Sesselja Omarsdottir

Cellular Mechanisms of the Anticancer Effects of the Lichen Compound Usnic Acid

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

In vitro and in vivo immunomodulating effects of traditionally prepared extract and purified compounds from Cetraria islandica.

Cetraria islandica (Iceland moss) has been used for centuries in folk medicine in many countries against a number of conditions, including inflammatory conditions, mainly as an aqueous extract. C. islandica contains many compounds, such as polysaccharides and secondary metabolites, some of which have established biological activity. However, very little is known about their effect on the immune system. Human monocyte-derived immature dendritic cells were cultured with an aqueous extract from C. islandica quantified with regard to the polysaccharides lichenan and isolichenan and secondary metabolites protolichesterinic and fumarprotocetraric acids. The purified compounds were also tested individually. Their effect on the maturation of the dendritic cells was assessed by measuring secretion of IL-10 and IL-12p40 and expression of surface molecules. In addition, the effect of the aqueous extract on antigen-induced arthritis in rats was investigated. The aqueous extract caused upregulated secretion of both IL-10 and IL-12p40, with IL-10 secretion being more prominent. Lichenan had similar effects, whereas isolichenan and the secondary metabolites were inactive, suggesting that the effect observed by the aqueous extract was mainly mediated by lichenan. Significantly less arthritis was observed for rats treated by the aqueous extract, administered subcutaneously, compared with rats treated with saline alone. These results suggest that the aqueous extract of C. islandica has anti-inflammatory effect, possibly by changing the cytokine secretion bias from IL-12p40 towards IL-10.

Rhamnopyranosylgalactofuranan,a new immunologically active polysaccharide from Thamnolia subuliformis

A complex polysaccharide, Ths-3, consisting mainly of rhamnopyranosyl and galactofuranosyl units, has been isolated from the water extract of the lichen Thamnolia subuliformis using ethanol fractionation, dialysis, ion-exchange chromatography, gel filtration and preparative GP-HPLC. The mean M(r) of Ths-3 was determined to be 1450 kD, and the monosaccharide composition is gal/rha/glc/xyl/man in the ratio of 40:31:13:10:6. The structure of Ths-3 was further elucidated by methylation analysis by GC-MS and NMR spectroscopy and found to be basically composed of (1-->3)-linked beta-D-galactofuranosyl units with branches on C6, and rhamnosyl units being predominantly (1-->2)-linked with branches on C3 and C4, while some units are (1-->3)-linked. Glucose, mannose and galactofuranose are found as terminal units and glucose and mannose are also (1-->4)-linked, while xylose is only present as terminal units. The trisaccharide xylglcglc was detected after partial hydrolysis of the polysaccharide. The immunomodulating activity of Ths-3 was tested in an in vitro phagocytosis assay and the classical anticomplementary assay, and proved to be active in both tests. The authors suggest the trivial name thamnolan for Ths-3.

Secondary metabolites from cetrarioid lichens: Chemotaxonomy, biological activities and pharmaceutical potential

BACKGROUND Lichens, as a symbiotic association of photobionts and mycobionts, display an unmatched environmental adaptability and a great chemical diversity. As an important morphological group, cetrarioid lichens are one of the most studied lichen taxa for their phylogeny, secondary chemistry, bioactivities and uses in folk medicines, especially the lichen Cetraria islandica. However, insufficient structure elucidation and discrepancy in bioactivity results could be found in a few studies. PURPOSE This review aimed to present a more detailed and updated overview of the knowledge of secondary metabolites from cetrarioid lichens in a critical manner, highlighting their potentials for pharmaceuticals as well as other applications. Here we also highlight the uses of molecular phylogenetics, metabolomics and ChemGPS-NP model for future bioprospecting, taxonomy and drug screening to accelerate applications of those lichen substances. CHAPTERS The paper starts with a short introduction in to the studies of lichen secondary metabolites, the biological classification of cetrarioid lichens and the aim. In light of ethnic uses of cetrarioid lichens for therapeutic purposes, molecular phylogeny is proposed as a tool for future bioprospecting of cetrarioid lichens, followed by a brief discussion of the taxonomic value of lichen substances. Then a delicate description of the bioactivities, patents, updated chemical structures and lichen sources is presented, where lichen substances are grouped by their chemical structures and discussed about their bioactivity in comparison with reference compounds. To accelerate the discovery of bioactivities and potential drug targets of lichen substances, the application of the ChemGPS NP model is highlighted. Finally the safety concerns of lichen substances (i.e. toxicity and immunogenicity) and future-prospects in the field are exhibited. CONCLUSION While the ethnic uses of cetrarioid lichens and the pharmaceutical potential of their secondary metabolites have been recognized, the knowledge of a large number of lichen substances with interesting structures is still limited to various in vitro assays with insufficient biological annotations, and this area still deserves more research in bioactivity, drug targets and screening. Attention should be paid on the accurate interpretation of their bioactivity for further applications avoiding over-interpretations from various in vitro bioassays.

Ethanol extract from birch bark (Betula pubescens) suppresses human dendritic cell mediated Th1 responses and directs it towards a Th17 regulatory response in vitro.

Extracts and fractions from birch bark have been used to treat various diseases, such as skin disorders and rheumatism, and for analgesic effects. Results from studies in vitro and in vivo have shown that birch bark extracts can have immunoregulatory effects. These effects have mainly been attributed to the various triterpenes found in birch bark. The effects of birch bark from Betula pubescens on immune responses have not been reported. Ethanol extract was prepared from dry birch bark (DBBEE) and five fractions made using various ratios of dichloromethane and methanol (fractions I-V). Human monocyte-derived dendritic cells (DCs) were matured with or without DBBEE or fractions I-V at several concentrations. The effects of the extract and fractions on DC maturation were determined by measuring cytokine secretion by ELISA and expression of surface molecules by flow cytometry. DBBEE and fractions III and IV reduced DC secretion of IL-6, IL-10 and IL-12p40 and expression of CD83, CD86, CCR7 and DC-SIGN compared with control DCs. Proliferation of allogeneic CD4(+) T cells co-cultured with DCs matured with fraction IV, as measured by (3)H-thymidine incorporation, was similar to proliferation of allogeneic CD4(+) T cells co-cultured with control DCs. However, IFN-γ secretion was reduced and IL-10 and IL-17 secretion was increased, a cytokine profile consistent with a Th17 regulatory phenotype. These results indicate that bark from Betula pubescens contains compound(s) that can modulate DCs so that their interaction with T cells leads to an immunoregulatory response.

Aqueous extracts from Menyanthes trifoliate and Achillea millefolium affect maturation of human dendritic cells and their activation of allogeneic CD4+ T cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Menyanthes trifoliate and Achillea millefolium have been used in traditional medicine to ameliorate chronic inflammatory conditions. The aim of this study was to identify the effects of ethanol and aqueous extracts of Menyanthes trifoliate and Achillea millefolium on maturation of dendritic cells (DCs) and their ability to activate allogeneic CD4(+) T cells. MATERIALS AND METHODS Human monocyte-derived DCs were matured in the absence or presence of lyophilised aqoueous or ethanol extracts from Menyanthes trifoliate or Achillea millefolium and their expression of surface molecules analysed with flow cytometry and cytokine secretion measured by ELISA. DCs matured in the presence of aqueous extracts from Menyanthes trifoliate and Achillea millefolium were co-cultured with allogeneic CD4(+) T cells and the expression of surface molecules by T cells and their cytokine secretion and cell proliferation determined. RESULTS Maturation of DCs in the presence of aqueous extracts from Menyanthes trifoliate or Achillea millefolium did not affect expression of the surface molecules examined but reduced the ratio of secreted IL-12p40/IL-10, compared with that by DCs matured in the absence of extracts. Allogeneic CD4(+) T cells co-cultured with DCs matured in the presence of aqueous extract from Menyanthes trifoliate secreted less IFN-γ, IL-10 and IL-17 than CD4(+) T cells co-cultured with DCs matured without an extract. Maturation of DCs in the presence of aqueous extract from Achillea millefolium decreased IL-17 secretion but did not affect IFN-γ and IL-10 secretion by allogeneic CD4(+) T cells. CONCLUSIONS Aqueous extract from Menyanthes trifoliate induces a suppressive phenotype of DCs that has reduced capacity to induce Th1 and Th17 stimulation of allogeneic CD4(+) T cells, whereas aqueous extract from Achillea millefolium reduces the capacity of DCs to induce a Th17 response.

Structural characterisation of a complex heteroglycan from the cyanobacterium Nostoc commune

An alkali-extractable O-methylated ribofuranose-containing heteroglycan, Nc-5-s, was isolated from wild-growing field colonies of the cyanobacterium Nostoc commune collected in Iceland, using ethanol fractionation and anion-exchange chromatography. The average molecular weight was estimated to be 1500 kDa. Structural characterisation of the heteroglycan was performed by high-field NMR spectroscopy (1D proton, 2D-COSY, 2D-NOESY, 2D-TOCSY, (1)H (13)C-HSQC, HMBC, H2BC and HSQC-NOESY) as well as monosaccharide analysis after methanolysis by GC and supported by linkage analysis by GC-MS. According to the data obtained, the structure of Nc-5-s is composed of repeating units of 1, 1a, 1b and 2 and 2a in approximate molar ratio of (10:25:50:5:10).

Marchantin A, a macrocyclic bisbibenzyl ether, isolated from the liverwort Marchantia polymorpha, inhibits protozoal growth in vitro.

In vitro anti-plasmodial activity-guided fractionation of a diethyl ether extract of the liverwort species Marchantia polymorpha, collected in Iceland, led to isolation of the bisbibenzyl ether, marchantin A. The structure of marchantin A (1) was confirmed by NMR and HREIMS. Marchantin A inhibited proliferation of the Plasmodium falciparum strains, NF54 (IC(50)=3.41μM) and K1 (IC(50)=2.02μM) and showed activity against other protozoan species Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani with IC(50) values 2.09, 14.90 and 1.59μM, respectively. Marchantin A was tested against three recombinant enzymes (PfFabI, PfFabG and PfFabZ) of the PfFAS-II pathway of P. falciparum for malaria prophylactic potential and showed moderate inhibitory activity against PfFabZ (IC(50)=18.18μM). In addition the cytotoxic effect of marchantin A was evaluated. This is the first report describing the inhibitory effects of the liverwort metabolite marchantin A against these parasites in vitro.

Effects of anti-proliferative lichen metabolite, protolichesterinic acid on fatty acid synthase, cell signalling and drug response in breast cancer cells

BACKGROUND The lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75. AIMS To test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated. MATERIALS AND METHODS Pure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)(®) assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software. RESULTS Treatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells. CONCLUSION Results suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.

Proton-shuttling lichen compound usnic acid affects mitochondrial and lysosomal function in cancer cells.

The lichen compound usnic acid (UA) is a lipophilic weak acid that acts as a proton shuttle and causes loss of mitochondrial inner membrane potential. In the current study we show that UA treatment induced the formation of autophagosomes in human cancer cells, but had minimal effects on normal human fibroblasts. However, autophagic flux was incomplete, degradation of autophagosomal content did not occur and acidification was defective. UA-treated cells showed reduced ATP levels and activation of AMP kinase as well as signs of cellular stress. UA is thus likely to trigger autophagosome formation both by energy depletion and stress conditions. Our findings indicate that the H+-shuttling effect of UA operates not only in mitochondria as previously shown, but also in lysosomes, and have implications for therapeutic manipulation of autophagy and pH-determined drug distribution.