Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts.

BACKGROUND: Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album "mother tinctures", MT) prepared from different host trees. METHODS: Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD. RESULTS: Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3. CONCLUSION: Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.

BACKGROUND: Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA). METHODS: Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay. RESULTS: In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected. CONCLUSIONS: In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.

Interaction of standardized mistletoe (Viscum album) extracts with chemotherapeutic drugs regarding cytostatic and cytotoxic effects in vitro.

BACKGROUND: Given the importance of complementary and alternative medicine (CAM) to cancer patients, there is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Mistletoe (Viscum album L.) belongs to the medicinal herbs that are used as supportive care during chemotherapy. In the in vitro study presented here the effect of standardized mistletoe preparations on the cytostatic and cytotoxic activity of several common conventional chemotherapeutic drugs was investigated using different cancer cell lines. METHODS: Human breast carcinoma cell lines HCC1937 and HCC1143 were treated with doxorubicin hydrochloride, pancreas adenocarcinoma cell line PA-TU-8902 with gemcitabine hydrochloride, prostate carcinoma cell line DU145 with docetaxel and mitoxantrone hydrochloride and lung carcinoma cell line NCI-H460 was treated with docetaxel and cisplatin. Each dose of the respective chemotherapeutic drug was combined with Viscum album extract (VAE) in clinically relevant concentrations and proliferation and apoptosis were measured. RESULTS: VAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of our experimental settings. At higher concentrations VAE showed an additive inhibitory effect. CONCLUSIONS: Our in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE simultaneously.

In vitro investigation into the potential of a mistletoe extract to alleviate adverse effects of cyclophosphamide.

OBJECTIVES: Mistletoe extracts have been shown to provide deoxyribonucleic acid (DNA)-stabilizing effects in human peripheral blood mononuclear cells (PBMC) in vitro. We investigated the effect of a mistletoe extract on PBMC with and without concomitant treatment with cyclophosphamide and compared mitochondrial activity and replication of normal PBMC with that of a T-cell leukemia cell line. DESIGN: The experiments were performed with PBMC of healthy blood donors and the T-cell leukemia Jurkat cell line. Cells were pre-incubated with mistletoe extract for 60 to 65 hours. 4-hydroperoxycyclophosphamide (4-hpc, precursor of 4-hydroxycyclophosphamide) was added for 2 hours, after which mitochondrial activity and replication were measured. All experiments were randomized and blinded. MAIN OUTCOME MEASURES: Cell mitochondrial activity and replication were assessed with spectrophotometric analysis of WST-1 reduction and BrdU incorporation. RESULTS: The application of 4-hpc consistently reduced mitochondrial activity and replication of PBMC and Jurkat cells. Mistletoe extract strongly enhanced PBMC mitochondrial activity and replication (with or without 4-hpc) and partially inhibited Jurkat cell replication (with 4-hpc only). Compared to mistletoe untreated cells, enhancement ofPBMC mitochondrial activity by mistletoe extract was independent of treatment with 4-hpc, but enhancement of PBMC replication by mistletoe extract was stronger when treated with 4-hpc. CONCLUSIONS: Mistletoe extract strongly stimulated healthy PBMC but not malignant Jurkat cells. In addition, mistletoe extract seemed to partially protect healthy PBMC-but not malignant Jurkat cells-from the cytostatic effect of 4-hpc. The results motivate further preclinical and clinical investigations of mistletoe extracts as an adjuvant medication in cancer therapy to alleviate side effects of conventional therapy.