Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro.

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4ß,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6-13.5 µM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1-3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258-propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation.

BACKGROUND: The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. PURPOSE: Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. METHODS: ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. RESULTS: In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rß expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. CONCLUSION: Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.

Epigallocatechin gallate, ellagic acid, and rosmarinic acid perturb dNTP pools and inhibit de novo DNA synthesis and proliferation of human HL-60 promyelocytic leukemia cells: Synergism with arabinofuranosylcytosine.

Epigallocatechin gallate (EGCG), ellagic acid (EA) and rosmarinic acid (RA) are natural polyphenols exerting cancer chemopreventive effects. Ribonucleotide reductase (RR; EC 1.17.4.1) converts ribonucleoside diphosphates into deoxyribonucleoside diphosphates being essential for DNA replication, which is why the enzyme is considered an excellent target for anticancer therapy. EGCG, EA, and RA dose-dependently inhibited the growth of human HL-60 promyelocytic leukemia cells, exerted strong free radical scavenging potential, and significantly imbalanced nuclear deoxyribonucleoside triphosphate (dNTP) concentrations without distinctly affecting the protein levels of RR subunits (R1, R2, p53R2). Incorporation of (14)C-cytidine into nascent DNA of tumor cells was also significantly lowered, being equivalent to an inhibition of DNA synthesis. Consequently, treatment with EGCG and RA attenuated cells in the G0/G1 phase of the cell cycle, finally resulting in a pronounced induction of apoptosis. Sequential combination of EA and RA with the first-line antileukemic agent arabinofuranosylcytosine (AraC) synergistically potentiated the antiproliferative effect of AraC, whereas EGCG plus AraC yielded additive effects. Taken together, we show for the first time that EGCG, EA, and RA perturbed dNTP levels and inhibited cell proliferation in human HL-60 promyelocytic leukemia cells, with EGCG and RA causing a pronounced induction of apoptosis. Due to these effects and synergism with AraC, these food ingredients deserve further preclinical and in vivo testing as inhibitors of leukemic cell proliferation.

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rß, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression.

In vitro anti-leukemic activity of the ethno-pharmacological plant Scutellaria orientalis ssp. carica endemic to western Turkey.

AIM OF THIS STUDY: Within the genus Scutellaria various species are used in different folk medicines throughout Asia. Traditional Chinese Medicine (TCM) uses S. baicalensis (Labiatae) to treat various inflammatory conditions. The root shows strong anticancer properties in vitro and was suggested for clinical trials against multiple myeloma. Further, S. barbata was successfully tested against metastatic breast cancer in a phase I/II trial. Therefore, we investigated the anti-cancer properties of S. orientalis L. ssp. carica Edmondson, an endemic subspecies from the traditional medicinal plant S. orientalis L. in Turkey, which is used to promote wound healing and to stop haemorrhage. MATERIALS AND METHODS: Freeze-dried plant material was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, and by investigating protein expression profiles specific for cell cycle arrest and apoptosis. RESULTS: The strongest anti-leukemic activity was shown by the methanol extract, which contained apigenin, baicalein, chrysin, luteolin and wogonin, with an IpC50 of 43 microg/ml (corresponding to 1.3mg/ml of dried plant material) which correlated with cyclin D1- and Cdc25A suppression and p21 induction. At 132 microg/ml (=4 mg/ml of the drug) this extract caused genotoxic stress indicated by substantial phosphorylation of the core histone H2AX (gamma-H2AX) followed by activation of caspase 3 and signature-type cleavage of PARP resulting in a 55% apoptosis rate after 48 hours of treatment. CONCLUSIONS: Here, we report for the first time that S. orientalis L. ssp. carica Edmondson exhibited potent anti-leukaemic properties likely through the anti-proliferative effect of baicalein and the genotoxic property of wogonin.

In vitro anti-neoplastic activity of the ethno-pharmaceutical plant Hypericum adenotrichum Spach endemic to Western Turkey.

Hypericum perforatum (St. John's wort) is well-established for its antidepressant activity throughout the world and also various other species within this genus are used in different folk medicines. Hyperforin of St. John's wort inhibited growth of cancer cell lines and the use of hypericin (another compound of H. perforatum) in cancer photodynamic therapy is proposed. Therefore, we investigated the anti-cancer properties of H. adenotrichum Spach (Guttiferae), an endemic species in Turkey called 'kantaron', which is used for wound healing and antiseptic effects. Freeze-dried plant was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, by investigating protein expression profiles specific for cell cycle arrest and apoptosis as well as composition by HPLC. The strongest anti-proliferative activity was determined for the petroleum ether extract with an IpC50 of approximately 5.8 microg/ml medium (referring to 1 mg dried plant) which correlated with cyclin D1 suppression and p21 induction. This extract also induced phosphorylation of H2AX, and activated caspase-3 followed by signature-type cleavage of PARP resulting in approximately 50% apoptosis at 23.2 microg/ml after 24 h of treatment. Neither hyperforin, hypericin, or amentoflavone contributed to these properties. To the best of our knowledge, we report for the first time that the endemic plant H. adenotrichum Spach exhibits potent p53-independent anti-neoplastic properties due to yet unexplored Hypericum constituents.

A polar extract of the Maya healing plant Anthurium schlechtendalii (Aracea) exhibits strong in vitro anticancer activity.

The Aracea Anthurium schlechtendalii and Syngonium podophyllum are traditional remedies for the treatment of severe and chronic inflammatory conditions. We cross-examined these plants regarding their anti-neoplastic properties, because several anti-inflammatory molecular targets are common for both pathologic conditions due to similar signalling pathways. Two malignant cell lines, HL-60 and MCF-7, were treated with increasing concentrations of plant extracts of increasing polarity. The potential of the extracts to inhibit the cell cycle and to induce cell death was investigated, because these are relevant endpoints to assess the anti-cancer potential in vitro and the protein expression and cell cycle distribution upon exposure to the strongest extract was analysed. Extracts from S. podophyllum were rather ineffective, but the freeze-dried (but not air-dried) roots of A. schlechtendalii exhibited strong growth inhibitory and apoptosis-inducing properties. In HL-60 cells 50% proliferation inhibition was achieved by 1.7 microg dichloromethane extract/ml medium and correlated with the activation of Chk2, down-regulation of Cdc25A, suppression of cyclin D1 level, and transient induction of p21. This extract efficiently triggered apoptosis, which was confirmed by caspase 3 activation. The polymerisation of alpha-tubulin and its subsequent degradation that depleted the cells from the G2/M contributed to apoptosis induction, because proper spindle-formation during mitosis is mandatory for survival. In conclusion, we demonstrated that A. schlechtendalii root extract specifically targeted carcinogenic mechanisms, because Cdc25A and cyclin D1 are oncogenes that are frequently overexpressed in a variety of cancer entities and further, this extract affected microtubule function reminiscent of taxol.

In vitro anti-cancer activity of two ethno-pharmacological healing plants from Guatemala Pluchea odorata and Phlebodium decumanum.

Many traditional healing plants successfully passed several hundred years of empirical testing against specific diseases and thereby demonstrating that they are well tolerated in humans. Although quite a few ethno-pharmacological plants are applied against a variety of conditions there are still numerous plants that have not been cross-tested in diseases apart from the traditional applications. Herein we demonstrate the anti-neoplastic potential of two healing plants used by the Maya of the Guatemala/Belize area against severe inflammatory conditions such as neuritis, rheumatism, arthritis, coughs, bruises and tumours. Phlebodium decumanum and Pluchea odorata were collected, dried and freeze dried, and extracted with five solvents of increasing polarity. We tested HL-60 and MCF-7 cells, the inhibition of proliferation and the induction of cell death were investigated as hallmark endpoints to measure the efficiency of anti-cancer drugs. Western blot and FACS analyses elucidated the underlying mechanisms. While extracts of P. decumanum showed only moderate anti-cancer activity and were therefore not further analysed, particularly the dichloromethane extract of P. odorata inhibited the cell cycle in G2-M which correlated with the activation of checkpoint kinase 2, and down-regulation of Cdc25A and cyclin D1 as well as inactivation of Erk1/2. In HL-60 and MCF-7 cells this extract was a very strong inducer of cell death activating caspase-3 followed by PARP signature type cleavage. The initiating death trigger was likely the stabilization of microtubules monitored by the rapid acetylation of alpha-tubulin, which was even more pronounced than that triggered by taxol. The dichloromethane extract of P. odorata contains apolar constituents which inhibit inflammatory responses and exhibit anti-cancer activity. The strong proapoptotic potential warrants further bioassay-guided fractionation to discover and test the active principle(s).

Avemar, a nontoxic fermented wheat germ extract, attenuates the growth of sensitive and 5-FdUrd/Ara-C cross-resistant H9 human lymphoma cells through induction of apoptosis.

Avemar (MSC) is a nontoxic fermented wheat germ extract, which has been shown to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in sensitive and 5-FdUrd/Ara-C cross-resistant H9 human lymphoma cells. After 48 and 72 h of incubation, Avemar inhibited the growth of sensitive H9 cells with IC50 values of 290 and 200 microg/ml, whereas the growth of 5-FdUrd/Ara-C cross-resistant H9 cells was attenuated with IC50 values of 180 and 145 microg/ml, respectively. Treatment with 300 microg/ml MSC for 48 h caused dose-dependent induction of apoptosis in 48% of sensitive H9 cells. In cross-resistant H9 cells, incubation with 200 microg/ml Avemar for 48 h led to 41% of apoptotic tumor cells. Growth arrest of sensitive H9 cells after exposure to various concentrations of MSC occurred mainly in the S phase of the cell cycle, thereby increasing the cell population from 54 to 73% while depleting cells in the G0-G1 phase from 40 to 19%. Growth arrest in cross-resistant H9 cells occurred also mainly in the S phase, increasing the cell population from 45 to 68% while depleting cells in the G0-G1 phase from 45 to 31%. As MSC treatment likely overcomes 5-FdUrd/Ara-C resistance, further investigations to elucidate the exact mechanisms are warranted. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.

Avemar, a nontoxic fermented wheat germ extract, induces apoptosis and inhibits ribonucleotide reductase in human HL-60 promyelocytic leukemia cells.

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in human HL-60 promyelocytic leukemia cells. After 24, 48, and 72 h of incubation, Avemar inhibited the growth of HL-60 cells with IC50 values of 400, 190, and 160 microg/ml, respectively. Incubation with MSC caused dose-dependent induction of apoptosis in up to 85% of tumor cells. In addition, Avemar attenuated the progression from G2-M to G0-G1 phase of the cell cycle and was also found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of de novo DNA synthesis. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.

Cytotoxic and biochemical effects of 3,3',4,4',5,5'-hexahydroxystilbene, a novel resveratrol analog in HL-60 human promyelocytic leukemia cells.

OBJECTIVE: Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, is an inhibitor of the proliferation-linked enzyme ribonucleotide reductase (RR) and shows a broad spectrum of cytotoxic effects against human cancer cells. In order to enhance these effects, we introduced additional hydroxyl moieties into the molecule. In the present study, the activity of a novel RV analog, 3,3',4,4',5,5'-hexahydroxystilbene (M8), was investigated in HL-60 human promyelocytic leukemia cells. METHODS: Cytotoxicity of M8 alone or in combination with Ara-C was assessed employing growth inhibition assays. Effects of M8 on nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) were examined by HPLC. The apoptotic potential of M8 and RV was compared using a specific double-staining method and inhibition of TNF-alpha-induced activation of NF-kappaB was studied. Cell-cycle distribution was analyzed by FACS. RESULTS: Addition of ascorbic acid decreased the IC(50) value of M8 from 6.25 microM to 2 microM. M8 depleted dATP and dTTP pools to 41% and 21% of control values, whereas dCTP pools increased to 199% of untreated controls. In addition, TTP, ATP, CTP, and GTP concentrations were decreased while UTP concentrations increased. M8 induced apoptosis at concentrations significantly lower than RV and could remarkably inhibit the activation of NF-kappaB. M8 arrested cells in the S phase of the cell cycle while depleting cells in the G2-M phase and exhibited synergistic combination effects when applied simultaneously with Ara-C. CONCLUSION: Due to these promising results, this novel polyhydroxylated stilbene derivative might become an additional option for the treatment of leukemia and therefore deserves further preclinical and in vivo testing.

Immunologic and biochemical effects of the fermented wheat germ extract Avemar.

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. Avemar has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. Avemar had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 microg/ml (7 days of incubation); this value could be decreased to 100 and 75 microg/ml in the presence of vitamin C. In the cell line examined, Avemar induced both necrosis and apoptosis, as demonstrated by Hoechst/propidium iodide staining. The incubation of cells with 3200 microg/ml Avemar for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. Avemar inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, Avemar inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of Avemar on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by Avemar with IC50 values of 100 and 300 microg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using Avemar.