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

Delebinski CI, Twardziok M, Kleinsimon S, Hoff F, Mulsow K, Rolff J, Jäger S, Eggert A, Seifert G - PLoS ONE (2015)

Bottom Line: Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatric Oncology/Haematology, Otto Heubner Centre for Paediatric and Adolescent Medicine (OHC), Charité -Universitaetsmedizin, Berlin, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Triterpene acid- and lectin-containing extracts induce apoptosis in leukaemia cells.A. To measure the induction of apoptosis by VAE, U937 and HL-60 cells were treated with different concentrations of the extracts for 18 h. Apoptosis was determined with Annexin V-APC and propidium iodide by flow cytometry. Values are given as percentages of Annexin V-positive/PI-negative cells (± SD, n = 3). The results indicate a dose-dependent induction of apoptosis. Webb´s fractional product was used to calculate the additive, antagonistic or synergistic effect of the combination viscumTT. A single asterisk indicates Fp>1 and reveals a synergistic effect of viscumTT when compared to the single agents TT and viscum. Fp = fractional product. To confirm apoptosis induction after VAE treatment, whole cell lysates of VAE-treated HL-60 cells were separated by SDS-PAGE followed by Western blotting and decrease of procaspase-3 and cleavage of PARP was detected. Equal loading was verified using ß-actin as internal protein control. The blots are representative of three independent experiments. B. VAEs induce mitochondrial membrane permeability. U937 and HL-60 cells were incubated with TT, viscum or viscumTT for 18 h. After incubation, the mitochondrial permeability transition was measured by JC-1 and flow cytometric analysis on single cell level. Values of mitochondrial permeability transition are given in percentages of cells with low mitochondrial potential (ΔΨm) ±SD, n = 3. A single asterisk indicates a synergistic effect of viscumTT compared to the product of the single agents (*Fp>1). The mitochondrial permeability transition did not change in the negative control. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used as mitochondrial membrane potential disrupter (positive control). Both cell lines show a dose-dependent loss of mitochondrial membrane potential after VAE treatment indicating the involvement of intrinsic apoptosis induction. ViscumTT shows a synergistic effect. C. VAEs induce cytochrome c release from mitochondria to cytosol. Cytochrome c release was analyzed after 18 h of VAE incubation. Western blots display a dose-dependent release of cytochrome c from mitochondria to cytosol after treatment with TT, viscum or viscumTT. The adjusted density value of cytochrome c was quantified by Quantity One software (Density Ctrl = 1; n = 3). C = cytosol.
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pone.0133892.g002: Triterpene acid- and lectin-containing extracts induce apoptosis in leukaemia cells.A. To measure the induction of apoptosis by VAE, U937 and HL-60 cells were treated with different concentrations of the extracts for 18 h. Apoptosis was determined with Annexin V-APC and propidium iodide by flow cytometry. Values are given as percentages of Annexin V-positive/PI-negative cells (± SD, n = 3). The results indicate a dose-dependent induction of apoptosis. Webb´s fractional product was used to calculate the additive, antagonistic or synergistic effect of the combination viscumTT. A single asterisk indicates Fp>1 and reveals a synergistic effect of viscumTT when compared to the single agents TT and viscum. Fp = fractional product. To confirm apoptosis induction after VAE treatment, whole cell lysates of VAE-treated HL-60 cells were separated by SDS-PAGE followed by Western blotting and decrease of procaspase-3 and cleavage of PARP was detected. Equal loading was verified using ß-actin as internal protein control. The blots are representative of three independent experiments. B. VAEs induce mitochondrial membrane permeability. U937 and HL-60 cells were incubated with TT, viscum or viscumTT for 18 h. After incubation, the mitochondrial permeability transition was measured by JC-1 and flow cytometric analysis on single cell level. Values of mitochondrial permeability transition are given in percentages of cells with low mitochondrial potential (ΔΨm) ±SD, n = 3. A single asterisk indicates a synergistic effect of viscumTT compared to the product of the single agents (*Fp>1). The mitochondrial permeability transition did not change in the negative control. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used as mitochondrial membrane potential disrupter (positive control). Both cell lines show a dose-dependent loss of mitochondrial membrane potential after VAE treatment indicating the involvement of intrinsic apoptosis induction. ViscumTT shows a synergistic effect. C. VAEs induce cytochrome c release from mitochondria to cytosol. Cytochrome c release was analyzed after 18 h of VAE incubation. Western blots display a dose-dependent release of cytochrome c from mitochondria to cytosol after treatment with TT, viscum or viscumTT. The adjusted density value of cytochrome c was quantified by Quantity One software (Density Ctrl = 1; n = 3). C = cytosol.

Mentions: To investigate whether apoptosis contributes to the anti-proliferative effect, the induction of apoptosis by viscum, TT or viscumTT in U937 and HL-60 cells was examined by annexin V-APC/PI and FACS analyses. Viscum, TT and viscumTT induced apoptosis in both cell lines dose-dependently (Fig 2A). Additionally, the incubation with the combination viscumTT induced apoptosis in a synergistic manner in both cell lines when compared to the single extracts viscum and TT. The synergism was calculated by Webb´s fractional product (*Fp>1).


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

Delebinski CI, Twardziok M, Kleinsimon S, Hoff F, Mulsow K, Rolff J, Jäger S, Eggert A, Seifert G - PLoS ONE (2015)

Triterpene acid- and lectin-containing extracts induce apoptosis in leukaemia cells.A. To measure the induction of apoptosis by VAE, U937 and HL-60 cells were treated with different concentrations of the extracts for 18 h. Apoptosis was determined with Annexin V-APC and propidium iodide by flow cytometry. Values are given as percentages of Annexin V-positive/PI-negative cells (± SD, n = 3). The results indicate a dose-dependent induction of apoptosis. Webb´s fractional product was used to calculate the additive, antagonistic or synergistic effect of the combination viscumTT. A single asterisk indicates Fp>1 and reveals a synergistic effect of viscumTT when compared to the single agents TT and viscum. Fp = fractional product. To confirm apoptosis induction after VAE treatment, whole cell lysates of VAE-treated HL-60 cells were separated by SDS-PAGE followed by Western blotting and decrease of procaspase-3 and cleavage of PARP was detected. Equal loading was verified using ß-actin as internal protein control. The blots are representative of three independent experiments. B. VAEs induce mitochondrial membrane permeability. U937 and HL-60 cells were incubated with TT, viscum or viscumTT for 18 h. After incubation, the mitochondrial permeability transition was measured by JC-1 and flow cytometric analysis on single cell level. Values of mitochondrial permeability transition are given in percentages of cells with low mitochondrial potential (ΔΨm) ±SD, n = 3. A single asterisk indicates a synergistic effect of viscumTT compared to the product of the single agents (*Fp>1). The mitochondrial permeability transition did not change in the negative control. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used as mitochondrial membrane potential disrupter (positive control). Both cell lines show a dose-dependent loss of mitochondrial membrane potential after VAE treatment indicating the involvement of intrinsic apoptosis induction. ViscumTT shows a synergistic effect. C. VAEs induce cytochrome c release from mitochondria to cytosol. Cytochrome c release was analyzed after 18 h of VAE incubation. Western blots display a dose-dependent release of cytochrome c from mitochondria to cytosol after treatment with TT, viscum or viscumTT. The adjusted density value of cytochrome c was quantified by Quantity One software (Density Ctrl = 1; n = 3). C = cytosol.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526680&req=5

pone.0133892.g002: Triterpene acid- and lectin-containing extracts induce apoptosis in leukaemia cells.A. To measure the induction of apoptosis by VAE, U937 and HL-60 cells were treated with different concentrations of the extracts for 18 h. Apoptosis was determined with Annexin V-APC and propidium iodide by flow cytometry. Values are given as percentages of Annexin V-positive/PI-negative cells (± SD, n = 3). The results indicate a dose-dependent induction of apoptosis. Webb´s fractional product was used to calculate the additive, antagonistic or synergistic effect of the combination viscumTT. A single asterisk indicates Fp>1 and reveals a synergistic effect of viscumTT when compared to the single agents TT and viscum. Fp = fractional product. To confirm apoptosis induction after VAE treatment, whole cell lysates of VAE-treated HL-60 cells were separated by SDS-PAGE followed by Western blotting and decrease of procaspase-3 and cleavage of PARP was detected. Equal loading was verified using ß-actin as internal protein control. The blots are representative of three independent experiments. B. VAEs induce mitochondrial membrane permeability. U937 and HL-60 cells were incubated with TT, viscum or viscumTT for 18 h. After incubation, the mitochondrial permeability transition was measured by JC-1 and flow cytometric analysis on single cell level. Values of mitochondrial permeability transition are given in percentages of cells with low mitochondrial potential (ΔΨm) ±SD, n = 3. A single asterisk indicates a synergistic effect of viscumTT compared to the product of the single agents (*Fp>1). The mitochondrial permeability transition did not change in the negative control. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used as mitochondrial membrane potential disrupter (positive control). Both cell lines show a dose-dependent loss of mitochondrial membrane potential after VAE treatment indicating the involvement of intrinsic apoptosis induction. ViscumTT shows a synergistic effect. C. VAEs induce cytochrome c release from mitochondria to cytosol. Cytochrome c release was analyzed after 18 h of VAE incubation. Western blots display a dose-dependent release of cytochrome c from mitochondria to cytosol after treatment with TT, viscum or viscumTT. The adjusted density value of cytochrome c was quantified by Quantity One software (Density Ctrl = 1; n = 3). C = cytosol.
Mentions: To investigate whether apoptosis contributes to the anti-proliferative effect, the induction of apoptosis by viscum, TT or viscumTT in U937 and HL-60 cells was examined by annexin V-APC/PI and FACS analyses. Viscum, TT and viscumTT induced apoptosis in both cell lines dose-dependently (Fig 2A). Additionally, the incubation with the combination viscumTT induced apoptosis in a synergistic manner in both cell lines when compared to the single extracts viscum and TT. The synergism was calculated by Webb´s fractional product (*Fp>1).

Bottom Line: Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatric Oncology/Haematology, Otto Heubner Centre for Paediatric and Adolescent Medicine (OHC), Charité -Universitaetsmedizin, Berlin, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus