Inhibition of A549 cell motility by acetone extracts of lichens.

<p>(A–B) Quantitative analysis of migration assay of A549 cells treated with 5 μg/ml of acetone extracts of <i>Alectoria samentosa</i>, <i>Flavocetraria nivalis</i>, <i>Alectoria ochroleuca</i>, <i>Bryoria capillaris</i>, <i>Hypogymnia physodes</i>, <i>Usnea florida</i> and <i>Evernia divaricata</i> (A), and representative images of migration assay of A549 cells treated with the extracts of <i>A</i>. <i>samentosa</i>, <i>F</i>. <i>nivalis</i>, <i>A</i>. <i>ochroleuca</i>, <i>U</i>. <i>florida</i> and <i>B</i>. <i>capillaris</i> (B). (C-D) Invasion assay of A549 cells treated with 5 μg/ml of acetone extracts of <i>A</i>. <i>samentosa</i>, <i>F</i>. <i>nivalis</i>, <i>A</i>. <i>ochroleuca</i>, <i>U</i>. <i>florida</i> and <i>B</i>. <i>capillaris</i> (C), and quantitative analysis of invaded cell numbers in each group (D). Representative images were shown from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). ***p<0.001; NS, no significant difference compared to 0.01% DMSO-treated A549 cells.</p

(+)-Usnic acid inhibits invasion of H1650 and H1975 human lung cancer cell.

<p>(A-B) Invasion assay of H1650, and H1975 cells treated with 5 μM of (+)-usnic acid (A), and quantitative analysis of invaded cell numbers in each cell line (B). Representative images are shown from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). **p<0.01; ***p<0.001; NS, no significant difference compared to 0.01% DMSO-treated A549 cells.</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

Regulation of RhoGTPases activity by (+)-usnic acid.

<p>(A-C) The levels of GTP-bound Rac1, Cdc42 and RhoA were measured in A549 cells treated with 5 μM of (+)-usnic acid. GTP-Rac1 and -Cdc42 were measured using GST-PBD, and GTP-RhoA was measured using GST-RBD. The total amounts of RhoA, Rac1, and Cdc42 were also shown. The relative activities of Rac1 (A), Cdc42 (B), and RhoA (C) were determined as described in Materials and Methods. The data represent the mean ± SEM (standard error of the mean), n = 3. **p<0.01; ***p<0.001; NS, no significant difference compared to 0.01% DMSO-treated A549 cells.</p

(+)-Usnic acid decreases mRNA level of downstream target genes of β-catenin/LEF and c-jun/AP-1.

<p>(A-D) Quantitative analysis of the mRNA level of CD44, c-myc, and Cyclin D1 in A549 (A), H1650 (B), H1975 (C), and H460 (D) cells treated with 5 μM of (+)-usnic acid. Data represent mean ± S.E.M. (standard error of the mean), n = 3. *p<0.05; ***p<0.001; NS, no significant difference when compared to the 0.01% DMSO-treated group in each cell line.</p

(+)-Usnic acid decreases β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity.

<p>(A) β-Catenin-mediated transcriptional activity of TOPFLASH promoter was decreased by (+)-usnic acid treatment. HEK 293T cells were transfected with β-catenin and TOPFLASH reporter plasmid. After 12 h transfection, cells were treated with (+)-usnic acid for 48h. (B) KITENIN-mediated transcriptional activity of AP1 promoter was decreased by (+)-usnic acid treatment. The HEK 293T cells were transfected with KITENIN and AP-1 reporter plasmid. After 12 h transfection, cells were treated with (+)-usnic acid for 48h with or without EGF. Experiments were performed in at least three independent cultures, n = 3. 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 0.01% DMSO-treated HEK 293T cells.</p

Identification of lichen secondary metabolite from candidate lichens.

<p>(A) High performance liquid chromatography (HPLC) analysis of lichen acetone extracts. The %intensity of peak for the usnic acid in the extract at a concentration of 5 mg/ml was obtained by comparing to that of peak for pure 5 mg/ml usnic acid. (B–C) Migration assay of A549 cells treated with 5 μM of (+)-usnic acid (B), and quantitative analysis of wound length (C). (D–E) Invasion assay of A549 cells treated with 5 μM of (+)-usnic acid (D), and quantitative analysis of invaded cell numbers in each group (E). Representative images are shown from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). ***p<0.001; NS, no significant difference compared to 0.01% DMSO-treated A549 cells.</p

Induction of Annexin V positivity and accumulation of sub G1 population on human cancer cells by the acetone extract of <i>F. cucullata</i> and usnic acid in lethal concentrations.

<p>(A) FITC-Annexin V staining of cells treated with the <i>F. cucullata</i> extract or usnic acid. Arrows indicate cells showing FITC positivity. (B–C) Flow cytometric analysis of cell-cycle distributions after <i>F. cucullata</i> extract (B) or usnic acid (C) treatment and graphical representation of the results. Representative images or results are shown from three independent experiments.</p

Reduction of phosphor-Akt level by the acetone extract of <i>F. cucullata</i> and usnic acid in sub-lethal concentrations.

<p>Phosphoprotein analysis for p(Ser⁶³)-c-jun, p(Ser⁴⁷³)-Akt, and p-(Thr²⁰²/Tyr²⁰⁴, Thr¹⁸⁵/Tyr¹⁸⁷)-ERK1/2 in A549 cells treated with the <i>F. cucullata</i> extract, usnic acid, or lichesterinic acid.</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