Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

Abstract

Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.

Fig 1. Inhibition of A549 cell motility by acetone extracts of lichens.

(A–B) Quantitative analysis of migration assay of A549 cells treated with 5 μg/ml of acetone extracts of Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, Bryoria capillaris, Hypogymnia physodes, Usnea florida and Evernia divaricata (A), and representative images of migration assay of A549 cells treated with the extracts of A. samentosa, F. nivalis, A. ochroleuca, U. florida and B. capillaris (B). (C-D) Invasion assay of A549 cells treated with 5 μg/ml of acetone extracts of A. samentosa, F. nivalis, A. ochroleuca, U. florida and B. capillaris (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.

Fig 2. Identification of lichen secondary metabolite from candidate lichens.

(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.

Fig 3. (+)-Usnic acid inhibits invasion of H1650 and H1975 human lung cancer cell.

(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.

Fig 4. (+)-Usnic acid decreases β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity.

(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.

Fig 5. (+)-Usnic acid decreases mRNA level of downstream target genes of β-catenin/LEF and c-jun/AP-1.

(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.

Fig 6. Regulation of RhoGTPases activity by (+)-usnic acid.

(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.

Fig 7. (+)-Usnic acid shows additive inhibitory activity with cetuximab.

(A-B) Invasion assay of A549 cells treated with 5 μM of (+)-usnic acid and/or various concentration of cetuximab (A), and quantitative analysis of invaded cell numbers in each group (B). Representative images are shown from three independent experiments, 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 between indicated group.