Lichen Secondary Metabolite, Physciosporin, Inhibits Lung Cancer Cell Motility

Abstract

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action.

Fig 1. Acetone extracts of lichens collected in Chile inhibited A549 cell motility.

(A) Quantitative analysis of migration assays of A549 cells treated with 5 μg/mL of acetone extracts of Pseudocyphellaria glabra, Pseudocyphellaria coriacea, Nephroma sp., Physcia sp., Flavoparmelia caperata, Hypotrachyna sinuosa, Rhizoplaca melanophthalma, Rhizoplaca melanophthalma, Pseudocyphellaria argyracea, Pseudocyphellaria verrucosa, Xanthoparmelia sp., Protousnea sp. and Xanthoparmelia sp.. (B) Representative images of migration assays of A549 cells treated with the extracts of R. melanophthalma, H. sinuosa, P. coriacea, P. glabra and Nephroma sp. (C-D) Invasion assays of A549 cells treated with 5 μg/mL of acetone extracts of R. melanophthalma, H. sinuosa, P. coriacea, P. glabra and Nephroma sp. (C) and quantitative analysis of invaded cell numbers in each treatment (D). Quantitative data were obtained from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). ***p<0.001 compared to DMSO-treated A549 cells.

Fig 2. Physciosporin was identified as an active lichen secondary metabolite from P. coriacea in the inhibition of lung cancer cell motility.

(A-B) Thin Layer Chromatography (TLC) analysis using (Toluene: Dioxin: Acetic acid = 180: 45: 5, v/v/v) solvent system for lichen extracts having inhibitory activity in A549 cell motility (A), the lichens in Pseudocyphellaria genus (B). ‘a’ denotes location of spot for usnic acid; ‘b’, for atranorin; ‘c’, for physciosporin; ‘d’, for tenuiorin. Lichen species L. cladonioides and U. longissimi were used as standard control for atranorin and usnic acid, respectively. (C) Chemical structure of physciosporin. (D-E) Migration assay of A549 cells treated with 5 μg/mL of acetone extract of P. coriacea or 5 μg/mL physciosporin (D), and quantitative analysis of wound length (E). (F–G) Invasion assays of A549 cells treated with 5 μg/mL of acetone extract of P. coriacea or physciosporin (F), and quantitative analysis of invaded cell numbers in each treatment (G). Quantitative data were obtained from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). ***p<0.001 compared to DMSO-treated A549 cells.

Fig 3. Acetone extract of P. coriacea and physciosporin inhibited lung cancer cell motility.

(A-B) Invasion assays of H1650 and H1975 lung cancer cells treated with 5 μg/mL of acetone extract of P. coriacea or 5 μg/mL physciosporin (A), and quantitative analysis of invaded cell number in each treatment (B). Quantitative data were obtained from three independent experiments, n = 3. Data represent mean ± S.E.M. (standard error of the mean). ***p<0.001 compared to DMSO-treated A549 cells.

Fig 4. Acetone extract of P. coriacea and physciosporin decreased the level of epithelial-mesenchymal transition marker.

(A-B) Western blot analysis of E-cadherin (A) and N-cadherin (B) in A549 cells treated with 5 μg/mL of acetone extracts of P. coriacea or physciosporin. (C) Quantitative analysis of the mRNA levels of N-cadherin, Snail and Twist in A549 cells treated with 5 μg/mL of acetone extracts of P. coriacea or physciosporin. Quantitative data were obtained from at least two independent experiments. Data represent mean ± S.E.M. (standard error of the mean). *p<0.05; **p<0.01; ***p<0.001 compared to DMSO-treated A549 cells.

Fig 5. Acetone extract of P. coriacea and physciosporin suppressed KITENIN-mediated AP-1 activity and affected the expression of KAI1 and KITENIN.

(A) AP-1 luciferase assay of HEK293T cells transfected with KITENIN and treated with 5 μg/mL of acetone extract of P. coriacea or physciosporin in the absence or presence of epidermal growth factor stimulation. (B-C) Quantitative analysis of the mRNA level of KITENIN (B) and KAI1 (C) in A549 cells treated with 5 μg/mL of acetone extract of P. coriacea or physciosporin. (D-E) KITENIN promoter (D) and 3’-UTR (E) luciferase assays of HEK293T cells treated with 5 μg/mL of acetone extract of P. coriacea or physciosporin. Quantitative data were obtained from at least three independent experiments. Data represent mean ± S.E.M. (standard error of the mean). *p<0.05; **p<0.01; ***p<0.001 compared to DMSO-treated cells.

Fig 6. Acetone extract of P. coriacea and physciosporin reduced RhoGTPase activity.

(A-B) The levels of GTP-bound Cdc42 (A) and Rac1 (B) were measured in A549 cells treated with 5 μg/mL of acetone extract of P. coriacea or physciosporin. GTP-Cdc42 and -Rac1 were measured using GST-PBD. The total amounts of Cdc42 and Rac1 were shown to measure relative activity. Quantitative data were obtained from three independent experiments, n = 3. Data represent the mean ± S.E.M. (standard error of the mean). ***p<0.001 compared to DMSO-treated A549 cells.