ViscumTT induces apoptosis and alters IAP expression in osteosarcoma in vitro and has synergistic action when combined with different chemotherapeutic drugs.

BACKGROUND: Osteosarcoma is the most common bone tumor and is associated with a poor prognosis. Conventional therapies, surgery and chemotherapy, are still the standard but soon reach their limits. New therapeutic approaches are therefore needed. Conventional aqueous mistletoe extracts from the European mistletoe (Viscum album L.) are used in complementary cancer treatment. These commercial extracts are water-based and do not include water-insoluble compounds such as triterpenic acids. However, both hydrophilic and hydrophobic triterpenic acids possess anti-cancer properties. In this study, a whole mistletoe extract viscumTT re-created by combining an aqueous extract (viscum) and a triterpene extract (TT) was tested for its anti-cancer potential in osteosarcoma. METHODS: Two osteosarcoma cell lines were treated with three different mistletoe extracts viscum, TT and viscumTT to compare their apoptotic potential. For this purpose, annexin/PI staining and caspase-3, -8 and -9 activity were investigated by flow cytometry. To determine the mechanism of action, alterations in expression of inhibitors of apoptosis (IAPs) were detected by western blot. Apoptosis induction by co-treatment of viscum, TT and viscumTT with doxorubicin, etoposide and ifosfamide was examined by flow cytometry. RESULTS: In vitro as well as ex vivo, the whole mistletoe extract viscumTT led to strong inhibition of proliferation and synergistic apoptosis induction in osteosarcoma cells. In the investigations of mechanism of action, inhibitors of apoptosis such as XIAP, BIRC5 and CLSPN showed a clear down-regulation after viscumTT treatment. In addition, co-treatment with doxorubicin, etoposide and ifosfamide further enhanced apoptosis induction, also synergistically. CONCLUSION: ViscumTT treatment results in synergistic apoptosis induction in osteosarcoma cells in vitro and ex vivo. Additionally, conventional standard chemotherapeutic drugs such as doxorubicin, etoposide and ifosfamide were able to dramatically enhance apoptosis induction. These results promise a high potential of viscumTT as an additional adjuvant therapy approach for osteosarcoma.

GADD45A and CDKN1A are involved in apoptosis and cell cycle modulatory effects of viscumTT with further inactivation of the STAT3 pathway.

ViscumTT, a whole mistletoe preparation, has shown synergistic induction of apoptosis in several pediatric tumor entities. High therapeutic potential has previously been observed in Ewing's sarcoma, rhabdomyosarcoma, ALL and AML. In this study, we analyzed modulatory effects on the cell cycle by viscumTT in three osteosarcoma cell lines with various TP53 statuses. ViscumTT treatment induced G1 arrest in TP53 wild-type and null-mutant cells, but S arrest in TP53 mutant cells. Blockage of G1/S transition was accompanied by down-regulation of the key regulators CDK4, CCND1, CDK2, CCNE, CCNA. However, investigations on the transcriptional level revealed secondary TP53 participation. Cell cycle arrest was predominantly mediated by transcriptionally increased expression of GADD45A and CDKN1A and decreased SKP2 levels. Enhanced CDKN1A and GADD45A expression further played a role in viscumTT-induced apoptosis with involvement of stress-induced MAPK8 and inactivation of MAPK1/3. Furthermore, viscumTT inhibited the pro-survival pathway STAT3 by dephosphorylation of the two sites, Tyr705 and Ser727, by down-regulation of total STAT3 and its direct downstream targets BIRC5 and C-MYC. Moreover, tests of the efficacy of viscumTT in vivo showing reduction of tumor volume confirmed the high therapeutic potential as an anti-tumoral agent for osteosarcoma.

Synergistic Antitumour Properties of viscumTT in Alveolar Rhabdomyosarcoma.

Aqueous mistletoe extracts from the European mistletoe (Viscum album) contain mainly mistletoe lectins and viscotoxins as cytotoxic compounds. Lipophilic triterpene acids, which do not occur in conventional mistletoe preparations, were solubilised with ß-cyclodextrins. The combination of an aqueous extract (viscum) and a triterpene-containing extract (TT) recreated a whole mistletoe extract (viscumTT). These extracts were tested on rhabdomyosarcoma in vitro, ex vivo, and in vivo with regard to anticancer effects. Viscum and viscumTT inhibited cell proliferation and induced apoptosis effectively in a dose-dependent manner in vitro and ex vivo, whereas TT showed only moderate inhibitory effects. viscumTT proved to be more effective than the single extracts and displayed a synergistic effect in vitro and a stronger effect in vivo. viscumTT induced apoptosis via the extrinsic and intrinsic pathways, evidenced by the loss of mitochondrial membrane potential and activation of CASP8 and CASP9. CASP10 inhibitor inhibited apoptosis effectively, emphasising the importance of CASP10 in viscumTT-induced apoptosis. Additionally, viscumTT changed the ratio of apoptosis-associated proteins by downregulation of antiapoptotic proteins such as XIAP and BIRC5, thus shifting the balance towards apoptosis. viscumTT effectively reduced tumour volume in patient-derived xenografts in vivo and may be considered a promising substance for rhabdomyosarcoma therapy.

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.

Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells.

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins ("TT" extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous "viscum" extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20% in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin.

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.

Annexin XIIIb guides raft-dependent and -independent apical traffic in MDCK cells.

Epithelial cells are characterized by a polarized organization of their plasma membrane that is divided into apical and basolateral domains. This architecture is maintained by a highly specific cargo sorting machinery that efficiently delivers components to their respective membrane domains. After TGN exit apical cargo is segregated by at least two distinct sorting mechanisms into lipid raft-dependent or lipid raft-independent apical pathways. Annexin XIIIb had been shown to be a member of the lipid raft-dependent trafficking machinery. We now identify this annexin also in raft-independent apical trafficking by mass spectrometry, immunoblotting and confocal microscopy. Annexin XIIIb accumulates in endosomal organelles that are traversed by raft-dependent and raft-independent apical cargo after TGN release. Finally, a specific reduction of annexin XIIIb expression by RNA interference results in a significant decrease in the apical delivery of raft as well as non-raft apical markers. Taken together, our data suggest that annexin XIIIb plays a general role in post Golgi apical trafficking early after TGN release, before the two apical pathways are segregated.