ABSTRACT
INTRODUCTION: Cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) belong to the group of keratinocyte carcinomas (KC). Actinic keratosis (AK) is a precursor lesion of cSCC. The incidences of cSCC, BCC, and AK are currently strongly increasing. Different standard therapies exist for these conditions but are not always applicable or successful. Hydrophilic Viscum album extracts have been used in anthroposophic cancer therapy since 1917. Viscum album lipophilic extract (VALE) is prepared by means of supercritical CO2 extraction. This retrospective case series assessed the safety and clinical effects of a topical application of 10% VALE in individual cases of cSCC, BCC, and AK. METHODS: For this retrospective case series, a positive vote was obtained from the Ethics Committee of the University of Witten/Herdecke (No. 146/2020). Eligible patients signed a declaration of consent prior to inclusion in the study. The main outcome parameters were the clinical response to treatment with VALE and adverse drug reactions. Risk factors, concomitant therapies and diseases, further diagnostic and therapeutic information were documented where available. Data analysis was performed on the level of patients and of individual lesions. RESULTS: The study population consisted of 55 patients with 74 skin lesions. Individual case analysis accompanied by photographic documentation revealed typical and promising treatment courses. Clinical response rates (complete + partial remissions) for individual lesions were 78% for cSCC, 70% for BCC, and 71% for AK. Complete remission rates for individual lesions were 56% for cSCC, 35% for BCC, and 15% for AK. In cSCC and BCC, shorter times to best clinical response were observed. Adverse drug reactions were reported in 5 patients including erythema and inflammatory reactions of mostly moderate severity that resolved completely. In one case, therapy was temporarily paused, in four cases it was continued without interruption. DISCUSSION/CONCLUSION: The results of this study suggest that VALE is a safe and tolerable extract under whose application complete and partial remissions of KC could be observed. To improve and assess the efficacy of VALE, prospective investigations are necessary.
Subject(s)
Carcinoma, Basal Cell , Carcinoma, Squamous Cell , Keratosis, Actinic , Plant Extracts , Skin Neoplasms , Viscum album , Humans , Retrospective Studies , Plant Extracts/therapeutic use , Carcinoma, Basal Cell/drug therapy , Male , Skin Neoplasms/drug therapy , Viscum album/chemistry , Female , Carcinoma, Squamous Cell/drug therapy , Aged , Keratosis, Actinic/drug therapy , Middle Aged , Aged, 80 and overABSTRACT
Mistletoe (Viscum album L.) is a hemiparasitic plant that absorbs water and nutrients from the host tree. Mistletoe contains two groups of cytotoxic, immunomodulatory and antitumor proteins, viscotoxins and lectins. This study evaluated the quantity and quality of viscotoxins and total lectins in the stems with leaves (foliage) and fruit of mistletoe on Parrotia persica and Carpinus betulus in September with immature green berries and in December with mature white berries. Viscum album L. plants were harvested from host species located in the Hyrcanian forests of Iran in 2019. The highest level of viscotoxins was detected in the December foliage of V. album settled on C. betulus (9.25 mg/g dry weight [DW]), and the highest content of lectins was found in the December foliage of V. album settled on P. persica (0.79 mg/g DW) and C. betulus (0.73 mg/g DW) respectively. The immature green berries of V. album from both host species contained much higher concentrations of viscotoxins and lectins than the mature white berries. Four isoforms of viscotoxins, viscotoxin A1, A2, A3 and B could be identified in all samples of both host species. Viscotoxin A3 was the predominant viscotoxin isoform followed by viscotoxin A1.
Subject(s)
Mistletoe , Toxins, Biological , Viscum album , Forests , Fruit , Lectins , Plant Preparations , Plant Proteins , Protein Isoforms , Ribosome Inactivating Proteins, Type 2 , TreesABSTRACT
Viscum album L., commonly known as European mistletoe, is a hemi-parasitic plant of the Santalaceae family. The in vitro and in vivo effects of V. album differ, according to its host tree. However, little is known about the host-dependent phytochemical diversity in V. album. In this study, the metabolic profiles of V. album ssp. album from Malus domestica Bork., Quercus robur L., and Ulmus carpinifolia Gled were compared. Leaves, stems, and berries were collected in Switzerland, by the same procedure, in September 2016 and 2017. The methanolic extracts were analyzed by ultra-performance liquid chromatography, coupled to electrospray quadrupole time-of-flight mass spectrometry in positive ionization mode. The data were submitted to partial-least square discriminant analysis (PLS-DA) and the results showed that the V. album ssp. album samples were clustered into three groups, according to the three distinct host trees. Seven compounds, with high VIP scores (variable importance in projection), were responsible for this differentiation. The following four compounds were detected in both the harvest years: arginine, pipecolic acid or lysine, dimethoxycoumarin, and sinapyl alcohol, suggesting their use as host specific V. album biomarkers. The present work highlights the importance of standardized harvest and analytical procedures for the reproducibility of the chemical results of herbal materials.
ABSTRACT
Fermented aqueous extracts of Viscum album L. are widely used for cancer treatment in complementary medicine. The high molecular weight compounds viscotoxins and lectins are considered to be the main active substances in the extracts. However, a vast number of small molecules (≤1500 Da) is also expected to be present, and few studies have investigated their identities. In this study, a comprehensive metabolome analysis of samples of fermented aqueous extracts of V. album from two host tree species (Malus domestica and Pinus sylvestris), both prepared by two pharmaceutical manufacturing processes, was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). A total of 212 metabolites were putatively annotated, including primary metabolites (e.g., amino acids, organic acids, etc.) and secondary metabolites (mostly phenolic compounds). A clear separation between V. album samples according to the host tree species, but not due to manufacturing processes, was observed by principal component analysis. The biomarkers responsible for this discrimination were assessed by partial least squares-discriminant analysis. Because V. album extracts from different host trees have different clinical applications, the present work highlights the possibility of characterizing the metabolome for identification and traceability of V. album fermented aqueous extracts.
Subject(s)
Fermentation , Metabolome , Metabolomics , Plant Extracts/metabolism , Tandem Mass Spectrometry , Viscum album/metabolism , Chromatography, Liquid , Discriminant Analysis , Least-Squares Analysis , Multivariate Analysis , Principal Component AnalysisABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Mistletoe/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , Viscum album/chemistry , Animals , Apoptosis/drug effects , Cell Line, Tumor , Humans , In Vitro TechniquesABSTRACT
Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies.
Subject(s)
Immune Tolerance/drug effects , Plant Extracts/pharmacology , Viscum album/chemistry , Adult , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Dendritic Cells/drug effects , Dendritic Cells/immunology , Humans , Interferon-gamma/metabolism , Lectins/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Cytokines/metabolism , Macrophages/drug effects , Monocytes/drug effects , Neoplasms/metabolism , Oleanolic Acid/pharmacology , Plant Extracts/pharmacology , Viscum album/chemistry , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line, Tumor , Chemokine CCL2/metabolism , Flow Cytometry , Humans , Interleukin-13/metabolism , Interleukin-4/metabolism , Macrophage Colony-Stimulating Factor/metabolism , PhenotypeABSTRACT
Extracts from European mistletoe (Viscum album L.) developed in anthroposophic medicine are based on specific pharmaceutical procedures to enhance remedy efficacy. One such anthroposophic pharmaceutical process was evaluated regarding effects on cancer cell toxicity in vitro and on colchicine tumor formation in Lepidium sativum. Anthroposophically processed Viscum album extract (APVAE) was produced by mixing winter and summer mistletoe extracts in the edge of a high-speed rotating disk and was compared with manually mixed Viscum album extract (VAE). The antiproliferative effect of VAE/APVAE was determined in five cell lines (NCI-H460, DU-145, HCC1143, MV3, and PA-TU-8902) by WST-1 assay in vitro; no difference was found between VAE and APVAE in any cell line tested (P > 0.14). Incidence of colchicine tumor formation was assessed by measurement of the root/shoot-ratio of seedlings of Lepidium sativum treated with colchicine as well as VAE, APVAE, or water. Colchicine tumor formation decreased after application of VAE (-5.4% compared to water, P < 0.001) and was even stronger by APVAE (-8.8% compared to water, P < 0.001). The high-speed mistletoe extract mixing process investigated thus did not influence toxicity against cancer cells but seemed to sustain morphostasis and to enhance resistance against external noxious influences leading to phenomenological malformations.
ABSTRACT
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.
Subject(s)
Herb-Drug Interactions , Neoplasms/drug therapy , Neoplasms/pathology , Plant Extracts/pharmacology , Plant Extracts/standards , Viscum album/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Cytostatic Agents/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Docetaxel , Doxorubicin/pharmacology , Humans , Male , Mitoxantrone/pharmacology , Plants, Medicinal/chemistry , Taxoids/pharmacology , GemcitabineABSTRACT
BACKGROUND: Preparations of mistletoe (Viscum album) are the form of cancer treatment that is most frequently used in the complementary medicine. Previous work has shown that these preparations are able to exert cytotoxic effects on carcinoma cells, the extent of which might be influenced by the host tree species and by the content of mistletoe lectin. METHODS: Using colorimetric assays, we have now compared the cytotoxic effects of Viscum album preparations (VAPs) obtained from mistletoe growing on oak (Quercus robur and Q. petraea, VAP-Qu), apple tree (Malus domestica,, VAP-M), pine (Pinus sylvestris, VAP-P) or white fir (Abies pectinata, VAP-A), on the in vitro growth of breast and bladder carcinoma cell lines. While MFM-223, KPL-1, MCF-7 and HCC-1937 were the breast carcinoma cell lines chosen, the panel of tested bladder carcinoma cells comprised the T-24, TCC-SUP, UM-UC-3 and J-82 cell lines. RESULTS: Each of the VAPs inhibited cell growth, but the extent of this inhibition differed with the preparation and with the cell line. The concentrations of VAP-Qu, VAP-M and VAP-A which led to a 50 % reduction of cell growth (IC50) varied between 0.6 and 0.03 mg/ml. Higher concentrations of VAP-P were required to obtain a comparable effect. Purified mistletoe lectin I (MLI) led to an inhibition of breast carcinoma cell growth at concentrations lower than those of VAPs, but the sensitivity towards purified MLI did not parallel that towards VAPs. Bladder carcinoma cells were in most cases more sensitive to VAPs treatment than breast carcinoma cells. The total mistletoe lectin content was very high in VAP-Qu (54 ng/mg extract), intermediate in VAP-M (25 ng/mg extract), and very low in VAP-P (1.3 ng/mg extract) and in VAP-A (1 ng/mg extract). As to be expected from the low content of mistletoe lectin, VAP-P led to relatively weak cytotoxic effects. Most remarkably, however, the lectin-poor VAP-A revealed a cytotoxic effect comparable to, or even stronger than, that of the lectin-rich VAP-Qu, on all tested bladder and breast carcinoma cell lines. CONCLUSION: The results suggest the existence of cytotoxic components other than mistletoe lectin in VAP-A and reveal an unexpected potential of this preparation for the treatment of breast and bladder cancer.
Subject(s)
Abies/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Urinary Bladder Neoplasms/drug therapy , Viscum album/chemistry , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Down-Regulation/drug effects , HumansABSTRACT
BACKGROUND: The aim of the study was to evaluate the antiproliferative potency of Viscum album extract (VA-E) in human bladder carcinoma cell lines with regard to its possible use for intravesical therapy of superficial bladder cancer. MATERIALS AND METHODS: Proliferation (MTT-test or 3H-thymidine incorporation), necrotic disintegration (3H-thymidine release of prelabelled cells) and portions of apoptotic and/or necrotic cells (Annexin-V binding, propidium iodide (PI) labelling and DNA-fluorescence profiles by flow cytometry) were measured in four different human bladder carcinoma cell lines (T24, TCCSUP, J82 and UM-UC3) cultured in vitro. RESULTS: Antiproliferative effects of VA-E were observed in the four bladder carcinoma cell lines tested. Metabolic activity could also be completely abrogated by short-time contact of the cells with VA-E. Apoptosis and necrosis, as underlying mechanisms of action, were differentially expressed by the different cell lines. CONCLUSION: VA-E and cytotoxic proteins, i.e., mistletoe lectins (ML) and viscotoxins (VT), were able to block the growth of bladder carcinoma cells. Together with the immunomodulating properties of VA-E, the observed antiproliferative potency might give a rationale for the topical intravesical application of VA-E for the treatment of superficial bladder cancer.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Transitional Cell/drug therapy , Plant Extracts/pharmacology , Urinary Bladder Neoplasms/drug therapy , Viscum album/chemistry , Administration, Intravesical , Carcinoma, Transitional Cell/pathology , Cell Growth Processes/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Jurkat Cells , Urinary Bladder Neoplasms/pathologyABSTRACT
The purpose of the following study was to evaluate the presence of the most frequently investigated pharmacologically active mistletoe compounds, viscotoxins (VT) and mistletoe lectins (ML), in European mistletoe Viscum album L. and in the pharmaceutical mistletoe preparations Iscador. Quantitative analysis of the VT isoforms A1, A2, A3, B, 1-PS, and U-PS in fresh mistletoe plant material from the three European subspecies of V. album, during fermentative extraction of mistletoe and in Iscador showed that the pharmaceutical proceeding specific for the preparation of Iscador warrants a high yield of VT. No degradation or transformation of VT during the production process became apparent. The VT compositions of the three host specific European subspecies of V. album, ssp. album, ssp. abietis, and ssp. austriacum, showed characteristic differences. They ensured the identification of the subspecies specific types of Iscador. ML contents of mistletoe extracts were reduced during fermentative extraction. The quantified contents of total ML were 261 +/- 9.3 ng/ml in Iscador M 5 mg spec. and 391 +/- 18.3 ng/ml in Iscador Qu 5 mg spec. Binding of ML to the glycoprotein asialofetuin (type 1) was found to be temperature dependent. Binding activity was increased to 250 % (ML I) and 410 % (ML II and ML III) respectively by decreasing temperature from 30 degrees C to 4 degrees C. 95% of ML could be eliminated from Iscador by affinity chromatography with immobilised glycoproteins at 0 degrees C. Quantitative extraction of ML from the crude extract and their analysis by SDS-PAGE revealed the presence of about 30 % ML I, 20% ML II, and 50% ML III in Iscador M 5 mg spec. and Iscador Qu 5 mg spec. The annual course of concentrations of ML and VT in the leaves of V. album showed maximal ML contents in December and culminaton of VT in June. Seasonal fluctuations of the composition of mistletoe imply the importance of fixed harvesting seasons.
Subject(s)
Plant Extracts/chemistry , Plant Proteins/chemistry , Viscum/chemistry , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Fermentation , Indicators and Reagents , Isomerism , Lectins/chemistry , Seasons , Viscum/growth & developmentABSTRACT
The subcutaneous application of lectin-rich mistletoe preparations such as Iscador Quercus (IQ; Weleda Company, Schwäbisch Gmünd, Germany) results in a peripheral eosinophilia. Our goal was to investigate whether this effect is related to mistletoe lectin (ML) and whether it is caused by a response of the specific immune system. In a double-blinded study, 43 volunteers were randomized to one of four treatment groups: (1) IQ, (2) ML that was derived from IQ, (3) IQ that was depleted of ML, and (4) placebo. The respective preparations were applied subcutaneously twice per week for 8 weeks, in increasing doses. Weekly the differential blood count was analyzed. Every 4 weeks interferon-gamma, interleukin-5 (IL-5), and granulocyte-macrophage colony stimulating factor (GM-CSF) were determined in cultures from peripheral mononuclear cells after stimulation with IQ. IQ and ML resulted in significant eosinophilia compared with placebo and ML-depleted IQ. Furthermore, the leukocyte and granulocyte counts were increased in the IQ and ML groups compared with placebo. GM-CSF, interferon-gamma, and IL-5 increased after ex vivo in vitro stimulation with IQ in the IQ and ML groups, and were significantly different from placebo in the IQ group but not in the ML group. Eosinophilia during therapy with mistletoe preparations is due to its content of ML. This effect might be related to a stimulation of IL-5 and/or GM-CSF, which was demonstrated ex vivo in vitro. ML resulted in a temporary increase of the granulocyte count, which is probably related to an acute-phase reaction.
Subject(s)
Eosinophils/drug effects , Leukocytes/drug effects , Plant Preparations/pharmacology , Plant Proteins/pharmacology , Toxins, Biological/pharmacology , Adult , Double-Blind Method , Eosinophilia/chemically induced , Eosinophilia/immunology , Eosinophils/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Humans , Interferon-gamma/blood , Interleukin-5/blood , Leukocytes/immunology , Lymphocyte Activation , Male , Prospective Studies , Ribosome Inactivating Proteins , Ribosome Inactivating Proteins, Type 2ABSTRACT
The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.
Subject(s)
Plant Preparations/chemistry , Plant Preparations/pharmacology , Plant Proteins , Toxins, Biological/chemistry , Toxins, Biological/pharmacology , Viscum album/chemistry , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Ribosome Inactivating Proteins, Type 2 , Static Electricity , Structure-Activity RelationshipABSTRACT
Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic alpha-helices, through which viscotoxins are supposed to interact with plasma membranes.
Subject(s)
Liposomes , Mistletoe , Plant Preparations/chemistry , Plant Preparations/pharmacology , Plant Proteins , Toxins, Biological/chemistry , Toxins, Biological/pharmacology , Amino Acid Sequence , Binding Sites , Calorimetry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/pharmacology , Protein Structure, Secondary , Ribosome Inactivating Proteins, Type 2 , Spectroscopy, Fourier Transform Infrared , Surface PropertiesABSTRACT
Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles.
