Phylogenetic tree of the isolated <i>L</i>. <i>salivarius</i> strains.

<p>(A) Hierarchical clustering of <i>L</i>. <i>salivarius</i> strains based on orthologous CDS contents. (B) Phylogenetic analysis of MLST sequences found in the strains. The bootstrap consensus tree inferred from 1,000 replicates is taken.</p

Comparison of RAST subsystems of the isolated genomes.

<p>The genomes were annotated and grouped by RAST with default options for bacteria. The different colors show the number of genes that incorporated in each subsystem (see color bar). Statistical analysis was carried out using Fisher's exact test between SBPs and SAPs, and the significantly different subsystems are shown in the figure (P < 0.05).</p

Phylogenetic clustering with extracellular protein genes of SAPs and SBPs.

<p>(A) Tree based on the presence of 30 extracellular protein genes of SAPs and SBPs. (B) Hierarchical clustering based on amino acid sequences of the 14 core genes for extracellular protein. The bootstrap consensus tree inferred from 1,000 replicates is taken.</p

Averages of genomic features in SBPs and SAPs (mean ± standard deviation).

<p>Averages of genomic features in SBPs and SAPs (mean ± standard deviation).</p

Lichen Secondary Metabolites in <i>Flavocetraria cucullata</i> Exhibit Anti-Cancer Effects on Human Cancer Cells through the Induction of Apoptosis and Suppression of Tumorigenic Potentials

<div><p>Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, <i>F. cucullata</i> exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of <i>F. cucullata</i> contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, <i>F. cucullata</i> and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.</p></div

Additional fie 1:

Figure S1. qRT-PCR analysis of RANK-RANKL signaling-related gene expression to optimize the concentration of commercial sRANKL. The mRNA expressions of TRAF6, NFATc1, and TRAP were analyzed at day 6 after exposure of commercial sRANKL (20-50 ng/ml) to RAW 264.7 cells. The mRNA levels were normalized by GAPDH expression, and expressed as relative gene expression compared to control. For significance tests, a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test were used, and expressed as follows; *P < 0.05, **P < 0.01, ***P < 0.001. Figure S2. Schematic illustration of IHC. Peyer’s patches were isolated from small intestine and fixed with 4 % (v/v) paraformaldehyde for 2 h at 4 °C. The tissues were blocked with 3 % goat serum at RT for 1 h and incubated with Alexa488-labeled GP-2 monoclonal antibody (1:400 dilution) at 4 °C overnight. The tissues were mounted on coverglass bottom dishes. (PDF 232 kb

Activation of apoptosis pathway on human cancer cells by the acetone extract of <i>F. cucullata</i> and usnic acid in lethal concentrations.

<p>(A and D) Western blot analysis of poly (ADP-ribose) polymerase (PARP) and caspase-3 in cells treated with the <i>F. cucullata</i> (A) or usnic acid (D). Arrowheads indicate cleaved fragments of each protein. (B–C and E–F) Quantificational analysis of Bax (B and E) and Bcl-xL (C and F) protein expression levels in cells treated with the F. cucullata or usnic acid, respectively. Data represent mean ± S.E.M. (standard error of the mean). *p<0.05; **p<0.01; ***p<0.001; NS, no significant difference compared to the dimethylsulfoxide-treated group.</p

Induction of nuclear condensation of human cancer cells by the acetone extract of <i>F. cucullata</i> and its main component, usnic acid in lethal concentrations.

<p>(A) Hoechst 33258 staining of AGS (human gastric cancer cell line) cells treated with the <i>F. cucullata</i> extract or its subcomponents, usnic acid and lichesterinic acid. Arrows indicate cells showing condensed or fragmented nuclear morphology. Representative images are shown from three independent experiments. (B) Quantificational analysis of condensed or fragmented nuclear morphology in various cells treated with <i>F. cucullata</i> extract or its subcomponents. Data represent mean ± S.E.M. (standard error of the mean), n = 3. **p<0.01; ***p<0.001; NS, no significant difference compared to the dimethylsulfoxide-treated group.</p

Cytotoxic effects of the acetone extract of <i>F. cucullata</i> and its main component, usnic acid, on human cancer cells.

<p>(A) Percent viability of cells treated with the acetone extract of <i>F. cucullata</i>. Cells were treated with the <i>F. cucullata</i> extract in a concentration ranging from 10–50 µg/mL for 48 hr, and cell viability was measured by an MTT assay. (B) High performance liquid chromatography chromatograms of the <i>F. cucullata</i> extract. The identity of each subcomponent is noted on the corresponding peak. (C–D) The percent viability of cells treated with either usnic acid (C) or lichesterinic acid (D). Cells were treated with the indicated subcomponent of <i>F. cucullata</i> in a concentration ranging from 12.5–50 µM for 48 hr, and cell viability was measured by an MTT assay. Data represent means ± S.E.M. (standard error of the mean), n = 3.</p

Inhibition of <i>in</i><i>vivo</i> tumorigenicity of A549 cancer cells pretreated by the acetone extract of F. cucullata and usnic acid in sub-lethal concentrations.

<p>A549 cells were pretreated with indicated concentration of <i>F. cucullata</i>, usnic acid, or lichesterinic acid before subcutaneous injection into Balb/c nude mouse (n = 8) and tumor free survival number in each group during 4 weeks were measured.</p