A NEW DEVELOPMENT of Triterpene Acids-CONTAINING Extracts FROM Viscum Album L. Displays Synergistic INDUCTION of Apoptosis In Childhood LEUKEMIA
Authors: Delebinski Catharina I. , Kemnitz-Hassanin Kristin , Jaeger Sebastian , Lode Holger N. , Seeger Karl-Heinz , Henze Guenter , Seifert Georg 
Published: October 2019

Abstract
Abstract 4296
Viscum album L. (mistletoe) is one of the most widely used complementary treatment in cancer therapy. Most investigations of Viscum album L. (VAE) are based on aqueous mistletoe extracts which contain cytotoxic and immunomodulatory proteins such as mistletoe lectins and viscotoxins. Mistletoe triterpene acids are poorly water soluble and therefore missing in mistletoe remedies though they are known to possess anti-cancer properties. Using cyclodextrins it was possible to solubilize the mistletoe triterpene acids, oleanolic acid (OA) and betulinic acid (BA), and to achieve a mistletoe extract with high levels of OA and mistletoe lectin-I, the predominant substances with anti-cancer properties within the mistletoe plant. Our basic aim was to test this novel composition as it occurs naturally in plants as well as the single components in different acute lymphoblastic (ALL, NALM-6) and myeloid leukemia (AML) cell lines (U937, HL60).
The experimental extracts contain either mistletoe lectin-I and viscotoxins (viscum) or solubilized OA and BA (TT) and more interestingly, a combination thereof (viscumTT). In these experiments, we investigated the apoptosis induction in ALL and AML cell lines and furthermore we analyzed the mechanism of apoptosis by caspase inhibitors, caspase activity assays and western blot analyses.
All three cell lines have shown distinct apoptosis induction for viscum, TT and viscumTT. Though, differences between ALL and AML cell lines toward the lectin and triterpene acids sensitivity were observed. NALM-6 cells were more sensitive to lectin-treatment, but less sensitive to triterpene acids than HL60 and U937 cells. Moreover, incubation with caspase-8 and -9 inhibitors only partially prevented apoptosis induction by TT, viscum and viscumTT, whereas the general caspase inhibitor Z-VAD-fmk prevented loss of cell viability induced by all three components. In combination with the caspase activity assay, the results revealed a caspase-8 and -9 mitochondrial dependent pathway for viscum, TT and viscumTT. Interestingly viscumTT, the combination of viscum and TT, acted synergistically compared with the sum of the single agent treatment in all cell lines.
In addition, we investigated whether triterpene-containing extracts can also induce dose-dependent apoptosis in primary patient cells with childhood leukemia ex vivo. Here, we also observed apoptosis induction, via caspase-8 and -9 signaling pathways, for viscum, TT and viscumTT.
Moreover, leukemia bearing mice were treated with Viscum album L. extracts. Recipients receiving PBS had a mean survival time of 38 days whereas viscumTT prolonged the mean survival to 50.5 days. Taken together, we were able to show that this new formulation “viscumTT” of aqueous mistletoe extracts and triterpene acids can induce apoptosis in leukemia cells via the intrinsic and extrinsic signaling pathways. Furthermore, we revealed a synergistic effect for the combination viscumTT compared to the single fractions viscum and TT. Based on these data we believe that Viscum album L. extracts containing triterpene acids may possess impressive therapeutic potential.

Disclosures:
No relevant conflicts of interest to declare.