Tumidulin, a Lichen Secondary Metabolite, Decreases the Stemness Potential of Colorectal Cancer Cells

Abstract

Lichens produce various unique chemicals that are used in the pharmaceutical industry. To screen for novel lichen secondary metabolites that inhibit the stemness potential of colorectal cancer cells, we tested acetone extracts of 11 lichen samples collected in Chile. Tumidulin, isolated from Niebla sp., reduced spheroid formation in CSC221, DLD1, and HT29 cells. In addition, mRNA expressions and protein levels of cancer stem markers aldehyde dehydrogenase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1 were reduced after tumidulin treatment. Tumidulin decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli) promoter in reporter assays, and western blotting confirmed decreased Gli1, Gli2, and Smoothened (SMO) protein levels. Moreover, the tumidulin activity was not observed in the presence of Gli and SMO inhibitors. Together, these results demonstrate for the first time that tumidulin is a potent inhibitor of colorectal cancer cell stemness.

Keywords: colorectal cancer cells; lichen; oncogene; secondary metabolites; stemness potential; transcriptional regulation; tumidulin.

Figure 1

Acetone extracts of lichens collected in Chile decrease CSC221 cell stemness. (a) Quantitative analysis of Gli-luc reporter assays of NIH 3T3 cells (stably incorporating Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters) treated with 5 μg/mL acetone extracts of Ramalina sp. (1), Everniopsis trulla, Neocatapyrenium sp., Niebla sp. (1), Roccella sp., Niebla sp. (2), Niebla sp. (3), Neocatapyrenium sp., Everniopsis trulla, Niebla sp. (4), and Ramalina sp. (2) for 48 h. (b) Representative images of spheroid formation of CSC221 cells treated with extracts of Niebla sp. (1) or Niebla sp. (4) for 14 days. (c) Quantitative analysis of the number of spheroids following each treatment. Quantitative data were obtained from three independent experiments (n = 3). Data represent mean ± standard error of the mean (SEM), and analysis was performed by one-way ANOVA. *** p < 0.001 compared with dimethyl sulfoxide (DMSO)-treated CSC221 cells.

Figure 1

Acetone extracts of lichens collected in Chile decrease CSC221 cell stemness. (a) Quantitative analysis of Gli-luc reporter assays of NIH 3T3 cells (stably incorporating Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters) treated with 5 μg/mL acetone extracts of Ramalina sp. (1), Everniopsis trulla, Neocatapyrenium sp., Niebla sp. (1), Roccella sp., Niebla sp. (2), Niebla sp. (3), Neocatapyrenium sp., Everniopsis trulla, Niebla sp. (4), and Ramalina sp. (2) for 48 h. (b) Representative images of spheroid formation of CSC221 cells treated with extracts of Niebla sp. (1) or Niebla sp. (4) for 14 days. (c) Quantitative analysis of the number of spheroids following each treatment. Quantitative data were obtained from three independent experiments (n = 3). Data represent mean ± standard error of the mean (SEM), and analysis was performed by one-way ANOVA. *** p < 0.001 compared with dimethyl sulfoxide (DMSO)-treated CSC221 cells.

Figure 2

Tumidulin is an active lichen secondary metabolite from Niebla sp. (1) that inhibits CSC221 cell stemness. (a) High-performance liquid chromatography (HPLC) analysis of the inhibitory activity of stemness in CSC221 cells by crude and active Niebla sp. (1) fractions using a methanol:water:phosphoric acid (80:20:1, v/v/v) solvent system. (b) Quantitative analysis of reporter assays of NIH 3T3 cells treated with a 5 μg/mL acetone extract of Niebla sp. and various concentrations of the active (tumidulin) fraction for 48 h. (c) Chemical structure of tumidulin. (d) Relative viability of CSC221 cells treated with tumidulin for 48 h by MTT assay. Quantitative data were obtained from three independent experiments (n = 3). Data represent mean ± SEM, and analysis was performed by one-way ANOVA. ** p < 0.01 and *** p < 0.001 compared with DMSO-treated CSC 221 cells.

Figure 3

Effects of crude acetone extracts of Niebla sp. (1) and tumidulin on spheroid formation of colorectal cancer (CRC) cells. CRC cells were treated with crude extracts of Niebla sp. (1) and various concentrations of tumidulin. After 14 days incubation, spheroid formation of CRC cells was calculated as described in the Materials and Methods. (a) Representative images of spheroid formation of CSC221, DLD1, and HT29 cells following treatment with crude extracts of Niebla sp. (1) and the purified tumidulin fraction at the indicated concentrations. (b–d) Quantitative spheroid assay data following treatment of CSC221 (b), DLD1 (c), and HT29 (d) cells with crude extracts of Niebla sp. (1) and the purified tumidulin fraction. Quantitative data were obtained from three independent experiments (n = 3). Data represent mean ± SEM, and analysis was performed by one-way ANOVA. ** p < 0.01; *** p < 0.001; NS: no significant difference compared with DMSO-treated CRC cells.

Figure 4

Effects of crude extracts of Niebla sp. (1) and tumidulin on cancer stemness-related gene expression and protein level. CSC221 cells were treated with 5 μg/mL acetone extracts of Niebla sp. (1) or various concentrations of tumidulin for 48 h. (a) Quantitative analysis of mRNA expression levels of cancer stem markers aldehyde dehydrigebase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1. Data represent mean ± SEM, and analysis was performed by one-way ANOVA. * p < 0.05; ** p < 0.01; NS: no significant difference compared with DMSO-treated CSC221 cells. (b) Representative immunoblots are shown with the indicated antibodies.

Figure 5

Effects of crude acetone extracts of Niebla sp. (1) and tumidulin on transcription factor activity and the expression of proteins related to cancer stemness. (a–c) Relative activities of promoters related to Hedgehog, Wnt, and Notch signaling pathways. HEK293T cells were co-transfected with the pRL-TK (Renilla) plasmid and the pGli-luc (a), pTOPFLASH (b), and pHES-luc (c) reporter plasmids (firefly). After 12 h, transfected cells were treated with crude extracts of Niebla sp. (1) or various concentrations of tumidulin and incubated for an additional 48 h. The relative firefly luciferase activity of treated vs. control groups is shown. Quantitative data were obtained from at least two independent experiments. (d–g) CSC221 cells were treated with 5 μg/mL Niebla sp. (1) or various concentrations of tumidulin. After incubation for 48 h, cells were lysed and total protein was subjected to immunoblot analyses with the indicated antibodies. (d,e) Representative immunoblots of Gli1/2 (d) and SMO (e). (f,g) Relative protein levels of Gli1/2 (f) and SMO (g) compared with the untreated group. Data represent the mean ± SEM, and analysis was performed by one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; NS: no significant difference compared with DMSO-treated CSC221 cells.

Figure 6

Effects of tumidulin on mRNA expression levels of ALDH1 in the presence of Gli, SMO, and mTOR inhibitors. CSC221 cells were treated with 5 μg/mL GANT61 (Gli inhibitor), GDC-0449 (SMO inhibitor), and rapamycin (mTOR inhibitor) together with or without 12.5 µM tumidulin for 48 h. Quantitative analysis of mRNA expression level of ALDH1 were presented as mean ± SEM, (n = 3). Analysis was performed by one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; NS: no significant difference compared with DMSO-treated CSC221 cells or between indicated groups.