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

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.


Introduction
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide [1], and its high mortality rate has made it a priority in clinical cancer therapy [2]. Large numbers of CRC patients present with metastatic disease [3], and the 5-year survival rate is only 12.5% [3]. Furthermore, resistance to chemotherapy occurs in 90% of patients with metastatic cancer, and this is believed to be the main reason for treatment failure, despite the development of numerous therapeutic agents targeting CRC in the past decade [4]. Mounting evidence indicates that cancer stem cells (CSCs) contribute to chemotherapy resistance [5][6][7], and recent studies suggest that the efficiency of chemotherapy could be improved by exploiting the therapeutic opportunities provided by the molecular properties of CSCs. Cluster of differentiation (CD) 133 [8,9], CD44 [10], and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) [11] are colorectal CSC markers related to proliferation, invasion, metastasis, and chemoresistance, and aldehyde dehydrogenase (ALDH) activity has been linked to the mechanism of chemoresistance in CSCs [12]. Therefore, it is important to seek new therapeutic strategies targeting CSC-like properties.
The extensive chemical diversity of lichen secondary metabolites makes them a powerful natural resource for candidate pharmaceutical reagents. In a previous study, we described the wide pharmaceutical application of lichen bioresources in anti-cancer chemotherapy reagent screening [13][14][15][16]. However, no attempt has been made to screen anti-cancer activity based on CSC-like traits, which has the potential to identify more efficient chemotherapeutic agents. Therefore, in the present work, we examined the inhibitory activity of 11 lichen species from Chile against the stemness potential of CRC cells, and investigated the possible molecular mechanisms underlying this inhibitory activity to identify novel compounds that could lower the chemoresistance of target therapeutic drugs.

Acetone Extracts of Lichens Collected in Chile Inhibit CRC Cell Stemness
The self-renewing ability of cancer stem cells (CSCs) can result in resistance to targeted therapies. To identify inhibitory substances among lichen secondary metabolites, Gli luciferase activity was tested against 11 acetone extracts of Chilean lichen thalli for in the NIH 3T3 cell line in which a Gli reporter gene was stably incorporated. For this, 5 µg/mL concentration of extracts was tested as cytotoxicity was not observed at this concentration ( Figure S1). As shown in Figure 1a, Niebla sp. (1) and Niebla sp. (4) both inhibited Gli-luc activity at a concentration of 5 µg/mL. To further check whether the above lichen extracts possessed inhibitory activity against the spheroid formation ability of CSC221 cells (human colorectal adenocarcinoma-enriched cancer stem cells [17]), spheroid formation assays were performed using ultra-low attachment 24-well plates with serum-free medium. As shown in Figure 1b, the number of spheroids in cells treated with acetone extracts was lower than in dimethyl sulfoxide (DMSO)-treated controls, and quantitative analysis revealed that the differences were significant (Figure 1c). These results showed that acetone extracts of Niebla sp. (1) and Niebla sp. (4) exhibited inhibitory activity against colorectal cancer (CRC) cell stemness. Molecules 2018, 23, x; doi: www.mdpi.com/journal/molecules chemotherapy could be improved by exploiting the therapeutic opportunities provided by the molecular properties of CSCs. Cluster of differentiation (CD) 133 [8,9], CD44 [10], and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) [11] are colorectal CSC markers related to proliferation, invasion, metastasis, and chemoresistance, and aldehyde dehydrogenase (ALDH) activity has been linked to the mechanism of chemoresistance in CSCs [12]. Therefore, it is important to seek new therapeutic strategies targeting CSC-like properties. The extensive chemical diversity of lichen secondary metabolites makes them a powerful natural resource for candidate pharmaceutical reagents. In a previous study, we described the wide pharmaceutical application of lichen bioresources in anti-cancer chemotherapy reagent screening [13][14][15][16]. However, no attempt has been made to screen anti-cancer activity based on CSC-like traits, which has the potential to identify more efficient chemotherapeutic agents. Therefore, in the present work, we examined the inhibitory activity of 11 lichen species from Chile against the stemness potential of CRC cells, and investigated the possible molecular mechanisms underlying this inhibitory activity to identify novel compounds that could lower the chemoresistance of target therapeutic drugs.

Acetone Extracts of Lichens Collected in Chile Inhibit CRC Cell Stemness
The self-renewing ability of cancer stem cells (CSCs) can result in resistance to targeted therapies. To identify inhibitory substances among lichen secondary metabolites, Gli luciferase activity was tested against 11 acetone extracts of Chilean lichen thalli for in the NIH 3T3 cell line in which a Gli reporter gene was stably incorporated. For this, 5 µg/mL concentration of extracts was tested as cytotoxicity was not observed at this concentration ( Figure S1). As shown in Figure 1a, Niebla sp. (1) and Niebla sp. (4) both inhibited Gli-luc activity at a concentration of 5 µg/mL. To further check whether the above lichen extracts possessed inhibitory activity against the spheroid formation ability of CSC221 cells (human colorectal adenocarcinoma-enriched cancer stem cells [17]), spheroid formation assays were performed using ultra-low attachment 24-well plates with serum-free medium. As shown in Figure 1b, the number of spheroids in cells treated with acetone extracts was lower than in dimethyl sulfoxide (DMSO)-treated controls, and quantitative analysis revealed that the differences were significant (Figure 1c). These results showed that acetone extracts of Niebla sp.

Tumidulin, A Lichen Secondary Metabolite from Niebla sp., Inhibits CRC Cell Stemness
The chemical compounds isolated from Niebla sp. (1; CH130494) and Niebla sp. (4; CH130414) were almost identical according to the results of thin layer chromatography (TLC) ( Figure S2); hence Niebla sp. (1) was chosen for subsequent research. To investigate the active compounds possessing inhibitory activity against CRC cell stemness, an acetone extract of Niebla sp. (1) was analyzed by high-performance liquid chromatography (HPLC) (Figure 2a), and the three main fractions were collected and tested in Gli-luc reporter assays using NIH 3T3 cell lines. The fraction II decreased Gliluc activity in a dose-dependent manner comparable to crude extract ( Figure 2b). This active fraction was therefore used for purification and structural identification of active components. The active, purified fraction was confirmed as tumidulin (molecular weight = 401.192 g/mol, purity > 99%) by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses ( Figure 2c; Figures S3-S7 in File S1). The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay revealed that cell viability was not significantly affected by tumidulin on CSC221 cells at less than 5 µg/mL (= 12.5 µM) concentration ( Figure 2d). These results indicate that tumidulin is the active compound responsible for the inhibitory activity against CRC cell stemness in the Niebla sp. (1) acetone extract.   (Figure 2a), and the three main fractions were collected and tested in Gli-luc reporter assays using NIH 3T3 cell lines. The fraction II decreased Gli-luc activity in a dose-dependent manner comparable to crude extract ( Figure 2b). This active fraction was therefore used for purification and structural identification of active components. The active, purified fraction was confirmed as tumidulin (molecular weight = 401.192 g/mol, purity > 99%) by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses ( Figure 2c; Figures S3-S7 in File S1). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that cell viability was not significantly affected by tumidulin on CSC221 cells at less than 5 µg/mL (= 12.5 µM) concentration ( Figure 2d). These results indicate that tumidulin is the active compound responsible for the inhibitory activity against CRC cell stemness in the Niebla sp. (1) acetone extract. . 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.

Tumidulin Inhibits Spheroid Formation in CRC Cells
To further confirm the CRC cell stemness inhibitory activity of tumidulin, the reduction in cancer stemness by acetone crude extracts of Niebla sp. (1) and tumidulin was evaluated by measuring spheroid formation in various CRC cell lines. As shown in Figure 3a, the number of spheroids following treatment with a 5 µg/mL crude extract of Niebla sp. (1) and various concentrations of tumidulin was significantly less than in DMSO-treated control cells for all CRC cell lines tested, including CSC221, DLD1, and HT29 cells. However, their profiles at the same concentrations 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.

Tumidulin Inhibits Spheroid Formation in CRC Cells
To further confirm the CRC cell stemness inhibitory activity of tumidulin, the reduction in cancer stemness by acetone crude extracts of Niebla sp. (1) and tumidulin was evaluated by measuring spheroid formation in various CRC cell lines. As shown in Figure 3a, the number of spheroids following treatment with a 5 µg/mL crude extract of Niebla sp. (1) and various concentrations of tumidulin was significantly less than in DMSO-treated control cells for all CRC cell lines tested, including CSC221, DLD1, and HT29 cells. However, their profiles at the same concentrations exhibited some differences. Tumidulin did not inhibit spheroid formation in CSC221 cells at 1.25 µg/mL (= 3.1 µM), but it did decrease the spheroid number in the other two CRC cell lines (DLD1 and HT29). Quantitative analysis confirmed that tumidulin inhibited spheroid formation in a dose-dependent manner for all three CRC cells (EC 50 CSC221 = 2.609 µg/mL = 6.523 µM; EC 50 DLD1 = 1.926 µg/mL = 4.815 µM; EC 50 HT29 = 1.944 µg/mL = 4.860 µM) (Figure 3b,c). It should be noted that cell viability was not significantly affected by acetone extracts of Niebla sp. (1) and tumidulin on tested CRC cells at 12.5 µM (= 5 µg/mL) concentration ( Figure S8), suggesting that cell death or blockade of cell proliferation is not involved in these effects. Taken together, these results indicate that tumidulin decreased the stemness of CRC cells.  (Figure 3b,c). It should be noted that cell viability was not significantly affected by acetone extracts of Niebla sp. (1) and tumidulin on tested CRC cells at 12.5 µM (= 5 µg/mL) concentration ( Figure S8), suggesting that cell death or blockade of cell proliferation is not involved in these effects. Taken together, these results indicate that tumidulin decreased the stemness of CRC cells. (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.

Tumidulin Decreases Cancer Stem Markers
ALDH1, CD133, CD44, Lgr5, and Msi-1 are markers for the acquisition of cancer stemness. Since tumidulin inhibited spheroid formation in CRC cells (Figure 3), we hypothesized that the expression of CSC markers could be downregulated at compound concentrations at which spheroid formation was reduced. To investigate whether tumidulin affected the expression of cancer stemness markers in CRC cells, mRNA expressions and protein levels of ALDH1, CD133, CD44, Lgr5, and Msi-1 were (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.

Tumidulin Decreases Cancer Stem Markers
ALDH1, CD133, CD44, Lgr5, and Msi-1 are markers for the acquisition of cancer stemness. Since tumidulin inhibited spheroid formation in CRC cells (Figure 3), we hypothesized that the expression of CSC markers could be downregulated at compound concentrations at which spheroid formation was reduced. To investigate whether tumidulin affected the expression of cancer stemness Molecules 2018, 23, 2968 6 of 13 markers in CRC cells, mRNA expressions and protein levels of ALDH1, CD133, CD44, Lgr5, and Msi-1 were measured by qRT-PCR and western blotting, respectively. As shown in Figure 4a,b, the inhibitory activity of tumidulin was similar to that of the crude acetone extract of Niebla sp. (1), and it reduced mRNA expressions and protein levels of all five cancer stem markers at concentrations of 2.5 and 5 µg/mL (= 6.25 and 12.5 µM).
Molecules 2018, 23, x 6 of 13 Molecules 2018, 23, x; doi: www.mdpi.com/journal/molecules measured by qRT-PCR and western blotting, respectively. As shown in Figure 4a,b, the inhibitory activity of tumidulin was similar to that of the crude acetone extract of Niebla sp. (1), and it reduced mRNA expressions and protein levels of all five cancer stem markers at concentrations of 2.5 and 5 µg/mL (= 6.25 and 12.5 µM).

Tumidulin Reduces Gli and SMO Protein Levels
To identify target signaling pathways involved in the inhibition of cancer stemness by tumidulin, reporter assays were performed using the HEK293T cell line transfected with Gli-luc, TOPFLASH-luc, and Hes-1-luc genes conjugated with firefly plasmids. Based on the relative values calculated with Renilla luciferase as an internal control, only Gli-luc activity was downregulated significantly by tumidulin treatment in a dose-dependent manner (Figure 5a-c). Since Gli is the main transcription factor in the sonic hedgehog (SHH) signaling pathway, this pathway is likely involved in the reduction of CRC stemness by tumidulin treatment. These results suggest that tumidulin might decrease CRC cancer stemness by downregulating the expression of CSC markers and inhibiting Gli transcription.
To confirm the involvement of the SHH signaling pathway in the CRC stemness reduction activity of tumidulin, Gli1/2, and smoothened (SMO) protein levels were measured. As shown in

Tumidulin Reduces Gli and SMO Protein Levels
To identify target signaling pathways involved in the inhibition of cancer stemness by tumidulin, reporter assays were performed using the HEK293T cell line transfected with Gli-luc, TOPFLASH-luc, and Hes-1-luc genes conjugated with firefly plasmids. Based on the relative values calculated with Renilla luciferase as an internal control, only Gli-luc activity was downregulated significantly by tumidulin treatment in a dose-dependent manner (Figure 5a-c). Since Gli is the main transcription factor in the sonic hedgehog (SHH) signaling pathway, this pathway is likely involved in the reduction of CRC stemness by tumidulin treatment. These results suggest that tumidulin might decrease CRC cancer stemness by downregulating the expression of CSC markers and inhibiting Gli transcription.
To confirm the involvement of the SHH signaling pathway in the CRC stemness reduction activity of tumidulin, Gli1/2, and smoothened (SMO) protein levels were measured. As shown in Figure 5d,f, both Gli1 and Gli2 protein levels were significantly downregulated by tumidulin at a concentration of 2.5 or 5 µg/mL (= 6.25 or 12.5 µM) in the CSC221 cell line. The Gli2 protein was decreased to a greater extent than Gli1 based on the quantification results shown in Figure 5f. Furthermore, SMO protein levels were also reduced by tumidulin treatment at 2.5 or 5 µg/mL (= 6.25 or 12.5 µM) (Figure 5e,g). These Gli proteins are the terminal effectors of SHH signaling, and SMO is a transcriptional regulator that acts upstream of the canonical hedgehog (Hh) pathway [18]. Together, these results suggest that tumidulin inhibits the Hh signaling pathway by suppressing Gli and SMO.  Figure 5d,f, both Gli1 and Gli2 protein levels were significantly downregulated by tumidulin at a concentration of 2.5 or 5 µg/mL (= 6.25 or 12.5 µM) in the CSC221 cell line. The Gli2 protein was decreased to a greater extent than Gli1 based on the quantification results shown in Figure 5f. Furthermore, SMO protein levels were also reduced by tumidulin treatment at 2.5 or 5 µg/mL (= 6.25 or 12.5 µM) (Figure 5e,g). These Gli proteins are the terminal effectors of SHH signaling, and SMO is a transcriptional regulator that acts upstream of the canonical hedgehog (Hh) pathway [18]. Together, these results suggest that tumidulin inhibits the Hh signaling pathway by suppressing Gli and SMO.  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.

Tumidulin Activity on Reducing ALDH1 Expression is Gli and SMO Dependent
To confirm the involvement of the Gli and SMO in the CRC stemness reduction activity of tumidulin, the mRNA expression level of ALDH1 were measured in the presence of Gli and SMO inhibitors [18]. As shown in Figure 6, Gli inhibitor (GANT61) and SMO inhibitor (GDC-0449) treatment decreased the mRNA expression of ALDH1, and GANT61 which targets downstream of pathway reduced the mRNA expression more than those of GDC-0449. In the presence of these inhibitors, tumidulin did not further decrease the ALDH1 expression, suggesting that the activity of tumidulin is dependent on Gli and SMO ( Figure 6). Given the extent of reduction in ALDH1 expression (Figure 4), tumidulin seems to act upstream of the pathway. mTOR inhibition by rapamycin has been reported to decreased pancreatic cancer stem cell population [19]. However, treatment of rapamycin did not affect the ALDH1 expression in CSC221 cells, and tumidulin decreased the ALDH1 expression in the presence of rapamycin (Figure 6), suggesting that the activity of tumidulin is independent on mTOR pathway. Together, these results showed that the lichen secondary metabolite tumidulin decreases CRC cell stemness by inhibiting the Hh signaling pathway, and has great potential for therapeutics development.

Tumidulin Activity on Reducing ALDH1 Expression is Gli and SMO Dependent
To confirm the involvement of the Gli and SMO in the CRC stemness reduction activity of tumidulin, the mRNA expression level of ALDH1 were measured in the presence of Gli and SMO inhibitors [18]. As shown in Figure 6, Gli inhibitor (GANT61) and SMO inhibitor (GDC-0449) treatment decreased the mRNA expression of ALDH1, and GANT61 which targets downstream of pathway reduced the mRNA expression more than those of GDC-0449. In the presence of these inhibitors, tumidulin did not further decrease the ALDH1 expression, suggesting that the activity of tumidulin is dependent on Gli and SMO ( Figure 6). Given the extent of reduction in ALDH1 expression (Figure 4), tumidulin seems to act upstream of the pathway. mTOR inhibition by rapamycin has been reported to decreased pancreatic cancer stem cell population [19]. However, treatment of rapamycin did not affect the ALDH1 expression in CSC221 cells, and tumidulin decreased the ALDH1 expression in the presence of rapamycin (Figure 6), suggesting that the activity of tumidulin is independent on mTOR pathway. Together, these results showed that the lichen secondary metabolite tumidulin decreases CRC cell stemness by inhibiting the Hh signaling pathway, and has great potential for therapeutics development. 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.

Discussion
Numerous novel chemicals isolated from lichens display anti-cancer activity and show great potential for pharmaceutical usage. In the present study, the active chemical compound tumidulin was isolated from the lichen Niebla sp. from Chile, and found to exhibit inhibitory activity against CRC cell stemness. Tumidulin, also known as methyl 3,5-dichlorolecanorate, was first discovered in Ramalina ceruchis var. tumidula [20,21]. However, surprisingly, this compound could not be found in the type specimen, and as previously reported, many specimens of Vermilacinia were erroneously claimed to contain methyl 3,5-dichlorolecanorate [22]. In the present work, we isolated tumidulin from the lichen Niebla sp., and found that (1) this lichen secondary metabolite displays inhibitory activity against CRC stemness, (2) mRNA levels of several cancer stem markers including ALDH1, CD133, CD44, Lgr5, and Musashi-1 are downregulated by tumidulin treatment, and (3) the Hh signaling pathway is involved in the suppression of Gli and SMO protein levels by tumidulin treatment. These results indicate that tumidulin inhibits CRC stemness and has great pharmaceutical potential.

Discussion
Numerous novel chemicals isolated from lichens display anti-cancer activity and show great potential for pharmaceutical usage. In the present study, the active chemical compound tumidulin was isolated from the lichen Niebla sp. from Chile, and found to exhibit inhibitory activity against CRC cell stemness. Tumidulin, also known as methyl 3,5-dichlorolecanorate, was first discovered in Ramalina ceruchis var. tumidula [20,21]. However, surprisingly, this compound could not be found in the type specimen, and as previously reported, many specimens of Vermilacinia were erroneously claimed to contain methyl 3,5-dichlorolecanorate [22]. In the present work, we isolated tumidulin from the lichen Niebla sp., and found that (1) this lichen secondary metabolite displays inhibitory activity against CRC stemness, (2) mRNA levels of several cancer stem markers including ALDH1, CD133, CD44, Lgr5, and Musashi-1 are downregulated by tumidulin treatment, and (3) the Hh signaling pathway is involved in the suppression of Gli and SMO protein levels by tumidulin treatment. These results indicate that tumidulin inhibits CRC stemness and has great pharmaceutical potential.
Since tumidulin was confirmed to decrease CRC cell stemness, several major signaling pathways related to this phenomenon, including Gli-mediated Hh, β-catenin-mediated Wnt, and Hes-1-mediated Notch signaling, were investigated using reporter assays. The Gli, TOPFLASH, and Hes-1 promoters were tested using firefly luciferase gene-conjugated plasmid transfection, and only Gli-luc activity was downregulated by tumidulin treatment, suggesting that Hh signaling may be involved in the inhibition of CRC stemness by tumidulin treatment. The Hh signaling pathway is a major regulator of cell differentiation, cell proliferation, and tissue polarity, and aberrant activation of the Hh signaling pathway is involved in various types of human cancers [18]. The gene encoding Gli was the first Hh signaling pathway gene found to be upregulated in human glioblastoma and derivative cell lines [23]. In the present study, Gli1/2 and SMO protein levels were downregulated following tumidulin treatment, which further confirmed that tumidulin decreases CRC cell stemness via suppression of the Hh signaling pathway. However, our results cannot determine whether other signaling pathways are involved in the downregulation of Gli protein expression, since Gli can be activated via both Hh-dependent and Hh-independent signaling pathways [18]. Thus, the exact molecular mechanisms require further investigation.
Together, the results of the present study suggest that tumidulin has great potential for development of a CRC cell stemness inhibitor, and further demonstrate the wide application of lichen bioresources for pharmaceutical reagent screening.

Preparation of Lichen Extracts
Thalli of lichens were collected in 2013 during field trips in the National Park of Torres Del Paine, Patagonia, Chile, organized by Dr. Pereira from Talca University, Talca

High-Performance Liquid Chromatography (HPLC) Analysis of Lichen Material
Acetone extracts of lichen thalli at a concentration of 5 mg/mL were subjected to high-performance liquid chromatography (HPLC) analysis using an Agilent 1260 instrument (Agilent Technology, Santa Clara, CA, USA) equipped with a YMC-Pack ODS reversed-phase column A (150 × 4.6 mm internal diameter) containing fully end-capped C18 material (particle size = 5 µm, pore size = 12 nm). Elution was performed at a flow rate of 1 mL/min with a solvent system of methanol:water:phosphoric acid (80:20:1, v/v/v) at a column temperature of 40 • C. Separation was monitored using an Agilent 1260 Infinity II Variable Wavelength Detector (1260 VWD; Agilent Technology) over a range of 190-800 nm. Observed peaks were detected at 254 nm. The sample injection volume was 50 µL. Salazinic acid (t R = 2.27 ± 0.2 min) isolated from the lichen Lobaria pulmonaria was used as a standard, and voucher specimens were deposited in the herbarium of KOLABIC at the KoLRI, Sunchon National University, Korea.

Separation and Identification of Tumidulin
Niebla sp. (1; CL130494) extract was separated by HPLC as described above, and the fraction identified as tumidulin was collected. The supernatant was dried and weighed after centrifugation, and the HPLC assay was performed to confirm that the purified sample yielded a single peak. The structure of tumidulin was then confirmed by liquid chromatography-mass spectrometry (LC-MS, Shimadzu measured using Multi-Gauge 3.0 software, and the relative density was calculated based on the density of the β-actin bands in each sample. Values are expressed as arbitrary densitometric units corresponding to signal intensity.

Statistical Analysis
All experiments were assayed in triplicate (n = 3). Results are reported as mean ± standard error of the mean (SEM). All statistical analyses were performed using the SPSS version 17 (IBM, Chicago, IL, USA). Treatment effects were determined using one-way ANOVA post-hoc analysis. The level of significance was set at p < 0.05.

Conclusions
In summary, tumidulin was isolated from Chilean lichen Niebla sp. (1; CH130494). Tumidulin exhibited significant inhibitory activity on spheroid formation in CRC cells and decreased the expression of CSC markers in CRC cells. The inhibitory activity of tumidulin against colorectal cancer stemness is through regulation of Hedgehog signaling pathways.