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

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have the following interests: Jana Rolff is employed by EPO GmbH and Sebastian Jäger by Birken AG. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. ViscumTT inhibits proliferation in Ewing sarcoma cell lines without early cytotoxicity.

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.

Fig 2. ViscumTT combined extract synergistically induces apoptosis in Ewing sarcoma cell lines.

A. 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. 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. C. 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 (ΔΨm, ± 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.

Fig 3. ViscumTT synergistically activates caspases in Ewing sarcoma cells lines above the levels activated by viscum alone.

A. 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. 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.

Fig 4. Viscum, TT and viscumTT alter apoptosis-related protein expression.

A. 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. 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.

Fig 5. ViscumTT and viscum induce apoptosis and inhibit proliferation ex vivo.

Primary Ewing sarcoma cells from a 15-year-old girl were grown as short-term cultures ex vivo, and treated with increasing concentrations of viscum, TT or viscumTT for 24h. Induction of apoptosis (n = 4), proliferation inhibition (n = 2), mitochondrial membrane potential (n = 2) and activation of CASP8 and CASP9 (n = 2) were measured flow cytometrically with the same assays described for the cell lines. Values are the means of n experiments ±SD (error bars). Controls were untreated cultures grown in parallel. A synergistic effect of the combined viscumTT extracts was calculated by Webb´s fractional product (*Fp > 1). Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively.

Fig 6. ViscumTT reduces the growth of human Ewing sarcoma xenograft tumors in mice.

Established Ewing sarcoma xenograft tumors were intratumorally (i.t.) injected with 40/50/60mg oleanolic acid/kg mouse weight (TT i.t.), 0.75/1.25/1.75μg mistletoe lectin /kg mouse weight (viscum i.t.) or a combination thereof (viscumTT i.t.) every 2 to 3 days in increasing concentrations. Each concentration was administered twice within 2–3 days. One treatment group received viscumTT intravenously (i.v.), and one positive control group was once intravenously (i.v.) treated with 4 mg/kg doxorubicin. The negative control group received the solubilizing agent, cyclodextrins (CD), which was injected either intratumorally (CD i.t.) or intravenously (CD i.v.). One-way ANOVA revealed a significant group effect (p = 0.007) and Fisher´s Least Significant Difference test comparing all groups with all groups showed a significant effect between the CD i.t. control group and viscumTT i.t. (p = 0.033), viscum i.t. (p = 0.002) and TT i.t. (p = 0.021) as well as between the CD i.v. control group and viscumTT i.v. (p = 0.014) or the positive control group, doxorubicin i.v. (p = 0.015). Box-and-wisker plots each represent one treatment group including 8 mice (*p

Fig 7. ViscumTT reduces the growth of patient-derived Ewing sarcoma xenograft tumors in mice.

Established Ewing sarcoma xenograft tumors in NMRI-nu/nu mice were intratumorally (i.t.) and intravenously (i.v.) injected with 40/60/80mg/kg oleanolic acid and 1.25/1.75/2.25μg/kg mistletoe lectin in combination (viscumTT, low dose). Additionally, mice were treated intratumorally (i.t.) with 50/70/90mg/kg oleanolic acid and 1.25/1.75/2.25μg/kg mistletoe lectin in combination (viscumTT, high dose). Viscum TT was given every 2–3 days in increasing extract concentrations. One positive control group was treated twice intravenously (i.v.) with 4 mg/kg doxorubicin. The negative control group received cyclodextrins (CD) intratumorally (CD i.t.). One-way ANOVA revealed a strongly significant group effect (p