and protein complexes. ViscumTT and viscum treated TC-71 cells also displayed an upregulation of proteins involved in protein folding, regulation of apoptosis, immune system and MAPK activation correlating with the transcriptomic results and suggesting that viscumTT and viscum have an impact on MAPK signalling and protein folding. Taken together, our transcriptomic and proteomic data showed that the triterpene extract TT had a lower influence on the transcriptome and proteome of TC-71 cells than viscum and viscumTT, which displayed similar profiles. The mistletoe extracts affect transcription, translation and the proteasome and they affect cellular stress and inflammatory responses. However, the similar proteomic and transcriptomic profiles of viscumTT and viscum treated TC-71 cells suggest that the synergistic effect of viscumTT is not created on transcriptional or translational level.
Our deeper assessment of the affected signalling pathways upon mistletoe treatment revealed the activation (phosphorylation) of stress-mediated MAP kinases MAPK8 and MAPK14 by viscumTT and viscum in TC-71 and MHH-ES-1 cells. In agreement with our results, others have shown the activation of MAPK8 signalling by an aqueous European and Korean mistletoe extract in leukaemia and hepatocarcinoma cells [
16,
54,
55]. Other ribosome-inactivating proteins as Ricin or Shiga toxins also activate MAPK8 and MAPK14 in human monocytes and macrophages [
56‐
58]. Contrary to our results, the triterpenes oleanolic and betulinic acid were also shown to activate MAPK8 and MAPK14 in melanoma, pancreatic cancer and osteosarcoma cells and hypertrophic scar fibroblasts [
19,
21,
59]. Since the activation of MAPK8 and MAPK14 is stress-mediated resulting in apoptosis and because our pathway analyses displayed response to oxidative stress and TLR signalling, we next investigated the impact of MAPK8, MAPK14 and TLR4 inhibitors and the anti-oxidant NAC on apoptosis induction in TC-71 cells upon treatment with the extracts. TT-mediated apoptosis was reduced when cells were pre-treated with the TLR4 antagonist LPS-RS suggesting the involvement of TLR4 signalling by TT treatment. TLR4 signalling has been described in mouse splenocytes after treatment with an oleanolic acid derivative [
60]. With regard to oxidative stress, NAC pre-treatment slightly reduced apoptosis by viscumTT and viscum indicating that oxidative stress is involved in viscumTT- and viscum-mediated apoptosis. In line with our results, the induction of oxidative stress by aqueous mistletoe extracts was shown in leukaemia and hepatocarcinoma cells [
16,
55]. However, an oleanolic acid derivative was also reported to induce oxidative stress in ovarian cancer cells [
61]. Notably, the applied MAPK14 and MAPK8 inhibitors (up to 10 μM SB203580, 5 μM SP600125) were not able to inhibit the activation of the kinases and to prevent the induction of apoptosis by viscumTT or viscum. Higher concentrations of the inhibitors resulted in a significant loss of cell viability demonstrating the cytotoxic potential of the inhibitors in TC-71 cells (data not shown). Others have used superior concentrations (up to 30 μM SB203580 or SP600125) to achieve a block in mistletoe mediated apoptosis induction without a loss of cell viability in leukaemia and hepatocarcinoma cells suggesting that higher concentrations might be necessary to effectively block apoptosis [
16,
55,
62]. However, it is likely that the MAPK pathway is not the major pathway engaged by the extracts since viscumTT and viscum displayed an impressive impact on the transcriptome and proteome of TC-71 cells suggesting that the reasons for the apoptosis induction by the extracts are manifold. In summary, our deeper assessment of the activated signalling pathways indicates the involvement of TLR4 signalling for the TT extract and the activation of the stress-mediated MAPK signalling and oxidative stress for viscum and viscumTT. However, a deeper investigation of the activated pathways is needed.
Since there is a crosstalk between oxidative stress and the unfolded protein response and ER stress is also linked to MAPK8 activation [
63‐
66], we analysed the protein expression of the ER chaperone protein HSPA5 [
67] and the ER stress sensor protein EIF2AK3 [
68,
69] in TC-71 and MHH-ES-1 Ewing sarcoma cells. We detected an upregulation of HSPA5 by viscumTT and both single extracts in both cell lines suggesting response to oxidative or ER stress by activation of the unfolded protein response. However, our results are preliminary and need further specific analyses for validation. Interestingly, the induction of HSPA5 and DDIT3 (formerly CHOP) leading to ER stress was already reported for a commercial aqueous extract in leukaemia cells [
16]. We also detected a transcriptional an upregulation of
DDIT3 after treatment with viscumTT or both single extracts. Other ribosome-inactivating proteins like Ricin and Shiga toxins were also shown to activate the unfolded protein response in human breast and colon cancer cell lines [
70] and a rat renal tubular epithelial cell line [
71]. Furthermore, the naturally occurring triterpenoid celastrol induced the unfolded protein response in head and neck cancer cell lines [
72]. Notably, viscumTT and viscum treatment concurrently displayed a decrease of EIF2AK3, whereas TT treatment showed a slight upregulation matching ER stress. Others demonstrated the induction of apoptosis by ER stress in HeLa cells after treatment with an oleanolic acid derivative [
73]. Proteomic investigation of betulinic acid-induced apoptosis in HeLa cells also displayed the induction of ER stress [
74]. Furthermore, an oleanolic acid derivative was shown to trigger ER stress leading to MAPK8-dependent apoptosis [
75]. Because ER stress is connected to autophagy [
46,
47,
76,
77], we also analysed the expression of the autophagy marker LC3B. The triterpene extract TT increased LC3B-II expression in both cell lines indicating enhanced autophagic activity. Consistent with our results, the triterpenes ursolic, betulinic or oleanolic acid and its derivatives were reported to induce autophagy in glioblastoma, breast cancer, prostate cancer, myeloma, KRAS transfected MCF10A breast epithelial cells as well as in Human Embryonic Kidney 293 cells, human hepatic cells, immortalized breast epithelial, gastric mucosal and primary bladder epithelial cells [
78‐
84]. Others, by contrast, reported autophagy induction by Korean mistletoe lectin in placenta-derived mesenchymal stem cells [
85]. In summary, viscumTT and the single extracts viscum and TT influence protein folding and indicate the involvement of the unfolded protein response and autophagy (TT).
In conclusion, we provide a first deep insight into the effects of single and combined mistletoe extracts in Ewing sarcoma cells in vitro. ViscumTT and both single extracts induce diverse cellular stress responses and influence protein folding. While viscumTT and viscum suggest the activation of the stress-mediated MAPK signalling and induction of oxidative stress, the TT extract indicates the induction of autophagy and TLR4 signalling. As viscumTT and viscum appear to activate the same signalling pathways differing from TT-activated pathways, the synergistic effect of viscumTT demonstrated in our previous work cannot be explained so far. However, the results suggest that the synergistic effect of viscumTT is created by other mechanisms. Nevertheless, viscumTT, which combines the anticancer effects of hydrophilic and hydrophobic mistletoe compounds, may represent a promising adjuvant phytopolychemotherapeutic therapy option for Ewing sarcoma patients.
Acknowledgements
The authors thank I. Lehmann and B. Lukaszewska-McGreal for RNA sequencing and MS sample preparation and K. Astrahantseff for comments on and editing of the manuscript.