Molecular determinants of the response of cancer cells towards geldanamycin and its derivatives.

Geldanamycin is an ansamycin-derivative of a benzoquinone isolated from Streptomyces hygroscopicus. It inhibits tyrosine kinases and heat shock protein 90 (HSP90). Geldanamycin and 11 derivatives were subjected to molecular docking to HSP90, and 17-desmethoxy-17-N,N-dimethylamino-geldanamycin (17-DMAG) was the compound with the highest binding affinity (-7.73 ± 0.12 kcal/mol) and the lowest inhibition constant (2.16 ± 0.49 µM). Therefore, 17-DMAG was selected for further experiments in comparison to geldanamycin. Multidrug resistance (MDR) represents a major problem for successful cancer therapy. We tested geldanamycin and 17-DMAG against various drug-resistant cancer cell lines. Although geldanamycin and 17-DMAG inhibited the proliferation in all cell lines tested, multidrug-resistant P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant, ΔEGFR-overexpressing tumor cells and p53 knockout cells were sensitive to these two compounds. COMPARE and hierarchical cluster analyses were performed, and 60 genes were identified to predict the sensitivity or resistance of 59 NCI tumor cell lines towards geldanamycin and 17-DMAG. The distribution of cell lines according to their mRNA expression profiles indicated sensitivity or resistance to both compounds with statistical significance. Moreover, bioinformatic tools were used to study possible mechanisms of action of geldanamycin and 17-DMAG. Galaxy Cistrome analyses were carried out to predict transcription factor binding motifs in the promoter regions of the candidate genes. Interestingly, the NF-ĸB DNA binding motif (Rel) was identified as the top transcription factor. Furthermore, these 60 genes were subjected to Ingenuity Pathway Analysis (IPA) to study the signaling pathway interactions of these genes. Interestingly, IPA also revealed the NF-ĸB pathway as the top network among these genes. Finally, NF-ĸB reporter assays confirmed the bioinformatic prediction, and both geldanamycin and 17-DMAG significantly inhibited NF-κB activity after exposure for 24 h. In conclusion, geldanamycin and 17-DMAG exhibited cytotoxic activity against different tumor cell lines. Their activity was not restricted to HSP90 but indicated an involvement of the NF-KB pathway.

Jozibrevine D from Ancistrocladus ileboensis, the fifth alkaloid in a series of six possible atropo-diastereomeric naphthylisoquinoline dimers, showing antiparasitic and antileukemic activities.

A new dimeric naphthylisoquinoline alkaloid, jozibrevine D (4e), was isolated from the Central-African liana Ancistrocladus ileboensis. It is a Dioncophyllaceae-type metabolite, being R-configured at C-3 and lacking an oxygen function at C-6 in both isoquinoline moieties. The two identical monomers of jozibrevine D are symmetrically linked via the sterically constrained 3',3''-positions of the naphthalene units so that the central biaryl linkage is rotationally hindered and the alkaloid is, thus, C2-symmetric. With the two outer biaryl bonds being chiral, too, 4e possesses three consecutive stereogenic axes. The absolute stereostructure of the new compound was assigned by 1D and 2D NMR, ruthenium-mediated oxidative degradation, and electronic circular dichroism (ECD) spectroscopy. Jozibrevine D (4e) is the fifth discovered isomer in a series of six possible natural atropo-diastereomeric dimers. It shows potent, and selective, antiprotozoal activity against P. falciparum (IC50 = 0.14 µM), and it also exhibits good cytotoxic activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells (IC50 = 11.47 µM) and their multidrug-resistant CEM/ADR5000 subline (IC50 = 16.61 µM).

In Silico and In Vitro Screening of 50 Curcumin Compounds as EGFR and NF-κB Inhibitors.

The improvement of cancer chemotherapy remains a major challenge, and thus new drugs are urgently required to develop new treatment regimes. Curcumin, a polyphenolic antioxidant derived from the rhizome of turmeric (Curcuma longa L.), has undergone extensive preclinical investigations and, thereby, displayed remarkable efficacy in vitro and in vivo against cancer and other disorders. However, pharmacological limitations of curcumin stimulated the synthesis of numerous novel curcumin analogs, which need to be evaluated for their therapeutic potential. In the present study, we calculated the binding affinities of 50 curcumin derivatives to known cancer-related target proteins of curcumin, i.e., epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB) by using a molecular docking approach. The binding energies for EGFR were in a range of −12.12 (±0.21) to −7.34 (±0.07) kcal/mol and those for NF-κB ranged from −12.97 (±0.47) to −6.24 (±0.06) kcal/mol, indicating similar binding affinities of the curcumin compounds for both target proteins. The predicted receptor-ligand binding constants for EGFR and curcumin derivatives were in a range of 0.00013 (±0.00006) to 3.45 (±0.10) µM and for NF-κB in a range of 0.0004 (±0.0003) to 10.05 (±4.03) µM, indicating that the receptor-ligand binding was more stable for EGFR than for NF-κB. Twenty out of 50 curcumin compounds showed binding energies to NF-κB smaller than −10 kcal/mol, while curcumin as a lead compound revealed free binding energies of >−10 kcal/mol. Comparable data were obtained for EGFR: 15 out of 50 curcumin compounds were bound to EGFR with free binding energies of <−10 kcal/mol, while the binding affinity of curcumin itself was >−10 kcal/mol. This indicates that the derivatization of curcumin may indeed be a promising strategy to improve targe specificity and to obtain more effective anticancer drug candidates. The in silico results have been exemplarily validated using microscale thermophoresis. The bioactivity has been further investigated by using resazurin cell viability assay, lactate dehydrogenase assay, flow cytometric measurement of reactive oxygen species, and annexin V/propidium iodide assay. In conclusion, molecular docking represents a valuable approach to facilitate and speed up the identification of novel targeted curcumin-based drugs to treat cancer.

Synthesis and Cytotoxicity of Thieno[2,3-b]Pyridine Derivatives Toward Sensitive and Multidrug-Resistant Leukemia Cells.

A new series of substituted ethyl 7-cyclopropyl-2-(2-aryloxo)-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylates 3a-e were prepared by utilizing ethyl 2-chloro-7-cyclopropyl-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate (1) and replacing of the 2-chlorine with anions obtained from phenol (2a), salicylaldehyde derivatives 2b-d or thiophenol (2e), leading to the respective ethyl 7-cyclopropyl-2-(2-aryloxo)-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylates 3a-e. The new compounds were evaluated for their in vitro cytotoxicity towards sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. The screening revealed that compounds 3a, 3b, and 3e inhibited the growth of both cell lines. Compound 3b, with a phenol moiety, exhibited the highest growth inhibitory activity against CEM/ADR5000 and CCRF-CEM cells with IC50 values 4.486 ± 0.286 and 2.580 ± 0.550 µM, respectively. Collectively, the presented results demonstrate that the synthesized thieno[2,3-b]pyridines warrant further exploration for potential use as anti-cancer agents.

Network pharmacology of triptolide in cancer cells: implications for transcription factor binding.

Background Triptolide is an active natural product, which inhibits cell proliferation, induces cell apoptosis, suppresses tumor metastasis and improves the effect of other therapeutic treatments in several cancer cell lines by affecting multiple molecules and signaling pathways, such as caspases, heat-shock proteins, DNA damage and NF-ĸB. Purpose We investigated the effect of triptolide towards NF-ĸB and GATA1. Methods We used cell viability assay, compare and cluster analyses of microarray-based mRNA transcriptome-wide expression data, gene promoter binding motif analysis, molecular docking, Ingenuity pathway analysis, NF-ĸB reporter cell assay, and electrophoretic mobility shift assay (EMSA) of GATA1. Results Triptolide inhibited the growth of drug-sensitive (CCRF-CEM, U87.MG) and drug-resistant cell lines (CEM/ADR5000, U87.MGΔEGFR). Hierarchical cluster analysis showed six major clusters in dendrogram. The sensitive and resistant cell lines were statistically significant (p = 0.65 × 10-2) distributed. The binding motifs of NF-κB (Rel) and of GATA1 proteins were significantly enriched in regions of 25 kb upstream promoter of all genes. IPA showed the networks, biological functions, and canonical pathways influencing the activity of triptolide towards tumor cells. Interestingly, upstream analysis for the 40 genes identified by compare analysis revealed ZFPM1 (friend of GATA protein 1) as top transcription regulator. However, we did not observe any effect of triptolide to the binding of GATA1 in vitro. We confirmed that triptolide inhibited NF-κB activity, and it strongly bound to the pharmacophores of IκB kinase ß and NF-κB in silico. Conclusion Triptolide showed promising inhibitory effect toward NF-κB, making it a potential candidate for targeting NF-κB.

A novel ligand of the translationally controlled tumor protein (TCTP) identified by virtual drug screening for cancer differentiation therapy.

Introduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

Cytotoxicity of fagaramide derivative and canthin-6-one from Zanthoxylum (Rutaceae) species against multidrug resistant leukemia cells.

In our continuous search for cytotoxic compounds from the genus Zanthoxylum, chromatographic separation of the MeOH/CH2Cl2 (1:1) extract of Z. chalybeum yielded one new alkamide; 4-(isoprenyloxy)-3-methoxy-3,4-deoxymethylenedioxyfagaramide (1) and a known one; fagaramide (2). Similarly, from the MeOH/CH2Cl2 (1:1) extract of the stem bark of Z. parachanthum four known compounds; canthin-6-one (3), dihydrochelerythrine (4), lupeol (5) and sesamin (6) were isolated. Characterization of the structures of these compounds was achieved using spectroscopic techniques (NMR and MS). Using resazurin reduction assay 1, 3 and 6 displayed moderate cytotoxicity with IC50 values below 50 µM against the drug sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines. It is interesting to note that 3 was more active than the standard drug, doxorubicin against CEM/ADR5000 leukemia cells. Compounds 3 and 6 showed good selectivity on leukemia cells than normal cells. In future studies 3 should be tested against a panel of drug resistant human cells.

AMG900 as novel inhibitor of the translationally controlled tumor protein.

INTRODUCTION: Cancer is one of the leading causes of death worldwide. Classical cytotoxic chemotherapy exerts high side effects and low tumor selectivity. Translationally controlled tumor protein (TCTP) is a target for differentiation therapy, a promising, new therapeutic approach, which is expected to be more selective and less toxic than cytotoxic chemotherapy. The aim of the present investigation was to identify novel TCTP inhibitors. METHODS: We performed in silico screening and molecular docking using a chemical library of more than 31,000 compounds to identify a novel inhibitor of TCTP. We tested AMG900 in vitro for binding to TCTP by microscale thermophoresis and co-immunoprecipitation. Additionally, we examined the effect of TCTP blockade on cell cycle progression by flow cytometry and Western blotting and cancer cell survival by resazurin assays in MCF-7, SK-OV3 and MOLT-4 cell lines. RESULTS: We identified AMG900 as new inhibitor of TCTP. AMG900 bound to the p53 binding site of TCTP with a free binding energy of -9.63 ±â€¯0.01 kcal/mol. This compound decreased TCTP expression to 23.4 ±â€¯1.59% and increased p53 expression to 194.29 ±â€¯24.27%. Furthermore, AMG900 induced G0/G1 arrest as shown by flow cytometry and Western blot of relevant cell cycle proteins. AMG900 decreased CDK2, CDK4, CDK6, cyclin D1 and cyclin D3 expression, whereas p18, p21 and p27 expression increased. Moreover, AMG900 disturbed TCTP-p53 complexation as shown by co-immunoprecipitation and increased expression of free p53. DISCUSSION: AMG900 may serve as novel lead compound for the development of differentiation therapy approaches against cancer.

Antiproliferative Properties of a Few Auranofin-Related Gold(I) and Silver(I) Complexes in Leukemia Cells and their Interferences with the Ubiquitin Proteasome System.

A group of triethylphosphine gold(I) and silver(I) complexes, structurally related to auranofin, were prepared and investigated as potential anticancer drug candidates. The antiproliferative properties of these metal compounds were assessed against two leukemia cell lines, i.e., CCRF-CEM and its multidrug-resistant counterpart, CEM/ADR5000. Interestingly, potent cytotoxic effects were disclosed for both series of compounds against leukemia cells, with IC50 values generally falling in the low-micromolar range, the gold derivatives being on the whole more effective than the silver analogues. Some initial structure-function relationships were drawn. Subsequently, the ability of the study compounds to inhibit the three main catalytic activities of the proteasome was investigated. Different patterns of enzyme inhibition emerged for the various metal complexes. Notably, gold compounds were able to inhibit effectively both the trypsin-like and chymotrypsin-like proteasome activities, being less effective toward the caspase-like catalytic activity. In most cases, a significant selectivity of the study compounds toward the proteasome proteolytic activities was detected when compared to other proteases. The implications of the obtained results are discussed.

Cytotoxic bufadienolides from the leaves of a medicinal plant Melianthus comosus collected in South Africa.

From the leaves of South African medicinal plant Melianthus comosus, four previously undescribed bufadienolides, 16ß-formyloxymelianthugenin (1), 2ß-acetoxymelianthusigenin (2), 2ß-hydroxy-3ß,5ß-di-O-acetylhellebrigenin (3), and 2ß-acetoxy-5ß-O-acetylhellebrigenin (4) were isolated together with two known bufadienolides. The structural elucidation of the compounds was based on 1D and 2D NMR spectroscopy, high-resolution mass spectrometry, and other spectroscopic methods. The relative configurations were determined by single-crystal X-ray crystallography analysis and NOESY correlations. The isolated compounds displayed strong cytotoxicity against MCF-7 breast cancer cells, sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Compound 1 showed the most potent activity, with IC50 values of 0.07 µM towards CCRF-CEM, 0.06 µM towards CEM/ADR5000 and 0.36 µM towards MCF-7 followed by compound 4 with IC50 values of 0.13 µM towards CCRF-CEM, 0.08 µM towards CEM/ADR5000 and 0.53 µM towards MCF-7.

Cytotoxic Bufadienolides from the Leaves of Melianthus major.

Melianthus major is a medicinal plant endemic to South Africa. Its leaf extract led to the isolation of five new bufadienolides, 2ß-acetoxy-3,5-di-O-acetylhellebrigenin (1), 2ß-acetoxy-3-O-acetylhellebrigenin (2), 2ß-acetoxy-14-deoxy-15ß,16ß-epoxymelianthugenin (4), 2ß-acetoxy-14-deoxy-15ß,16ß-epoxymelianthusigenin (5), and 2ß-hydroxymelianthusigenin (6), and four known analogues. The structures of the compounds were elucidated using NMR and HRESIMS data analyses. The relative configurations were defined by single-crystal X-ray crystallography and NOESY correlations. The isolated compounds exhibited strong cytotoxicity against MCF-7 breast cancer cells and sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Compound 1 showed the most potent activity, with IC50 values of 0.1 µM toward CCRF-CEM and CEM/ADR5000 and 0.3 µM toward MCF-7.

Collateral sensitivity of natural products in drug-resistant cancer cells.

Cancer chemotherapy is frequently hampered by drug resistance. Concepts to combine anticancer drugs with different modes of action to avoid the development of resistance did not provide the expected success in the past, because tumors can be simultaneously non-responsive to many drugs (e.g. the multidrug resistance phenotype). However, tumors may be specifically hypersensitive to other drugs - a phenomenon also termed collateral sensitivity. This seems to be a general biological mechanism, since it also occurs in drug-resistant Escherichia coli and Saccharomyces cerevisiae. Here, we give a timely and comprehensive overview on hypersensitivity in resistant cancer cells towards natural products and their derivatives. Since the majority of clinically established anticancer drugs are natural products or are in one way or another derived from them, it is worth hypothesizing that natural products may deliver promising lead compounds for the development of collateral sensitive anticancer drugs. Hypersensitivity occurs not only in classical ABC transporter-mediated multidrug resistance, but also in many other resistance phenotypes. Resistant cancers can be hypersensitive to natural compounds from diverse classes and origins (i.e. mitotic spindle poisons, DNA topoisomerase 1 and 2 inhibitors, diverse phytochemicals isolated from medicinal plants, (semi)synthetic derivatives of phytochemicals, antibiotics, marine drugs, recombinant therapeutic proteins and others). Molecular mechanisms of collateral sensitivity include (1) increased ATP hydrolysis and reactive oxygen species production by futile cycling during ABC transporter-mediated drug efflux, (2) inhibition of ATP production, and (3) alterations of drug target proteins (e.g. increased expression of topoisomerases and heat shock proteins, inhibition of Wnt/ß-catenin pathway, mutations in ß-tubulin). The phenomenon of hypersensitivity needs to be exploited for clinical oncology by the development of (1) novel combination protocols that include collateral sensitive drugs and (2) novel drugs that specifically exhibit high degrees of hypersensitivity in resistant tumors.

Ruthenium(ii) and palladium(ii) homo- and heterobimetallic complexes: synthesis, crystal structures, theoretical calculations and biological studies.

Four Ru-Pd heterobimetallic complexes, each one in two different coordination modes (NNSS and NS) having metals connected by a binucleating dialkyldithiooxamidate [N(R)SC-CS(R)N] [R = methyl, ethyl, n-butyl and isopropyl], were prepared by reacting the monochelate [(trinpropyl-phosphine)ClPd(HR2C2N2S2κ-S,S-Pd)] with [(η6-p-cymene)RuCl2]2. Furthermore, two palladium homobimetallic complexes having two (trinpropyl-phosphine)ClPd moieties joined by a diethyldithiooxamidate in both κ-N,S Pd, κ-N',S' Pd' and κ-N,N' Pd, κ-S,S' Pd' coordination modes were synthesized. For both kinds of complexes, homo- and heterobimetallic, at room temperature and in chloroform solution, the NNSS coordination mode (kinetic compounds) turns out to be unstable and therefore the resulting complexes rearrange into a thermodynamically more stable form (NS coordination mode). The crystal structures of [(trinpropyl-phosphine)ClPd]2[µ-(ethyl)2-DTO κ-N,S Pd, κ-N',S' Pd'] (2) and [(η6-p-cymene)ClRu][µ-(methyl)2-DTO κ-N,S Ru, κ-N,S Pd] [(trinpropyl-phosphine)ClPd] (1c) were determined by solid state X-ray crystallography. Moreover, the higher stability of the thermodynamic species in the heterobimetallic complexes (Ru-Pd) was evaluated by means of computational studies in accordance with the maximum hardness principle. All stable NS complexes (i.e.1c-4c, 2 and the previously reported homobimetallic Ru complex 3) were tested against two leukemia cell lines, namely the drug-sensitive CCRF-CEM cell line and its multidrug-resistant sub-cell line CEM/ADR5000 showing anti-proliferative activity in the low micromolar range (∼1-5 µM) and micromolar range (∼10-25 µM), respectively. In addition, these complexes efficaciously block at least two out of the three proteolytic activities of the tumor target 20S proteasome, with heterobimetallic complex 3c and homobimetallic complex 3 possessing the best inhibitory profile.

Effects of anti-inflammatory and adaptogenic herbal extracts on gene expression of eicosanoids signaling pathways in isolated brain cells.

INTRODUCTION: The adaptogens modulate expression of genes playing key roles in development of aging-related disorders, which are considered as low-grade systemic inflammatory conditions characterized by an imbalance between pro-and anti-inflammatory eicosanoids. AIM OF THE STUDY: We compared the effects of anti-inflammatory and adaptogenic plant extracts on the expression of genes involved in biosynthesis of eicosanoids with the purpose to find those plants, which selectively upregulated the expression of anti-inflammatory lipoxins signaling pathways and inhibited pro-inflammatory signaling pathways associated with biosynthesis of leukotrienes, prostaglandins and thromboxanes. MATERIALS AND METHODS: We conducted transcriptome-wide RNA sequencing to profile gene expression alterations in T98G neuroglia cells upon treatment of plant extract and analyzed the relevance of deregulated genes to eicosanoids signaling pathways using in silico models. RESULTS: For the first time, we demonstrated that Rhodiola rosea, Withania somnifera and Eleutherococcus senticosus downregulate the expression of key genes (ALOX5AP, DPEP2, LTC4S) involved biosynthesis of leukotrienes A, B, C, D and E, resulting in inhibition of leukotriene signaling pathway suggesting their potential benefits in Alzheimer disease. The common feature for all tested anti-inflammatory plants extracts was related to downregulation of ALOX12, which was also associated with neuroprotective action of these medicinal plants as well as their potential benefits in neurodegenerative diseases. None of tested anti-inflammatory and adaptogenic plants selectively activated the ALOX15-mediated signaling pathway, which is associated with generation anti-inflammatory lipoxins. Almost all tested plants upregulated the expression of the prostaglandin E receptor 3 gene (PTGER3) suggesting their potential benefits in the treatment of cancer. CONCLUSION: Every single plant tested in this study revealed a specific "signature" on eicosanoid signaling-related gene expression, regardless of their common features as anti-inflammatory or adaptogenic activity. Further studies of the combination of Rhodiola with Withania (Adaptra) for the treatment of Alzheimer disease are required.

Adaptogens in chemobrain (Part II): Effect of plant extracts on chemotherapy-induced cytotoxicity in neuroglia cells.

BACKGROUND: Cancer chemotherapy-induced cognitive impairments are apparently associated with harmful effects on physiological functions of brain cells. Adaptogens, are known to exhibit neuroprotective effects and to increase cognitive functions in clinical studies. In our previous study (Seo et al., 2018), we demonstrated that selected adaptogenic extracts significantly attenuate cytostatic-induced regulation of more than 100 genes involved in the activation of neuronal death and inhibiting neurogenesis. Neuroprotective and cytoprotective activities of adaptogens rise the question about their possible impact on cytostatic effects of a chemotherapeutic combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC). AIM: The aim of this study was to assess the effects of selected adaptogenic herbal extracts, namely of andrographolide (AND), Herba Andrographidis (AP), Radix Eleutherococci (ES) genuine extracts, their fixed combination (AE), and the combination of three adaptogenic herbs, Rhodiola Radix, Shisandra Fructus and Eleutherococci Radix (RSE) on the cytotoxicity of a fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC) on neuroglia cells. METHODS: Cytotoxicity of FEC, adaptogenic extracts and their combination with FEC was tested on isolated T98G cells in a wide range of concentrations of all tested compounds. RESULTS: FEC reproducibly inhibited the proliferation of T98G cells by 50% at concentrations of 5 × 10-1 µg/ml epirubicin, 500 × 10-1 µg/ml 5-fluorouracil and 20 × 10-1 µg/ml 4-hydroperoxycyclophosphamide after 24 h incubation of cells. These concentrations were subsequently used for experiments with adaptogenic extracts. The cytotoxic activity of FEC was not significantly changed in the presence of AND, ES and AE. Furthermore, it was potentiated by AP extract and RSE in concentrations of 0.06-6 µg/ml and 17.6-26.4 µg/ml. CONCLUSION: The neuroprotective effect of adaptogens did not attenuate the cytotoxic activity of FEC. Application of cytostatic drugs in combination with adaptogenic plant extracts likely have no impact in cytotoxic effect of FEC. Furthermore, AP and RSE potentiated the cytotoxic effects of FEC.

Adaptogens in chemobrain (Part III): Antitoxic effects of plant extracts towards cancer chemotherapy-induced toxicity - transcriptome-wide microarray analysis of neuroglia cells.

BACKGROUND: Toxicity of chemotherapeutics is a serious problem in cancer therapy. Adaptogens are known to increase adaptability and survival organisms. AIM: The aim of this study was to assess the effects of selected adaptogenic herbal extracts on FEC (fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide) induced changes in transcriptome-wide microarray profiles of neuroglia cells. Another task of the study was to identify those genes, which are associated with FEC-induced hepato-, cardio- and nephrotoxicity to predict potential effects of andrographolide (AND), Andrographis herb, Eleutherococcus roots genuine extracts (ES), their fixed combination (AE) and the combination of Rhodiola roots, Schisandra berries and Eleutherococcus roots (RSE) on the organismal level. METHODS: Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human T98G neuroglia cells after treatment with adaptogens. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULT: Significant differences of transcriptome-wide microarray profiles were observed after treatment of T98G cells with FEC and after co-incubation with adaptogens. FEC induced deregulation of certain genes with suggested toxicity associated with liver fibroses, necrosis and congenital heart diseases. Co-incubation of AE with FEC prevented FEC-induced deregulation of 66 genes increasing organismal death, 37 genes decreasing cell survival, 37 genes decreasing DNA repair, 37 genes decreasing viral infection and some other functions, indicating on potential beneficial effects of AE. Furthermore, FEC-induced hepato-, nephro- and cardiotoxicity related to deregulation of genes was predictably attenuated by AE. Moreover, co-incubation of AE with FEC caused differential expression of genes, which presumably are beneficial for an organism during chemotherapy. They include predicted activation of DNA repair, activation of movement of antigen presenting cells and inhibition of muscle cells death. The main active constituent of AE is AND. Co-incubation of FEC only with AND results in deregulation of 10 genes causing death of breast cancer cells, decrease of liver toxicity and attenuation of organismal death. Co-incubation of ES extract with FEC showed that ES suppressed FEC-induced deregulation of genes, which inhibit organismal death and fertility. Co-incubation of FEC with RSE indicated potential hepatoprotective effect against FEC-induced apoptosis of liver cells presumably due to suppression of FEC-induced expressions of genes, which increased liver cell apoptosis. Simultaneously, RSE activated expression of genes inhibiting tumor growth. Though, microarray analysis did not provide final proof that the genes induced by the AE, AP and ES are responsible for the physiological effects observed in human patients following their oral administration, it provided insights into putative genes and directions for future research and possible implementation into practice. CONCLUSION: Application of cytostatic drugs in combination with adaptogenic plant extracts induced significant changes in transcriptome-wide microarray profiles of neuroglial cells. These changes indicate on potential beneficial effects of adaptogens on FEC induced adverse events in cancer chemotherapy.

Adaptogens in chemobrain (Part I): Plant extracts attenuate cancer chemotherapy-induced cognitive impairment - Transcriptome-wide microarray profiles of neuroglia cells.

BACKGROUND: Cancer chemotherapy-induced cognitive impairments are presumably associated with undesirable effects of chemotherapy on physiological functions of brain cells. Adaptogens are natural compounds or plant extracts increasing an organism's adaptability and survival in stress. They exhibited neuroprotective effects and increased cognitive functions in clinical studies in human beings. HYPOTHESIS: We hypothesized that selected adaptogenic plant extracts attenuate or prevent cancer chemotherapy-induced cognitive impairments. AIM: We assessed the effects of selected adaptogenic herbal extracts on FEC (fixed combination 5-fluorouracil, epirubicin and cyclophosphamide) induced changes in transcriptome-wide RNA microarray profiles of neuroglia cells. The aim of the study was to predict potential effects of andrographolide, Andrographis herb, Eleutherococcus root genuine extracts, their fixed combination (AE) and the combination of Rhodiola roots, Schisandra berries and Eleutherococcus roots (RSE) on cellular and physiological, mostly cognitive functions. METHODS: Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human T98G neuroglia cells after treatment with adaptogens. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULTS: FEC deregulated 67 genes involved in decrease of neuronal development, 37 genes involved in development of the sensory system, 12 genes in extension of axons, and 3 genes in migration of neurons. Co-incubation with Andrographis paniculata (AP) suppressed FEC-induced deregulation of a large number of genes involved in predicted activation of neuronal death and inhibition of neurogenesis, and 16 genes related to inhibition of several functions in the nervous system. Co-incubation with AE suppressed FEC-induced deregulation of a number of genes involved in predicted inhibition of axon extension, migration of T98G neuroglia cells, conduction of nerves and other genes related to regulations of some other functions in the nervous system. CONCLUSION: Application of cytostatic drugs in combination with apoptogenic plant extracts induced significant changes in transcriptome-wide mRNA microarray profiles of neuroglial cells. These changes indicate on potential beneficial effects on neuronal functions associated with mild cognitive impairments in cancer chemotherapy.

Biopiracy versus One-World Medicine-From colonial relicts to global collaborative concepts.

BACKGROUND: Practices of biopiracy to use genetic resources and indigenous knowledge by Western companies without benefit-sharing of those, who generated the traditional knowledge, can be understood as form of neocolonialism. HYPOTHESIS: The One-World Medicine concept attempts to merge the best of traditional medicine from developing countries and conventional Western medicine for the sake of patients around the globe. STUDY DESIGN: Based on literature searches in several databases, a concept paper has been written. Legislative initiatives of the United Nations culminated in the Nagoya protocol aim to protect traditional knowledge and regulate benefit-sharing with indigenous communities. The European community adopted the Nagoya protocol, and the corresponding regulations will be implemented into national legislation among the member states. Despite pleasing progress, infrastructural problems of the health care systems in developing countries still remain. Current approaches to secure primary health care offer only fragmentary solutions at best. Conventional medicine from industrialized countries cannot be afforded by the impoverished population in the Third World. Confronted with exploding costs, even health systems in Western countries are endangered to burst. Complementary and alternative medicine (CAM) is popular among the general public in industrialized countries, although the efficacy is not sufficiently proven according to the standards of evidence-based medicine. CAM is often available without prescription as over-the-counter products with non-calculated risks concerning erroneous self-medication and safety/toxicity issues. The concept of integrative medicine attempts to combine holistic CAM approaches with evidence-based principles of conventional medicine. CONCLUSION: To realize the concept of One-World Medicine, a number of standards have to be set to assure safety, efficacy and applicability of traditional medicine, e.g. sustainable production and quality control of herbal products, performance of placebo-controlled, double-blind, randomized clinical trials, phytovigilance, as well as education of health professionals and patients.

Ancistrobreveines A-D and related dehydrogenated naphthylisoquinoline alkaloids with antiproliferative activities against leukemia cells, from the West African liana Ancistrocladus abbreviatus.

A unique series of six biaryl natural products displaying four different coupling types (5,1', 7,1', 7,8', and 5,8') were isolated from the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae). Although at first sight structurally diverse, these secondary metabolites all have in common that they belong to the rare group of naphthylisoquinoline alkaloids with a fully dehydrogenated isoquinoline portion. Among the African Ancistrocladus species, A. abbreviatus is so far only the second one that was found to produce compounds with such a molecular entity. Here, we report on four new representatives, named ancistrobreveines A-D (12-14, and 6). They were identified along with the two known alkaloids 6-O-methylhamateine (4) and ent-dioncophylleine A (10). The two latter naphthylisoquinolines had so far only been detected in Ancistrocladus species from Southeast Asia. All of these fully dehydrogenated alkaloids have in common being optically active despite the absence of stereogenic centers, due to the presence of the rotationally hindered biaryl axis as the only element of chirality. Except for ent-dioncophylleine A (10), which lacks an oxygen function at C-6, the ancistrobreveines A-D (12-14, and 6) and 6-O-methylhamateine (4) are 6-oxygenated alkaloids, and are, thus, typical 'Ancistrocladaceae-type' compounds. Ancistrobreveine C (14), is the first - and so far only - example of a 7,8'-linked fully dehydrogenated naphthylisoquinoline discovered in nature that is configurationally stable at the biaryl axis. The stereostructures of the new alkaloids were established by spectroscopic (in particular HRESIMS, 1D and 2D NMR) and chiroptical (electronic circular dichroism) methods. Ancistrobreveine C (14) and 6-O-methylhamateine (4) exhibited strong antiproliferative activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000.

Novel molecular mechanisms for the adaptogenic effects of herbal extracts on isolated brain cells using systems biology.

INTRODUCTION: Adaptogens are natural compounds or plant extracts that increase adaptability and survival of organisms under stress. Adaptogens stimulate cellular and organismal defense systems by activating intracellular and extracellular signaling pathways and expression of stress-activated proteins and neuropeptides. The effects adaptogens on mediators of adaptive stress response and longevity signaling pathways have been reported, but their stress-protective mechanisms are still not fully understood. AIM OF THE STUDY: The aim of this study was to identify key molecular mechanisms of adaptogenic plants traditionally used to treat stress and aging-related disorders, i.e., Rhodiola rosea, Eleutherococcus senticosus, Withania somnifera, Rhaponticum carthamoides, and Bryonia alba. MATERIALS AND METHODS: To investigate the underlying molecular mechanisms of adaptogens, we conducted RNA sequencing to profile gene expression alterations in T98G neuroglia cells upon treatment of adaptogens and analyzed the relevance of deregulated genes to adaptive stress-response signaling pathways using in silico pathway analysis software. RESULTS AND DISCUSSION: At least 88 of the 3516 genes regulated by adaptogens were closely associated with adaptive stress response and adaptive stress-response signaling pathways (ASRSPs), including neuronal signaling related to corticotropin-releasing hormone, cAMP-mediated, protein kinase A, and CREB; pathways related to signaling involving CXCR4, melatonin, nitric oxide synthase, GP6, Gαs, MAPK, neuroinflammation, neuropathic pain, opioids, renin-angiotensin, AMPK, calcium, and synapses; and pathways associated with dendritic cell maturation and G-coupled protein receptor-mediated nutrient sensing in enteroendocrine cells. All samples tested showed significant effects on the expression of genes encoding neurohormones CRH, GNRH, UCN, G-protein-coupled and other transmembrane receptors TLR9, PRLR, CHRNE, GP1BA, PLXNA4, a ligand-dependent nuclear receptor RORA, transmembrane channels, transcription regulators FOS, FOXO6, SCX, STAT5A, ZFPM2, ZNF396, ZNF467, protein kinases MAPK10, MAPK13, MERTK, FLT1, PRKCH, ROS1, TTN), phosphatases PTPRD, PTPRR, peptidases, metabolic enzymes, a chaperone (HSPA6), and other proteins, all of which modulate numerous life processes, playing key roles in several canonical pathways involved in defense response and regulation of homeostasis in organisms. It is for the first time we report that the molecular mechanism of actions of melatonin and plant adaptogens are alike, all adaptogens tested activated the melatonin signaling pathway by acting through two G-protein-coupled membrane receptors MT1 and MT2 and upregulation of the ligand-specific nuclear receptor RORA, which plays a role in intellectual disability, neurological disorders, retinopathy, hypertension, dyslipidemia, and cancer, which are common in aging. Furthermore, melatonin activated adaptive signaling pathways and upregulated expression of UCN, GNRH1, TLR9, GP1BA, PLXNA4, CHRM4, GPR19, VIPR2, RORA, STAT5A, ZFPM2, ZNF396, FLT1, MAPK10, MERTK, PRKCH, and TTN, which were commonly regulated by all adaptogens tested. We conclude that melatonin is an adaptation hormone playing an important role in regulation of homeostasis. Adaptogens presumably worked as eustressors ("stress-vaccines") to activate the cellular adaptive system by inducing the expression of ASRSPs, which then reciprocally protected cells from damage caused by distress. Functional investigation by interactive pathways analysis demonstrated that adaptogens activated ASRSPs associated with stress-induced and aging-related disorders such as chronic inflammation, cardiovascular health, neurodegenerative cognitive impairment, metabolic disorders, and cancer. CONCLUSION: This study has elucidated the genome-wide effects of several adaptogenic herbal extracts in brain cells culture. These data highlight the consistent activation of ASRSPs by adaptogens in T98G neuroglia cells. The extracts affected many genes playing key roles in modulation of adaptive homeostasis, indicating their ability to modify gene expression to prevent stress-induced and aging-related disorders. Overall, this study provides a comprehensive look at the molecular mechanisms by which adaptogens exerts stress-protective effects.