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Apoptosis-inducing Activity of Helleborus niger in ALL and AML

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Abstract

Helleborus niger is used in the adjuvant treatment of different tumors in anthroposophical medicine. Indications include various types of brain tumors in children, as well as prostate cancer, leukemia and lymphoma. Our aim was to investigate the therapeutic effects of these extracts apart from the traditional use. : We used an aqueous whole plant extract of H. niger in different cancer and leukemia cell lines and primary cells of patients with childhood ALL and AML and identified the main mechanisms of action. A strong inhibition of proliferation is caused by specific apoptosis induction, which is executed via the mitochondrial pathway and caspase-3 processing. Apoptosis could be detected in lymphoma (BJAB), leukemia (Reh, Nalm6, Sup-B15) and melanoma (Mel-HO) cells and overcomes a Bcl-2-mediated block of apoptosis. In primary cells of patients with childhood ALL and AML, which were partly poor responding to doxorubicin and daunorubicin, a strong apoptosis induction was determined. In combination with the vinca alkaloid vincristine, strong synergistic effects were detected in BJAB cells. We demonstrate in vitro efficacy of H. niger extract in cells of hematological malignancies; these studies should encourage in vivo experiments.

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... H. niger preparations possess immunomodulatory properties (Bogdan et al., 1990) and affect the DNA stability of lymphocytes (Büssing and Schweizer, 1998). In addition, cytotoxic and apoptosis-inducing effects on leukaemia cells have been described (Jesse et al., 2009). Still, few publications dealing with the distinct activity of H. niger extracts exist. ...
... Extracts from H. niger are used therapeutically in anthroposophic medicine for the adjuvant treatment of different tumour entities (Jesse et al., 2009). Therefore, the influence of different concentrations of a standardised H. niger extract, registered in Germany as a herbal homeopathic remedy for the proliferation of tumour cells of different origin, was tested (Fig. 2). ...
... The cell growth of MOLT4 (IC 50 : 171 mg/mL) was most sensitive to H. niger treatment, followed by SK-UT-1b (IC 50 : 304 mg/mL) and HT-144 cells (IC 50 : 569 mg/mL). As mentioned above, only Jesse et al. (2009) demonstrated dose-dependent in vitro cytotoxicity of H. niger on a cell line of systemic origin (EBV-negative Burkitt's lymphoma cells; BJAB). This effect was mediated through an apoptosisinducing mechanism, and these results are in line with our experiments demonstrating an anti-proliferative effect of H. niger on the investigated tumour cells, which was mediated due to early and late apoptosis induction. ...
Article
Ethnopharmacological relevance: In Romanian folk medicine, Helleborus niger L. is used for the treatment of rheumatoid arthritis or viral infections and in complementary therapy, especially in anthroposophic medicine (AM), where the plant is administered as an adjuvant to treat malignant diseases. In the present study, we investigated the differential cytotoxic effects of H. niger on human tumour and healthy cells of the human immune system in vitro. Material and methods: Protoanemonin and saponins, as significant constituents of H. niger extracts, were quantified in five individual batches using validated HPLC methods. Further, the impact of H. niger on proliferation capacity (MTT assay) as well as on apoptosis and necrosis induction in a panel of tumour cell lines and human lymphocytes (combined annexin V and propidium iodide staining) was monitored. In addition, NK cell function (degranulation-CD107a assay and IFN-gamma secretion) was also investigated since these immunocompetent cells are important for the control of malignancies within the human body. Results: Extracts of H. niger induced proliferation inhibition not only of lymphoblastic leukaemia cells (MOLT4; IC50: 171 µg/mL) but also of myosarcoma (SK-UT-1b; IC50: 304 µg/mL) and melanoma cells (HT-144; IC50: 569 µg/mL) due to the induction of apoptosis. Purified T cells or NK cells were significantly affected through the presence of high H. niger concentrations while bulk lymphocytes were not affected. NK cells' anti-tumour functions expressed by degranulation capacity as well as IFN-y production were unaffected by the presence of the H. niger extract. Since protoanemonin and saponins have been reported in the literature to exert cytotoxic effects, their content was also determined. Conclusions: H. niger extracts exhibit differential cytotoxicity towards tumour cell lines and healthy human T- and NK-cells.
... Therefore, phytopreparations that have cytotoxic activity could potentially be used as medications for a number of health conditions that primarily feature uncontrolled cell proliferations. Numerous studies point to the bioactive properties of extracts of some Helleborus species (Büssing & Schweizer 1998; Lindholm et al. 2002; Jesse et al. 2009). Extracts of some Helleborus species have immunostimulatory and anti-inflammatory properties (Linke et al. 1998). ...
... possesses different anticancer properties. Also, it has been demonstrated that the aqueous extract of Helleborus niger L. induces apoptosis in various types of tumour cell lines (Jesse et al. 2009). Aqueous extracts of H. niger L. have immunomodulating properties by inducing sister chromatid exchange in lymphocyte cultures of healthy individuals (Büssing & Schweizer 1998 *Corresponding author. ...
... Based on these findings, it can be presumed that treatments with hellebore extracts have both a clastogenic and aneugenic effects, and that an induction of apoptosis is the main mechanism of antiproliferative activity of extracts of the three Helleborus taxa. These findings confirm earlier research that explains the mechanisms of apoptosis induction in five tumour cell lines treated with aqueous extracts of H. niger (Jesse et al. 2009). Further studies demonstrated that apoptosis was the result Natural Product Researchof activation of capsase-3 that is present in mammalian cells, and that it catalyses the degradation of many essential cell enzymes resulting in cell death (Porter & Jänicke 1999). ...
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Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Alliumcepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction.
... The main clinical indications are brain tumors and lymphomas [5]. Furthermore H. niger is related to the renal system because of its diuretic effect and its ability to support elimination of edemas through kidney activation [6]. ...
... Black hellebore has only recently regained scientific interest as a plant exhibiting an interesting multi-component profile [1,2,[4][5][6].With respect to pharmacologically relevant ingredients, four compound classes are in the forefront, i.e. the steroidal saponins, the ranunculin derivatives (including protoanemonin), beta-ecdysone and the flavonoids [4,7,8]. The latter two exert anabolic and cell protective actions [9,10] while saponins possess the ability to disintegrate membrane structures [11][12][13]. ...
... A chemiluminescent cell proliferation assay (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) was performed according to the manufacturer's instructions. HNE was diluted in 1:20, 1:100, 1:500, 1:2500, 1:12,500, 1:62,500 and 1:312,500 according to previous investigations [2,5,6]. Paclitaxel was tested as positive reference in concentrations of 8539.06-0.0085 ...
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Background The therapeutic use of Helleborus niger L. is manifold due to its specific phytochemical composition. Two compound groups, the ranunculin derivates including protoanemonin and the steroidal saponins, are also associated with toxicity (genotoxicity, disintegration of membrane structures). Therefore, in vitro investigations were performed on safety aspects of a Helleborus niger aqueous fermented extract (HNE). In addition its therapeutic potential against various cancer cell lines was assessed to gain insight into the respective mechanisms of action. Methods To evaluate the safe use of HNE, Ames and hemolytic tests were carried out. Two angiogenesis assays in 2D and 3D design were conducted to assess the anti-angiogenetic potential, for which human umbilical vein endothelial cells (HUVEC) were chosen. A panel of tumor cell lines was used in 2D and 3D proliferation assays as well in the migration- and invasion-assay. All investigations were performed with HNE compared to reference substances. The 2D proliferation assay was additionally performed with isolated compounds of HNE (characteristic steroidal saponins). Results HNE did not exhibit any genotoxic potential. Concentrations up to 10 μl/ml were classified as non-hemolytic. HNE exerted anti-angiogenetic effects in HUVEC and anti-proliferative effects in five cancer cell lines, whereas hellebosaponin A and D as well macranthosid I did not show comparable effects neither singly nor in combination. Due to the inherent instability of protoanemonin in isolated form, parallel investigations with protoanemonin could not be performed. HNE (600–1000 μg/ml) inhibited the migration of certain cancer cells by > 80% such as Caki-2, DLD-1 and SK-N-SH. Conclusion HNE exhibit neither genotoxic nor hemolytic potential. The present investigations verify the anti-angiogenetic effects on HUVEC, the anti-proliferative effects and migration-inhibiting properties on tumor cells. The lower effect of the relevant steroidal saponins compared to the whole extract underlines the fact that the latter is more effective than a blend of isolated pharmacologically active components. Electronic supplementary material The online version of this article (10.1186/s12906-019-2517-5) contains supplementary material, which is available to authorized users.
... Moreover, 20-hydroxyecdysone also stimulates CD2 presentation on T-lymphocytes and aids in the function of the immune system. Since Helleborus niger has other active components besides β-ecdysone (20-Hydroxyecdysone), it cannot be concluded that the plant extracts' apoptotic properties are solely the result of ecdysteroids and this requires further studies [30]. ...
... In contrast to their pro-apoptotic effect on leukaemic cells [30], ecdysteroids exert an anti-apoptotic effect on non-cancerous cell lines. In this context, most refer to the phosphatidylinositol-3-kinase/protein kinase B signal transduction (PI3K/Akt) pathway, which is responsible for the anti-apoptotic effects of ecdysteroids [29,31]. ...
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Background: In 2018, leukaemia accounted for 2.6% of all new cancers, it being the 13th most common cause of cancer and the 10th most common cause of cancer death. Glucocorticoids are commonly used in lymphoid leukaemia treatment, where they are cytotoxic. The aim of this review is to highlight ongoing research of steroid use in myeloid leukaemias. Main text: Glucocorticoids increase infection risks in acute myeloid leukaemia, but with adequate antifungal cover, they can help in hyperleucocytic disease. They also show some benefits in sensitising multidrug-resistant AML cell lines to cytotoxic agents, induce differentiation marker expression and can also induce CD38 expression, making AML cells possible targets of daratumumab. Cardiotonic steroids, like digitalis, are being recognised as sensitising AML cells to the chemotherapeutic effects of many cytotoxic agents, primarily by inhibiting efflux pumps, thus minimising AML resistance. Ecdysteroids enhance sensitivity in multidrug-resistant AML, but also in non-resistant AML cell lines, through pathways including the activation of mitochondrial apoptosis. Their anti-apoptotic effects on non-malignant cell lines help their target specificity. Sensitisation is chemotherapy-specific, enhancing the effects of doxorubicin and tubulin inhibitors but increasing resistance to cisplatinum. Short conclusion: Cardiotonic steroids and ecdysteroids both show chemosensitisation to the cytotoxic effects of chemotherapy on AML cell lines. It is likely time to consider clinical trials to assess whether these, as well as traditional glucocorticoids, can contribute to the AML armamentarium, particularly in chemo-resistant disease.
... In recent literature, immunomodulating, antihepatotoxic, antiviral, and antifungal activities were reported for the genus Helleborus [2]. Most interestingly, an H. niger plant extract showed significant cytotoxic activity against leukemia cell lines [4]. This considerable variety of biological activities was attributed to steroidal saponins which occur in the leaves and roots from Helleborus [2,5]. ...
... In contrast, the first eluting polyhydroxy saponins started from 51 min. These compounds (2)(3)(4)(5) are constituted of additional hexoses which contribute to a more polar character (Fig. 1). ...
Article
Steroidal saponins comprise a substantial part of the secondary metabolite spectrum in the medicinal plant Helleborus niger L. (black hellebore). The saponin fraction from the roots was investigated by LC–MSn resulting in 38 saponins and β-ecdysone. Nine diosgenyl-type glycosides, mainly furostanols consisting of the aglycones diosgenin, macranthogenin, sceptrumgenin, and sarsasapogenin were accompanied by 5 diosgenyl-type saponins exhibiting an aglycone with an additional OH group. However, the most relevant compounds were 24 acetylated polyhydroxy saponins including hellebosaponins A and E.The enzymes glucuronidase, β-glucosidase, and pectinase were used to obtain an idea on potential fermentative transformation reactions by incubation of the isolated model saponins macranthosid I and hellebosaponin A. In a second step, aqueous H. niger extracts containing a much greater range of saponins were monitored during fermentation and 12 months of storage. The metabolites were examined and assigned by LC–MSn and targeted extracted ion current (EIC) scan analyses. Good agreement was found among the results from the model compounds and the whole aqueous fermented extracts.The native diosgenyl-type furostanol saponins were converted to spirostanols under scission of hexoses. Alteration of the acetylated polyhydroxy saponins, exclusively spirostanols, took place following cleavage of acetyl groups and terminal deoxyhexoses. Most interestingly, the pentoses of the sugar chain at C(1) were not affected. Conversion of acetylated polyhydroxy saponins resulted in a final structure type which was stable and detectable, even after 12 months of fermentation and storage.
... Thus, by decreasing Bcl-2 expression simultaneously with increasing Bax and Bad gene expression, HPex increases the susceptibility of tumor cells to the process of apoptosis. Jesse et al. evaluated the effect of an extract of H. niger on the effect on genes involved in apoptosis, mainly the Bcl-2 gene, noting that it induces Bcl-2-dependent cellular apoptosis [26]. Regarding the species H. cyclophyllus, Yfanti and collaborators determined that it has pronounced cytotoxic effects in the lung adenocarcinoma cell line, A549. ...
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In the field of oncology, the plant kingdom has an inexhaustible supply of bioactive compounds. Phytochemical compounds isolated from Helleborus species have been found to be useful in various chronic diseases. This has brought Helleborus to the attention of medical researchers. H. purpurascens is a plant characteristic of the Carpathian area, known since ancient times for its beneficial effects. The aim of the study was to evaluate the flavonoids composition of a hydroalcoholic extract of H. purpurascens, as well as to assess its antioxidant activity and antitumor potential at the level of two healthy cell lines and four tumor cell lines. In addition, the expression of the genes involved in the apoptotic process (Bcl-2, Bad, and Bax) were evaluated. The results indicated that the extract has a high concentration of flavonoids, such as epicatechin, quercetin, and kaempferol. The extract has an increased antioxidant activity, very similar to that of the standard, ascorbic acid and cytotoxic effects predominantly in the breast cancer cell line, being free of cytotoxic effects in healthy cell lines. Underlying the cytotoxic effect is the induction of the process of apoptosis, which in the present study was highlighted by decreasing the expression of anti-apoptotic genes (Bcl-2) and increasing the expression of pro-apoptotic genes (Bad and Bax). In conclusion, the hydroalcoholic extract of H. purpurascens can be considered an important source for future medical applications in cancer therapy.
... malignant versus benign skin tumors), prevalence (e.g. more versus less prevalent cancer types in Westernized societies), and the sites (e.g. occult pancreatic cancer versus breast cancer invading the skin) of Palestinian traditional medicine [28] Inhibition of tumor promotion in mice model [29] Breast [31] -Colorectal adenoma [32] Gastric [33] Inhibition of DMBA-induced mouse skin tumorgenesis [30] Prostate [34] Stomach and esophagus [35] Endometrial [ Yemenite Jews' traditional medicine [45] Apoptosis induction in prostate cells [46] Cervical precancerous lesions [48] May impair Bortezomib activity [ Growth inhibition of colorectal cancer cells [70] Colorectal cancer: safetybioavailability study [71] Augments gemcitabine cytotoxic effect on pancreatic adenocarcinoma cell lines [73] Pancreatic cancer [72] Cyperus longus (sweet cyperus, Cyperaceae) Apoptosis-inducing activity in lymphoma, leukemia, and melanoma cells [83] -- Chemopreventive effects of sage oil on skin papillomas in mice [106] Diet including salvia improved quality of life in patients with advanced breast cancer [108] -Apoptosis in human colon cancer cell lines [107] different cancer types reported in Middle Eastern historical medicinal texts. Other limitations include possible inaccuracy in identifying the herb's scientific name, language limitations (e.g. ...
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Based on traditional, historical, ethnobotanical, laboratory, and clinical findings, we present research framework aiming to identify Middle Eastern herbs that are worthy of further research for their anticancer potential. A comprehensive research project was developed by a multinational team comprising family physicians, medicine specialists, oncologists, an Islamic medicine history specialist, a traditional medicine ethnobotanist, and a basic research scientist. The project followed two consecutive phases: (i) historical and ethnobotanical search for cancer-related keywords and (ii) Medline search for in vitro and in vivo studies. This search yielded 44 herbs associated with cancer care. The Medline search yielded 34 herbs of which 9 herbs were reported in various clinical studies. This multidisciplinary survey was found to be a valuable way to identify herbs with potential clinical significance in cancer care. Based on this pilot study, it is suggested that the Middle East can serve as a valuable region for future multicultural-oriented cancer research.
... The successful treatment of acute myeloid leukemia (aMl) is frequently impeded by the development of resistance to a wide spectrum of cytotoxic drugs. Previous studies have shown that adM resistance is a consequence of the failure of leukemic cells to engage apoptosis (19,20). apoptosis is an energy-requiring suicide programme that is normally activated in response to cellular damage (21). ...
Article
Adriamycin (ADM) is a drug used in the treatment of various types of cancer and exerts an antineoplastic effect mainly through the induction of apoptosis. Phosphoinositide 3 kinase (PI3K)/Akt and MAPK are fundamental survival pathways activated by exposure to most chemotherapeutical agents. However, the role of these pathways in the ADM-induced apoptosis of leukemia cells remains unclear. In the present study, ADM triggered dose-dependent cytotoxicity and resulted in a significant loss of cell viability in HL-60 cells. Moreover, treatment with ADM significantly reduced mitochondrial membrane potential (ΔΨm) in the cells. Akt and ERK activation was also detected, and the inhibition of these two pathways resulted in the enhancement of ADM-induced apoptosis. These results indicate that the PI3K/Akt and ERK survival pathways antagonize the chemotherapeutic effect of ADM. Thus, inhibiting these pathways may serve to enhance the effect of ADM.
... On the other hand, in vitro administration of the aqueous extracts from Helleborus niger has been correlated with induction of sister chromatid exchanges, which is the molecular mechanism of their property to destabilize the DNA and to cause apoptotic cell death in various cancerous cell lines (Büssing. and Schweitzer, 1988, Jesse et al., 2009). A last in vitro experimental model was used for investigation of the consequences of hellebore's interaction with the HeLa neoplastic cells upon the cell cultures development degree, the percentage values of this index being included in Figures 3 and 4. The neoplastic cell cultures incubated with the total or fractionated hydrous hellebore extracts have reached various levels of cultures development process. ...
Article
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The in vitro action of some total aqueous and hydroalcoholic extracts and of their concentrate or permeate fractions, separated by membranary micro- and ultrafiltrations of the primary homogenates, obtained from Helleborus purpurascens ethnomedicinal plant, upon the cell protein biosynthesis, proliferation, viability and development of the HeLa cancerous cells cultures was investigated. The significant proteinsynthesis alteration, protein dynamics modification, decrease of total cell number, cell viability diminution, inhibitory impact upon the cell cultures development, during studied evolution period, suggest the behaviour of these polyphenolic hellebore extracts as in vitro active cytostatic and cytotoxic agents. Our preliminary characterization of these vegetal biopreparations as protein, mitotic, growth inhibitors offers the informational background for further investigations, on many other cancerous and normal cell lines and adequate experimental models to in vitro prescreening, as well as for their introduction in the in vivo antitumoral screening program on different experimental tumoral systems.
... Synergistic effects were also observed when a cytostatic drug -vincristine -was added to the BJAB cell culture in combination with H. niger whole plant extract. Although apoptosis in Mel-OH cell line was correlated with Bcl-2 protein overexpression, sensitivity to H. niger extract was clearly recorded [105]. Another extract from roots of H. bocconei ssp. ...
... Although apoptosis in Mel-OH cell line was correlated with Bcl-2 protein overexpression, sensitivity to H. niger extract was clearly recorded [105]. Another extract from roots of H. bocconei ssp. ...
Article
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Helleborus (family Ranunculaceae) are well-known as ornamental plants, but less known for their therapeutic benefits. Over the past few years, Helleborus sp. has become a subject of interest for phytochemistry, pharmacology and other medical research areas. On the basis of their usefulness in traditional medicine, it was assumed that their biochemical profile could be a source of metabolites with the potential to overcome critical medical issues. There are studies involving natural extracts from these species which demonstrate that Helleborus plants are a valuable source of chemical compounds with great medical potential. Some phytochemicals produced by these species have been separated and identified a few decades ago: hellebrin, deglucohellebrin, 20-hydroxyecdysone and protoanemonin. Lately, many other active compounds have been reported and considered as promising remedies for severe diseases such as cancer, ulcer, diabetes and also for common medical problems such as toothache, eczema, low immunity and arthritis. This paper is an overview of the Helleborus genus focusing on some recentlydiscovered compounds and their potential for finding new drugs and useful biochemicals derived from these species.
... Helleborus niger is another plant used as adjuvant treatment for different tumors in anthroposophic medicine, and may be added to Viscum album (mistletoe) treatment. Jesse et al. studied the whole plant extract in different cancer and leukemia cell lines and primary cells from patients with childhood acute lymphoblastic and myeloblastic leukemias, and reported inhibition of proliferation induced via apoptosis induction in leukemia (Reh, Nalm6, Sup-B15) and lymphoma (BJAB) cells, and in the latter cells synergistic effects were also evident when used in combination with vincristine [73]. ...
Article
Herbal remedies are clearly a complementary and alternative modality used frequently by patients with hemato-oncological neoplasias during the course of their specific treatment. This review focuses on the potential safety and efficacy of herbs which are either used often or even on a daily basis by patients with hematological malignancies or indicated in the herbal pharmacopeias utilized by various traditional systems of medicine, in order to improve the well-being of patients with these cancers. Traditional medicine worldwide is a source for ongoing laboratory research related to the activity of herbs on cultured cell lines derived from patients with leukemia, lymphoma, and myeloma. Although the number of clinical studies in the field of hemato-oncology is limited, there appears to be potential efficacy in studies of mistletoe (Viscum album), green tea, Indian and Middle-Eastern spices, and some traditional Chinese, American, and European herbs. In addition to the potential efficacy of herbs, safety issues are also reviewed here, particularly, the documented and potential side effects, herb-drug interactions, and matters of quality control. Based on the above issues, the authors suggest enhancing doctor-patient communication regarding herbal use by adopting a patient-centered attitude based on scientific perspective.
... The combined organic extracts were dried over magnesium sulfate, and volatiles were removed under reduced pressure. The crude product required multiple purification steps by flash column chromatography (silica, Cy/EtOAc/EtOH 6:1:1), giving 43 mg (0.12 mmol, 40%) of rac-10 as yellow foam. 1 79 Human hepatocytes were obtained from a patient at the Children's Hospital Amsterdamer Straße, Cologne, Germany. Healthy leukocytes were donated by the authors of this paper. ...
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Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a B-ring contracted 6-exo-methylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6-derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of β-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.
... All in vitro experiments were performed at least in three sets of independent experiments, for which means ± standard error were calculated and plotted in bar graphs. Webb's fractional product (*Fp) >1 was calculated for the synergistic effect of viscumTT and the chemotherapeutic agents on apoptotic induction in vitro as described earlier [18,30]. Briefly, Webb's fractional product based on the formula: ...
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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. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1545-7) contains supplementary material, which is available to authorized users.
... Many other active compounds have been reported and considered to be promising remedies for serious diseases such as cancer, ulcers, diabetes as well as common medical problems such as toothache, eczema, low immunity and arthritis [Maior and Dobrota 2013]. In addition, cytotoxic and apoptosisinducing effects on leukaemia cells have been described [Jesse et al. 2009]. In Germany, H. niger is used in homeopathy and as an adjuvant therapy in the treatment of tumour patients in anthroposophical medicine [Büssing and Schweizer 1998]. ...
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The genus Helleborus belongs to the family Ranunculaceae and comprises about 22 species, which are distributed over different parts of Europe and West Asia. In Poland, only H. purpurascens is native and it occurs in the Western Bieszczady Mountains. Hellebores are popular as ornamental cut flowers and medicinal plants in Europe and the USA. Conventional propagation by seeds or division has a low multiplication rate and is time-consuming. Vegetative propagation is necessary to maintain the desirable characteristic of a particular hellebore cultivar. Although some research on tissue culture of hellebores has been published, effective commercial micropropagation of these species has not been attained because cultivation in vitro is still very difficult. This review presents the progress in Helleborus species propagation in vitro for its commercial production. The efficacy of hellebore micropropagation (initiation and stabilization of culture, multiplication and rooting in vitro and acclimatization ex vitro) has been influenced by several factors, such as: type of initial explants, genotype, growth regulators, and environmental factors (temperature, sucrose, nitrogen salts, phosphorus). The genotype-dependence of multiplication and rooting in vitro, and acclimati-zation ex vitro of some Helleborus species has been presented.
... HNE has not been investigated in MPM but has demonstrated proliferation inhibition and cytotoxic effects in lymphoma (MOLT4), sarcoma (SK-UT-1b), and melanoma (HT-144) cell lines, as well as specific apoptosis induction in lymphoma (BJAB), leukemia (Reh, Nalm6, Sup-B15), and melanoma (Mel-HO) cell lines (20,30). This suggests interesting properties regarding potential tumor control. ...
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Malignant pleural mesothelioma (MPM) is a rare cancer with a dismal prognosis. Viscum album extracts (VAE) have strong immune stimulatory properties, cytotoxic effects, can downregulate cancer genes and inhibit angiogenesis. VAE are often used as an adjunct treatment in cancer patients but have rarely been investigated in MPM. Helleborus niger extracts (HNE) have been used in anticancer therapy since antiquity, and also show tumor specific cytotoxic effects. We present a case of a 64-year old woman with epithelioid MPM of the right chest with node involvement (T2N1M0, stage III). Deciding against the recommended radio-chemotherapy, surgery and pleurodesis, she opted for an integrative treatment approach and was treated with VAE and HNE. After 6 weeks' treatment, the pleural and nodal MPM manifestations were reduced by about 15%. Subsequent tumor growth was slow, and the patient remained in good health, enabling her to remain physically active until shortly before her death 56 months after the initial diagnosis. This is a rare case of an MPM patient not receiving any standard anticancer treatment; it still shows an extraordinary long survival and good performance status. We presume that VAE and HNE might had an impact on this clinically relevant outcome and therefore should be further investigated in MPM.
... Helleborus preparations in particular have become important for supportive treatment of different tumors and haematological malignancies [21,22]. Recent in vitro experiments showed cytotoxic or antiproliferative and apoptosis-inducing effects on leukaemia cells [28], lymphoblastic leukaemia, myosarcoma, and melanoma cell lines [23,29], and renal, gastric, and colorectal tumor cell lines [21]. P. vulgaris exhibits antimicrobial and antifungal, sedative (motility-inhibiting), analgesic, antipyretic and antispasmodic effects mainly due to its most active ingredient protoanemonin [30][31][32]. ...
Article
The concentration levels and stability of protoanemonin, a characteristic constituent of Ranunculaceae species with antimicrobial and fungicidal properties, were studied for the first time in plant extracts prepared from Helleborus nigerL. and Pulsatilla vulgarisMill. using fermentative production processes. Protoanemonin levels quantified by HPLC-DAD analysis were 0.0345 and 0.0662 mg/g in two freshly prepared Helleborus (whole, flowering plant) extracts and 0.3875 mg/g and 0.4193 mg/g in Pulsatilla (flowers) extracts. Protoanemonin proved to be rather instable in aqueous-fermented extracts stored at 15 °C in the dark, and its concentration decreased rapidly over 12 months of storage independently of the plant species. The decrease was most pronounced when initial concentrations were high (decrease by about 70%). Source in contrast, low protoanemonin levels remained stable in solution for more than 12 months. Anemonin, the dimer of protoanemonin, was detected in increasing concentrations only in Pulsatilla samples, but its concentration only accounted for less than 50% of the theoretically expected amount. With respect to fermented extracts, both physical processes such as self-polymerization and adsorption/binding to other extract constituents as well as biodegradation were concluded to be responsible for protoanemonin decline. As opposed to plant extracts, both protoanemonin and anemonin levels decreased in 0.22 μm-filtered samples stored in vials. This may be explained by a reduced release from plant material in combination with physicochemically induced degradation. Reduction was most pronounced upon light exposure and elevated temperatures, clearly indicating that photochemical degradation is involved. Contents of protoanemonin in a set of extract batches were 0.0896 ± 0.0125 mg/g and 0.0618 ± 0.0180 mg/g in Helleborus and Pulsatilla extracts, and anemonin levels were 0.0230 ± 0.0076 mg/g and 0.0482 ± 0.0282 mg/g, respectively. Due to its antibiotic effects, but also its reactivity, protoanemonin is a therapeutically and toxicologically relevant constituent, and its concentration should therefore be carefully monitored.
... Compared to this the MelHO Bcl-2 cells had the pIRES-Bcl-2-vector included. They strongly over-express the antiapoptotic Bcl-2 protein (Jesse et al. 2009). Figure 4b clearly demonstrates that WQF 044 had no effect on the MelHO Bcl-2 cell line, but induced apoptosis in the MelHO pIRES cells with a high significance compared to the MelHO Bcl-2 cells, respectively. ...
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Purpose Since the discovery of the well-known cis-platin, transition metal complexes are highly recognized as cytostatic agents. However, toxic side effects of the metal ions present in the complexes may pose significant problems for their future development. Therefore, we investigated the metal-free salalen ligand WQF 044. Methods DNA fragmentations in leukemia (Nalm6) and solid tumor cells (BJAB, MelHO, MCF-7, RM82) proved the apoptotic effects of WQF 044, its overcoming of resistances and the cellular pathways that are affected by the substance. The apoptotic mechanisms finding were supported by western blot analysis, measurement of the mitochondrial membrane potential and polymerase chain reactions. Results A complex intervention in the mitochondrial pathway of apoptosis with a Bcl-2 and caspase dependence was observed. Additionally, a wide range of tumors were affected by the ligand in a low micromolar range in-vitro. The compound overcame multidrug resistances in P-gp over-expressed acute lymphoblastic leukemia and CD95-downregulated Ewing’s sarcoma cells. Quite remarkable synergistic effects with vincristine were observed in Burkitt-like lymphoma cells. Conclusion The investigation of a metal-free salalen ligand as a potential anti-cancer drug revealed in promising results for a future clinical use.
... 27 This finding was supported by two independent studies, which both found that the proliferation of tumor cells can be inhibited by Helleborus niger root and whole plant extracts. 28,29 The chemistry of the active ingredients in the above-ground parts of Helleborus niger has been investigated in a few studies. In 1973, Martinek 30 isolated a crystalline compound from the ethanol extract of dried stems, leaves, and flowers of Helleborus niger L. which had been collected in the Austrian Alps (South Carinthia). ...
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The use of medicinal herbs as remedies reaches back to the Stone Age, and their importance as a source of drugs has continuously increased since then. Herbal ingredients can serve as active pharmaceuticals themselves or as lead substances for the development of synthetic pharmaceuticals with less toxicity, higher effectiveness or with new properties. To date, only 6% of the ∼600,000 plants on earth have been tested pharmacologically. Among these, the medicinal plant Helleborus niger L. (Christmas rose) is especially promising because its leaves contain ( + )-ranuncoside 1, characterized by a spiroacetal ring system, a motif which is responsible for the biological activity of a multitude of natural products. Structure-activity relationship studies of ( + )-ranuncoside 1 are lacking and no synthesis of 1 has been described yet. Therefore, we developed a protocol for the rapid and efficient isolation of 1 from the leaves of cultivated Christmas rose. Crystals of high purity were obtained that enabled us to study the stereochemistry of 1 by NMR spectroscopy in solution for the first time. The spiro configuration, the absolute stereochemistry, and the geometry of all three rings was then confirmed by x-ray structure analysis. Our data will enable future structure-activity relationship studies to assess the potential of 1 as a lead substance for the development of novel antibiotics and anticancer agents.
Article
The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MS(n) ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non-acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, β-ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and β-ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plant's defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.
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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.
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Cardiac glycosides (CGs) have a long history of treating cardiac diseases. However, recent reports have suggested that CGs also possess anticancer and antiviral activities. The primary mechanism of action of these anticancer agents is by suppressing the Na+/k+-ATPase by decreasing the intracellular K+ and increasing the Na+ and Ca2+. Additionally, CGs were known to act as inhibitors of IL8 production, DNA topoisomerase I and II, anoikis prevention and suppression of several target genes responsible for the inhibition of cancer cell proliferation. Moreover, CGs were reported to be effective against several DNA and RNA viral species such as influenza, human cytomegalovirus, herpes simplex virus, coronavirus, tick-borne encephalitis (TBE) virus and Ebola virus. CGs were reported to suppress the HIV-1 gene expression, viral protein translation and alters viral pre-mRNA splicing to inhibit the viral replication. To date, four CGs (Anvirzel, UNBS1450, PBI05204 and digoxin) were in clinical trials for their anticancer activity. This review encapsulates the current knowledge about CGs as anticancer and antiviral drugs in isolation and in combination with some other drugs to enhance their efficiency. Further studies of this class of biomolecules are necessary to determine their possible inhibitory role in cancer and viral diseases.
Article
Aqueous Viscum album L. extracts are widely used for anti-cancer therapies. Due to their low solubility, triterpenes (which are known to act on cancers), do not occur in aqueous extracts in significant amounts. Using cyclodextrins, we have found it possible to solubilize mistletoe triterpene acids and to determine their effects on acute lymphoblastic leukaemia (ALL) in vitro and in vivo. A C.B-17/SCID model of pre-B ALL (NALM-6) was used to test efficacy and mechanisms of treatment with lectin- and triterpene acid containing preparations in vivo. Cytotoxicity of increasing concentrations of V. album L. preparations was assessed in vitro. Apoptosis was determined using mitochondrial membrane potential measurements, annexin V/PI, western blot analyses and caspase inhibitor assays. Solubilized triterpene acid- or lectin-containing V. album L. extracts inhibited cell proliferation and demonstrated cytotoxic properties in vitro. Annexin V/PI and mitochondrial membrane potential assays indicated that dose-dependent induction of apoptosis was the main mechanism. Combination (viscumTT) of lectin- (viscum) and triterpene-containing (TT) extracts resulted in greatest induction of apoptosis. Furthermore, caspase activity demonstrated that these extracts were able to induce apoptosis through both caspase-8 and -9 dependent pathways. In vivo experimentation showed that treatment of mice with viscumTT combination prolonged mean survival to 50.5 days compared to 39.3 days in the phosphate-buffered saline group. Here for the first time, we have demonstrated that either solubilized triterpene acids or lectins and combinations thereof, induce dose-dependent apoptosis in the ALL cell line NALM-6 via caspase-8 and -9 dependent pathways.
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Mucuna macrocarpa Wallich (Leguminosae) is believed to hold blood circulation activating effects, and has been used as a folk remedy in Southeast Asia for the treatment of various hematologic and circulatory-related ailments. The objective of this study was to investigate whether crude methanolic extract of M macrocarpa (CMEMM) possessed antileukemic effects on HL-60, human leukemia cells. CMEMM was prepared from dried stems of this plant, and its apoptosis-inducing effects were investigated using HL-60 cells in vitro and in vivo. With treatment of 25 to 75 μg/mL CMEMM, the in vitro antiproliferative effect on HL-60 cells increased in a dose- and time-dependent manner during the 72-hour treatment period. The concentration of CMEMM that exhibited a 50% growth inhibition (IC(50)) for 72-hour exposure was 36.4 μg/mL. Apoptosis triggered by CMEMM in HL-60 cells was confirmed by the following observations: ( a) characteristic apoptotic nuclear fragmentation, (b) dose-dependent accumulation of sub-G(1) phase in cell cycle analyses, (c) increased percentages of annexin V-positive apoptotic cells, and (d) dose-dependent elevation of active caspase-3. Furthermore, an in vivo tumor growth suppression effect by CMEMM (500 mg/kg/d intraperitoneally) was observed in mouse xenografts. The results suggest that CMEMM exerts antileukemic effects via an apoptotic pathway in HL-60 cells, and could be a candidate for developing antileukemic agents in the future.
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Helleborus niger L (HN) is widely used in the treatment of diabetes mellitus in the Indian traditional system of medicine. Therefore, the present study was done to evaluate the antihyperglycemic, antihyperlipidemic and antioxidant activities of ethanol extract of HN root (EHN) in streptozotocin (STZ) - nicotinamide (NC) induced diabetic rats. in vitro α-amylase inhibition assay, normoglycemic study and oral glucose tolerance test were carried out in different root extracts of HN. Antihyperglycemic effect was assessed in EHN using diabetic rats, which was identified as the most effective extract by initial screening. EHN (50, 100 and 200 mg/kg) and glibenclamide (5 mg/kg) orally administered daily for 28 days and the animal was observed in next 14 days. Fasting blood glucose (FBG) and body weight was determined weekly basis up to 42 days. On the 42nd day, various biochemical parameters were estimated. The three doses of EHN shows significantly decrease (p<0.01) in blood glucose levels. The effect was more pronounced in 200mg/kg (71.53%) than 100mg/kg (67.46%) and 50mg/kg (66.63%). In addition, decreased HbA 1 C and improved Hb level were 1 evidenced clearly in diabetic rats. Simultaneously, improvements in serum lipid profile, serum liver profile in diabetic rats were also evidenced clearly. Moreover, body weight and protein levels were increased in diabetic rats. On the other hand antioxidant activity was restored in diabetic rats. Increased glycogen content, glucokinase and decreased glucose - 6 phosphatase, fructose 1, 6 - biphosphatase effects in liver tissues were observed. EHN preserved islet architecture and prevented hypertrophy of β-cells. The EHN is capable of managing hyperglycemia and complications of diabetes in STZ-NC induced diabetic rats. Hence this plant may be considered as one of the potential sources for the isolation of new oral antihyperglycemic agent(s).
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Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.
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Criteria for the immunological classification of acute leukemias are proposed by a recently established European group designated EGIL. The main aims of EGIL are to establish guidelines for the characterization of acute leukemias based on marker expression and provide a uniform basis for the diagnosis of the various types of these hemopoietic malignancies which should be helpful for future multinational clinical and laboratory investigations. Within the two major types (B and T cell lineage) of acute lymphoblastic leukemia (ALL), several groups are delineated according to the degree of cell differentiation. Within the acute myeloid leukemias (AML), only three subtypes as defined by the FAB classification: M0-AML, M6-AML and M7-AML, can be unequivocally defined by immunological markers; prospective studies are undertaken to see whether characteristic immunological profiles are associated with particular AML subtypes defined by specific cytogenetic abnormalities. Criteria for the definition of biphenotypic acute leukemia (BAL) are devised and a scoring system is outlined aimed to distinguish BAL from those acute leukemias with expression of a marker from another lineage. In addition, an uncommon subset of acute leukemias with no evidence of lymphoid or myeloid differentiation is recognized and the useful panel of markers to investigate and establish the cell nature of the acute leukemias is outlined. EGIL will focus in the future on testing the reproducibility of the proposed guidelines, particularly those for BAL, assessing their clinical value within a framework of multicentric trials and setting up uniform methodological criteria.
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Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.
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Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-alpha in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified.
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20-hydroxyecdysone (1 microM) was found to activate in vitro T-cell CD2 presentation, which is suppressed both in secondary immunodeficient persons and pharmacologically by increasing intracellular cAMP levels. The compound was found to act like a synthetic psycho-immunomodulator 1-oxy-4-oxoadamantane (1 microM) and to exceed the effects of the thymomimetic agent levamisol (1 microM). In addition, 20-hydroxyecdysone (1 microM) was also revealed to modulate the fluoride-stimulated respiratory burst of human neutrophils in the same manner as water soluble antioxidants. Arch.
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Pro- and anti-apoptotic members of the Bcl-2 family control the permeability of the outer mitochondrial membrane. They could do this either by forming autonomous pores in the membrane or by collaborating with components of the permeability transition pore. Here we discuss why we favour the first of these possibilities.
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We examined the biological effects of the ceramide analogues (1S,2R)-2-N-myristoylamino-1-phenyl-1-propanol (D-e-MAPP) and (1R,2R)-2-N-myristoylamino-1-(4-nitrophenyl)-1,3-propandiol (D-NMAPPD) on human HaCaT keratinocytes and human melanoma cells. We could demonstrate that D-e-MAPP and D-NMAPPD are able to suppress acid ceramidase activity. The elevation of the endogenous level of ceramide is followed by induction of apoptosis and suppression of proliferation in HaCaT keratinocytes. Moreover, we recently identified a group of human melanoma cell populations which are heterogeneously susceptible to C2-ceramide-mediated apoptosis. Studies with these melanoma cells revealed correlation between ceramide-mediated apoptosis and D-NMAPPD-induced apoptosis, confirming the effect of this inhibitor on ceramide signaling in human melanoma cells. These findings suggest ceramidase inhibitors as a potential new therapeutical class of antiproliferative and cytostatic drugs.
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Thionins are relatively small-sized multiple-cystine peptides that are probably involved in the plant defense against pathogens. As such, these peptides constitute promising candidates for engineered plant resistance in the agricultural industry. More recently, thionins have been proposed as potential immunotoxins in tumor therapy. In the search for pharmacologically active natural products, a new family of thionins was recently discovered in the roots of Helleborus purpurascens that accordingly were termed hellethionins. The structural characterization by NMR of one representative member of this family, i.e., of hellethionin D, clearly reveals that thionins from different sources share a highly conserved overall fold. In fact, the well-defined 3D structure of hellethionin D is very similar to those reported so far for viscotoxins, purothionins, or crambin, although distinct differences could be detected in the C-terminal portion, especially for loop 36-39. These differences may derive from the unusual distribution of charged residues in the C-terminal half of the peptide sequence compared to other thionins and from the uncommon occurrence of four contiguous threonine residues in loop 36-39. As expected, reduction of the disulfide bonds in hellethionin D leads to complete unfolding, but upon oxidative refolding by air oxygen in the presence of glutathione the correct isomer is recovered in high yields, confirming the very robust fold of this class of bioactive cystine peptides.
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The rhizomes of Helleborus orientalis have been analyzed for the bufadienolide glycoside and spirostanol saponin constituents, resulting in the isolation of a new bufadienolide rhamnoside (1), along with two known bufadienolide glycosides (2 and 3) and five new spirostanol saponins (4-8). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against cultured tumor and normal cells.
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Studies in Drosophila have provided a detailed understanding of how programmed cell death is regulated by steroid hormones during development. This work has defined a two-step hormone-triggered regulatory cascade that results in the coordinate induction of central players in the death pathway, including the reaper and hid death activators, the Apaf-1 ortholog dark, and the dronc apical caspase gene. Recent transcriptional profiling studies have identified many new players in this pathway. In addition, genetic studies are providing new insights into the control of autophagic cell death and revealing how this response is related to, but distinct from, apoptosis.
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A method is described for the extraction and high-performance liquid chromatographic separation and quantitation of the lactone protoanemonin in leaves of Ranunculaceae. For samples with a protoanemonin concentration over 10 microg/g W. W., a reversed-phase technique using a Lichrosorb RP 18 column and a binary solvent system was developed. Alternatively, for samples with a lower lactone concentration, a normal-phase technique with a Lichrosorb Si 60 and quaternary elution system was elaborated. Protoanemonin was detected at 258 nm; its calibration curves were established, and its response factor was calculated using, as standard, the pure compound extracted from HELLEBORUS NIGER. A survey of ten different taxa of Ranunculaceae was performed and it showed the suitability of the method for routine work with high sensitivity limit.
Effects of more than one inhibitor. Enzymes and metabolic inhibitors
  • Webb
Webb. Effects of more than one inhibitor. Enzymes and metabolic inhibitors, Vol. 1. New York, USA: Academic Press. 1963. pp. 66– 79, 487–512.