A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML

Wirkmechanismus von Viscum album L. Extrakten beim Ewing-Sarkom

1 Introduction 1 1.1 Viscum album L. 1 1.1.1 Mistletoe lectins 2 1.1.2 Viscotoxins 4 1.1.3 Triterpene acids 4 1.2 Ewing sarcoma 6 1.2.1 Pathogenesis 6 1.2.2 Origin 8 1.2.3 Therapy and prognosis 10 1.3 Aim of the work 12 2 Material and methods 13 2.1 Material 13 2.1.1 Equipment 13 2.1.2 Consumables 13 2.1.3 Chemicals and reagents 14 2.1.4 Viscum album L. extracts 16 2.1.5 Triterpene standards 16 2.1.6 Buffers 16 2.1.7 SDS-PAGE gels 17 2.1.8 Kits 17 2.1.9 Antibodies 18 2.1.10 Primers 18 2.1.11 Cell lines 19 2.1.12 Primary cells 19 2.2 Methods 19 2.2.1 Cell culture 19 2.2.1.1 Cell lines 19 2.2.1.2 Ewing sarcoma primary cells 20 2.2.1.3 Cryopreservation of cells 20 2.2.2 Ewing sarcoma xenografts 21 2.2.3 Cell biological analyses 22 2.2.3.1 Measurement of cell proliferation 22 2.2.3.2 LDH assay 22 2.2.3.3 Measurement of apoptotic cell death 23 2.2.3.4 Measurement of mitochondria membrane potential 23 2.2.3.5 Measurement of active caspases 23 2.2.3.6 Inhibitor assays 24 2.2.3.7 CD99 immunostaining 24 2.2.4 Molecular biological analyses 25 2.2.4.1 Antibody array 25 2.2.4.2 Western blotting 25 2.2.4.3 Proteome profiling, pathway and protein network analysis 26 2.2.4.4 RNA isolation 27 2.2.4.5 Two-step real-time PCR 27 2.2.4.6 Transcriptome profiling and pathway analysis 28 2.2.5 Statistical analyses 28 3 Results 30 3.1 Viscum album L. extracts inhibit proliferation in vitro 30 3.2 Viscum album L. extracts show no early cytotoxicity via necrosis in vitro 31 3.3 Viscum album L. extracts induce apoptosis in vitro 32 3.4 Viscum and viscumTT induce depolarization of mitochondria membrane in Ewing sarcoma cell lines 34 3.5 TT-mediated apoptosis induction is driven by the combination of oleanolic and betulinic acid 35 3.6 Viscum and viscumTT activate caspases in vitro 36 3.7 Viscum and viscumTT induce apoptosis and inhibit proliferation ex vivo 38 3.8 Viscum album L. extracts reduce tumor volume in a Ewing sarcoma mouse model 40 3.9 Viscum album L. extracts alter the expression of apoptosis related proteins in Ewing sarcoma cell lines 42 3.10 Viscum album L. extracts alter the transcriptomic profile of TC-71 cells 44 3.11 Viscum album L. extracts alter the proteomic profile of TC-71 cells 50 3.12 Viscum album L. extracts induce cellular stress and activate the unfolded protein response in vitro 58 4 Discussion 61 4.1 Viscum album L. extracts inhibit proliferation and induce apoptosis in Ewing sarcoma cells in vitro and ex vivo 61 4.2 Viscum and viscumTT are effective in Ewing sarcoma xenografts in vivo 63 4.3 Viscum album L. extracts alter the expression of many apoptosis-related proteins in vitro 64 4.4 Viscum album L. extracts alter the transcriptomic profile of TC-71 cells 66 4.5 Viscum album L. extracts alter the proteomic profile of TC-71 cells 67 4.6 Viscum album L. extracts induce cellular stress and activate the unfolded protein response in vitro 69 4.7 Final conclusion 73 5 Summary 74 6 Zusammenfassung 76 7 Supplementary data 78 8 References 82 9 Publications 105 9.1 Research articles 105 9.2 Oral presentations 106 9.3 Poster 106Ewing sarcoma is the second most common bone cancer in children and adolescents. It has a poor prognosis and outcome. Therefore, drug discovery of new anti-tumor agents is still crucial to improve survival of Ewing sarcoma patients. Combining active substances may be beneficial since some drug combinations are able to enhance each other creating synergistic effects. Natural substances in plant extracts contain diverse cytotoxic compounds and provide potential active ingredients for tumor therapy. The European mistletoe (Viscum album L.) contains hydrophobic triterpene acids and hydrophilic lectins and viscotoxins, which all possess anti-cancer properties. Standardized commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. However, triterpene acids can be solubilized by using cyclodextrins. By combining a solubilized mistletoe triterpene extract (TT) with an aqueous mistletoe extract (viscum) a total mistletoe effect was obtained (viscumTT) and the mechanism of action of viscum, TT and viscumTT was analyzed in Ewing sarcoma cells. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent manner, while the TT extract displayed only moderate pro- apoptotic properties. Importantly, viscumTT combination treatment generated a synergistic effect. Viscum- and viscumTT-induced apoptosis occurred via the intrinsic and extrinsic apoptotic pathway, evidenced by the depolarization of mitochondria membrane and the activation of caspase 8 and 9. Additionally, the antitumor activity of viscum and viscumTT was validated in a Ewing sarcoma mouse model in vivo. A deeper assessment of the molecular mechanisms of viscum, TT and viscumTT treatment leading to apoptosis in vitro revealed a balance shift of anti-apoptotic/ pro-survival regulatory proteins towards the pro-apoptotic state, mainly via MCL1, CLSPN, BIRC5 and XIAP downregulation. Moreover, the transcriptomic and proteomic profile of Ewing sarcoma cells showed strong alterations evoking a transcriptional upregulation of stress- activated and apoptosis-associated genes accompanied with an upregulation of proteasomal proteins and a downregulation of ribosomal and spliceosomal proteins. Furthermore, the extracts triggered the activation of the unfolded protein response. While viscum and viscumTT displayed also response to oxidative stress and the activation of stress-mediated MAPK signalling, the TT extract indicated the involvement of TLR signalling and autophagy. Since the combinatory viscumTT extract displayed phytopolychemotherapeutic properties and demonstrated a potent pro-apoptotic impact, it may represent an adjuvant therapy option for paediatric patients with Ewing sarcoma. In any case, this work contributes to an improved understanding on the mechanism of action of Viscum album L. extracts.Das Ewing-Sarkom stellt den zweithäufigsten Knochentumor bei Kindern und Jugendlichen dar und ist mit einer ungünstigen Prognose assoziiert. Somit ist die Erforschung und Entwicklung neuer therapeutischer Agenzien weiterhin essentiell, um das Überleben von Patienten mit einem Ewing-Sarkom zu verbessern. Die Kombination mehrerer Wirkstoffe kann vorteilhaft sein, da einige Kombinationen sich gegenseitig verstärken können und so synergistische Effekte hervorrufen. Pflanzen enthalten diverse natürlich vorkommende zytotoxische Substanzen und liefern dadurch potentielle Wirkstoffe für die Tumortherapie. Die europäische Mistel (Viscum album L.) enthält als anti- kanzerogene Substanzen die lipophilen Triterpensäuren sowie die hydrophilen Mistellektine und Viscotoxine. Standardisierte, kommerziell erhältliche Viscum album L. Extrakte sind jedoch auf wässriger Basis und enthalten deshalb keine Triterpensäuren. Diese können aber durch die Verwendung von Cyclodextrinen solubilisiert werden. Durch die Kombination eines triterpenhaltigen Mistelextrakts (TT) mit einem wässrigen Mistelextrakt (viscum) wurde ein Mistel-Gesamtextrakt (viscumTT) erhalten und der Wirkmechanismus von viscum, TT und viscumTT im Ewing-Sarkom-Modell untersucht. Die Behandlung der Ewing- Sarkom Zellen mit den Extrakten in vitro und ex vivo zeigte, dass viscum konzentrationsabhängig die Proliferation inhibierte und die Apoptose induzierte, während TT nur schwache pro-apoptotische Eigenschaften aufwies. Zudem zeigte sich, dass der kombinatorische Extrakt viscumTT synergistisch Apoptose induziert. Die durch viscum und viscumTT vermittelte Apoptose erfolgte dabei sowohl durch den intrinsichen als auch den extrinsichen Apoptoseweg, was durch die Absenkung des Mitochondrienmembranpotentials und die Aktivierung der Caspase-8 und Caspase-9 belegt wurde. Die anti- kanzerogenen Eigenschaften von viscum und viscumTT wurden auch in einem Ewing- Sarkom-Mausmodell in vivo nachgewiesen. Tiefer gehende Untersuchungen des molekularen Wirkmechanismus von viscum, TT und viscumTT in vitro zeigten eine Verschiebung von anti-apoptotischen Proteinen zugunsten der Apoptose, die sich in der Abnahme der Proteinexpression von MCL1, CLSPN, BIRC5 und XIAP äußerte. Zudem führten die Extrakte zu einer starken Veränderung des Transkriptom- und Proteom-Profils, wobei sie die transkriptionelle Hochregulation von Stress- aktivierten und Apoptose-assoziierten Genen bewirkten, sowie die Zunahme von proteasomalen Proteinen und die Abnahme von ribosomalen und spliceosomalen Proteinen auslösten. Außerdem lösten die Extrakte eine Antwort auf ungefaltete Proteine (unfolded protein response) aus. Weiterhin wurde gezeigt, dass viscum und viscumTT oxidativen Stress und die Aktivierung des Stress-vermittelten MAPK Signalwegs verursachen, während die Behandlung mit dem TT Extrakt auf die Beteiligung des TLR Signalwegs und der Autophagie hindeutet. Da vor allem der kombinatorische Extrakt viscumTT potente pro-apoptotische Eigenschaften in Ewing-Sarkom Zellen zeigte, könnte dieser als eine Art Phytopolychemotherapie eine zusätzliche, adjuvante Therapieoption für pädiatrische Patienten mit einem Ewing-Sarkom darstellen. In jedem Fall trägt die vorliegende Arbeit zu einem besseren Verständnis des Wirkmechanismus von Viscum album L. Extrakten bei

Multiple Active Compounds from Viscum album L. Synergistically Converge to Promote Apoptosis in Ewing Sarcoma.

Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10-15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (Viscum album L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT in vitro, ex vivo and in vivo. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. ViscumTT also demonstrated strong antitumor activity in a cell line- and patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma

Viscum, TT and viscumTT alter apoptosis-related protein expression.

<p><b>A.</b> Whole-cell protein extracts from TC-71 cells after 24h of treatment with viscum, TT or viscumTT concentrations approximating the IC50 were analyzed using the R&D systems human proteome profiler apoptosis array (n = 1). Bars represent the n-fold change in apoptosis-related protein expression relative to untreated control expression. <b>B.</b> TC-71 and MHH-ES-1 cells were treated with viscum, TT or viscumTT at the concentrations shown for 24h, then expression of the apoptosis-related proteins indicated was examined in whole-cell lysates using western blotting. Representative pictures are shown from 3 independent experiments. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.</p

ViscumTT inhibits proliferation in Ewing sarcoma cell lines without early cytotoxicity.

<p>TC-71 and MHH-ES-1 cells were treated with increasing concentrations of TT, viscum or viscumTT for 24h and proliferation was estimated from total cell numbers in cultures started from defined cell numbers compared to control cultures (upper graphs). Cells were counted using a CASY^® Cell Counter. TC-71 and MHH-ES-1 cells were incubated with increasing concentrations of viscum, TT or viscumTT for 2h, before assessing early cytotoxicity via LDH release into the culture medium (lower graphs). All results are presented as the percentage of untreated control (Ctrl) cultures ± SD, and are the mean of 3 independent experiments. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.</p

ViscumTT combined extract synergistically induces apoptosis in Ewing sarcoma cell lines.

<p><b>A.</b> TC-71 and MHH-ES-1 cells were incubated for 24h with increasing concentrations of viscum, TT and viscumTT. Cultures were then stained with annexin V and propidium iodide and examined by flow cytometry. The percentage of dead cells in each treatment group are shown (±SD) from 3 independent experiments. A synergistic effect of the combined viscumTT extract above single extracts was calculated by Webb´s fractional product (*Fp > 1). <b>B.</b> PARP1 and CASP3 cleavage was assessed in whole-cell extracts from cells treated as described above using western blotting. β-actin was used as loading control, and images shown are representative for the results in 3 independent experiments. <b>C.</b> TC-71 and MHH-ES-1 cells treated as above for 24h and were assessed for mitochondrial involvement in apoptosis using JC-1 staining and flow cytometry. Bars depict the percentage of cells with low mitochondrial membrane potential (ΔΨ_m, ± SD, n = 3) in each treatment group averaged from 3 independent experiments (±SD, error bars). A synergistic effect of combined viscumTT extracts was calculated by Webb´s fractional product (*Fp > 1). Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was used as positive control. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.</p

Multiple Active Compounds from <i>Viscum album L</i>. Synergistically Converge to Promote Apoptosis in Ewing Sarcoma

<div><p>Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10–15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (<i>Viscum album</i> L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial <i>Viscum album</i> L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i>. <i>In vitro</i> and <i>ex</i> vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. ViscumTT also demonstrated strong antitumor activity in a cell line- and patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma.</p></div

ViscumTT synergistically activates caspases in Ewing sarcoma cells lines above the levels activated by viscum alone.

<p><b>A.</b> TC-71 and MHH-ES-1 cells treated 24h with either viscum, TT or viscumTT. CASP9, CASP8, CASP3 activation was measured using FITC-LEHD-FMK, FITC-IETD-FMK and FITC-DEVD-FMK staining followed by flow cytometry. Bars represent mean activation (±SD, error bars) in treatment groups from 3 independent experiments relative to untreated control cultures. A synergist effect of the combined viscumTT extracts was calculated by Webb´s fractional product (*Fp > 1). <b>B.</b> TC-71 cells were treated with viscum, TT and viscumTT for 24h with or without 40μM Z-VAD-FMK (pan-caspase inhibitor), then apoptotic cells were detected using annexin V/propidium iodide staining and flow cytometry. Bars represent the mean (±SD, error bars) of 3 independent experiments. Results are expressed as percentages of the untreated control cultures. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.</p

Caspase activity of HL-60 cells after VAE treatment.

<p><b>A.</b> HL-60 cells were incubated for 18 h with VAE. Caspase-8 and-9 activity was measured by FITC-LEHD and FITC-IETD and flow cytometry, ±SD, n = 3. VAEs show a dose-dependent activation of caspase-8 and -9, viscumTT activates the caspases in a synergistic manner. * Fp>1, displays the synergistic effect. <b>B.</b> The cells were treated with TT, viscum or viscumTT for 18 h in the presence or absence of 100 μM z-VAD-fmk, z-IETD-fmk or z-LEHD-fmk. Effects of caspase inhibitors were analyzed by Annexin V/ propidium iodide staining and flow cytometry, ±SD, n = 3. Inhibition of caspase-8 reduces VAE-induced apoptosis by up to 55%, treatment with pan-caspase inhibitor shows an inhibition of apoptosis by up to 75%.</p

Mistletoe extracts induce apoptosis in acute myeloid leukaemia cells <i>ex vivo</i>.

<p>Mononuclear cells obtained from bone marrow aspirates of two patients with acute myeloid leukaemia (patient 1 (m; 17 years); patient 2 (m, 10 years)) were incubated with the depicted VAEs for 18 h. Induction of apoptosis (n = 2), mitochondrial transmembrane potential (n = 1) and activation of caspase-8 / -9 (n = 1) were measured by flow cytometry. VAEs induce apoptosis with involvement of mitochondria and activation of caspases in AML patients <i>ex vivo</i>, viscumTT shows synergistic apoptosis induction.</p