Katarina Hostanska

Hyperforin a constituent of St John's wort (Hypericum perforatum L.) extract induces apoptosis by triggering activation of caspases and with hypericin synergistically exerts cytotoxicity towards human malignant cell lines

Hyperforin (HP) is an abundant component of St Johns wort with antibiotic and antidepressive activity. We report here the ability of HP and that of polyphenolic procyanidin B2 (PB-2) to inhibit the growth of leukemia K562 and U937 cells, brain glioblastoma cells LN229 and normal human astrocytes. HP inhibited the growth of cells in vitro with GI(50) values between 14.9 and 19.9 microM. The growth inhibitory effect of PB-2 was more pronounced in leukemia cell lines K562 and U937, the GI(50) concentrations being about 12.5 microM established after 48 h incubation differed significantly (P<0.05) from those of LN229 and normal human astrocytes (103.1 and 96.7 microM), respectively. Further, HP and hypericin (HY) (a naphthodianthrone from St Johns wort) acted synergistically in their inhibitory effect on leukemic (K562, U937) cell growth. Cell death occurred after 24 h treatment with HP and PB-2 by apoptosis. A dose-dependent loss of membrane phospholipid asymmetry associated with apoptosis was induced in all cell lines as evidenced by the externalization of phosphatidylserine (PS) and morphological changes in cell size and granulosity by scatter characteristics. In leukemia U937 cells, HP increased the activity of caspase-9 and caspase-3 and in K562 cells caspase-8 and caspase-3. In addition, the broad spectrum caspase inhibitor z-VAD-fmk inhibited both the appearance of PS exposure and the activation of caspases, illustrating the functional relevance of caspase activation during HP-induced apoptosis. Cytocidal effects of HP and its cooperation with HY on tumor growth inhibition in a synergistic manner make the St Johns wort an interesting option in cancer warranting further in vitro and in vivo investigation.

Cimicifuga racemosa Extract Inhibits Proliferation of Estrogen Receptor-positive and Negative Human Breast Carcinoma Cell Lines by Induction of Apoptosis

Hormone replacement therapy is contraindicated in women with breast cancer. Extracts from the rhizomes of Cimicifuga racemosa, have gained acceptance as a natural alternative for the treatment of menopausal symptoms. In the present study we investigated the antiproliferative activity of C. racemosa extracts (isopropanolic and ethanolic) on the estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB231 breast cancer cells by WST-1 assay. Down regulation of the proliferative activity and cell killing by isopropanolic and ethanolic extracts occurred in a clear dose-dependent response with a 50% growth inhibitory concentration of 54.1 ± 11.4 and 80.6 ± 17.7 µg/ml in MCF-7 cells and of 29.5 ± 3.0 and 58.6 ± 12.6 µg/ml in MDA-MB231 cells, respectively. Further, the mode of cell death was identified as apoptosis by microscopic inspection and confirmed by light scatter characteristics and by detection of Annexin V adherence to phosphatidylserine by flow cytometry. In addition, the involvement of activated caspases was supported by the cleavage of cytokeratin 18 detected with M30 antibody. Increases in the level of M30-antigen of about 4-fold and 2-fold over untreated controls were observed in C. racemosa-treated MCF-7 and MDA-MB231 cells. These results indicate that C. racemosa extract exerts no proliferative activity, but kills the estrogen receptor positive MCF-7 as well as estrogen receptor negative MDA-MB231 cells by activation of caspases and induction of apoptosis.

Mechanisms Involved in Spontaneous and Viscum album Agglutinin-I-Induced Human Neutrophil Apoptosis: Viscum album Agglutinin-I Accelerates the Loss of Antiapoptotic Mcl-1 Expression and the Degradation of Cytoskeletal Paxillin and Vimentin Proteins Via Caspases

Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodulatory activities. We have recently found that VAA-I is a potent inducer of human neutrophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H2O2, fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91phox, indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin A, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compared with neutrophils that undergo spontaneous apoptosis, but not vinculin or α- and β-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and that its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.

Effect of a Recombinant Lectin, Viscum album Agglutinin on the Secretion of Interleukin-12 in Cultured Human Peripheral Blood Mononuclear Cells and on NK-Cell-Mediated Cytotoxicity of Rat Splenocytes in vitro and in vivo

A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.

Efficacy and safety of mistletoe preparations (Viscum album) for patients with cancer diseases. A systematic review.

Background: Mistletoe is often used as a complementary approach in oncology. Despite experimental anti-tumour effects and several reviews there remains controversy about its clinical role. Patients and Methods: Potentially relevant trials were identified to perform a systematic review (databases: e.g. EMBASE, MEDLINE; hand search: e.g. bibliographies; search terms: e.g. mistletoe). To be included, randomised or comparative clinical trials at least had to examine mistletoe preparations standardized according to manufacturing process and to describe interventions explicitly. Additionally, cohort studies were included for reasons of external validity. Results were summarised in tables. Results: 18 clinical trials (>6,800 participants) were included. Their internal quality was mostly low. Due to heterogeneity between trials a meta-analysis was impossible. Regarding efficacy, findings were inconsistent regarding life expectancy, relation to tumour entity, dosing and treatment duration. Yet, studies indicate that quality of life (QoL) is improved. As these findings do not seem to be limited to one of the different parenteral mistletoe preparations reviewed the treatment may be summarised under the umbrella term ‘mistletoe therapy’. Regarding safety, 1 serious adverse event (AE) related to mistletoe was described; non-serious AEs were local reactions at injection site. Allergic reactions were rare. Conclusion: Supportive ‘mistletoe therapy’ seems safe and beneficial for QoL in adult patients with solid tumours. But there is an urgent need to confirm its efficacy in patient-centred care in a complex oncological setting. This has to be evaluated systematically in prospective observational trials with validated, multidimensional patient-rated QoL questionnaires and comparisons of different preparations and dosages.

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis

The plant lectin Viscum album agglutinin‐I (VAA‐I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA‐I induces de novo protein synthesis of metabolically 35S‐labeled human neutrophils when used at low concentrations (<100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC‐Annexin‐V/PI labeling) and cytology (Diff‐Quick staining) approaches, we found that VAA‐I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA‐I was also found to reverse the delaying effect of GM‐CSF on neutrophil apoptosis and to inhibit GM‐CSF‐inducedde novo protein synthesis. In contrast to GM‐CSF, VAA‐I does not induce tyrosine phosphorylation by itself and does not alter the GM‐CSF‐induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA‐1077, and z‐VAD‐FMK, only the latter (inhibitor of caspases‐1, ‐3, ‐4, and ‐7) was found to inhibit VAA‐I‐induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 ± 1.3 to 54 ± 3.2% (n =4). Furthermore, we confirm that caspases are involved in VAA‐I‐induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase‐3‐dependent. Such degradation was reversed by the z‐VAD‐FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA‐I is a caspase‐dependent mechanism that does not involve tyrosine phosphorylation events, G‐proteins, PKCs, and PLA2. In addition, we conclude that at least caspase‐3 is involved. Correlation between VAA‐I‐induced neutrophil apoptosis and VAA‐I‐induced inhibition of de novo protein synthesis is discussed.

Oncopharmacological Perspectives of a Plant Lectin (Viscum album Agglutinin-I): Overview of Recent Results from In vitro Experiments and In vivo Animal Models, and Their Possible Relevance for Clinical Applications

An old goal of natural complementary medical therapy has been to aim at a long-term stimulation of natural resistance in order to restrain cancer progression or improve defective immunological conditions without toxic side effects. Mistletoe extracts were applied to a large number of cancer patients because of their modulatory effect on the natural immune system. By carefully removing lectins, an essential group of components, from mistletoe extracts, a significant reduction of their effectiveness on several cellular immune parameters could be observed in vivo (1). That is the reason why, for the last 14 years, biological research of mistletoe extracts has focused on lectins. Meanwhile, the quantitatively dominant lectin, Viscum album agglutinin (VAA)-I has become available in a recombinant form (rVAA). Other constitiuents of plant extracts such as viscotoxins (2,3), poly- and oligosaccharides (4), flavonoids (5,6), chitin-binding mistletoe lectin (7) and arginine have also been investigated in connection with the effects of mistletoe extracts on the host defense. However, little evidence has been found that these substances contribute to the effects of mistletoe in vivo (8).

Immunomodulatory effects of Viscum album agglutinin-I on natural immunity.

In 24 h cultured human peripheral blood mononuclear cells, treated with various (1 µg/ml to 1 ng/ml) concentrations of Viscum album agglutinin-l, quantitative assessment of DNA breaks labelled with terminal deoxynucleotidyl transferase revealed a dose-dependent Viscum album agglutinin-l-induced apoptosis above a lectin concentration of 10 ng/ml. After 24 h incubation of peripheral blood mononuclear cells with non-cytotoxic concentrations of Viscum albumagglutinin- l (10 and 1 ng/ml), messenger (m)RNA expression and secretion of a panel of cytokines were evaluated by reverse polymerase chain reaction and by enzyme-linked immunosorbent assay (ELISA), respectively. The lectin induced expression of interleukin-1α interleukin-1 β, interleukin-6, tumour necrosis factor-α, interferon-γ, granulocyte-monocyte colony stimulating factor and interleukin-10 genes, but no expression of interleukin-2 or interferon-y production could be detected. In addition, cellular components of the natural immune system (such as monocytes and granulocytes) bound Viscum album agglutinin-l molecules to a higher degree than lymphocytes.To establish the modulatory potency of Viscum album agglutinin-l on the natural immunity of human subjects, four randomized, double-blind crossover trials were performed on healthy volunteers. In contrast to the significant lectin-induced increases in number and activity of natural killer cells observed in animal models, in the first and second trial human healthy individuals showed no significant differences between their natural killer responses following an injection of lectin-enriched preparation or saline. Due to considerable intrinsic fluctuation of these parameters, a third and fourth double-blind trial with freshly isolated Viscum album agglutinin-l was performed using a more rapidly detectable parameter, the priming of granulocytes. Here, significant lectin-induced increases were found.

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside‐specific plant lectin, Viscum album agglutinin (VAA‐I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA‐I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA‐I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA‐I induced a significant increase in the cytokine‐dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non‐metastatic breast cancer using fluorescence‐activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence‐conjugated VAA‐I. Co‐incubation with d‐galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin‐3, granulocyte/monocyte colony‐stimulating factor and granulocyte colony‐stimulating factor. VAA‐I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA‐I increased the cytokine‐dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA‐I binds to haematopoietic progenitor cells and has a co‐stimulatory effect on their proliferation.

A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

BackgroundDrugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro.MethodsWe investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments.ResultsNone of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712–3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712–1), which caused 22.1% wound closure.ConclusionResults of this study showed that the low potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.