*3.1. The Novel Compound, B, Isolated from Crude Extract, EL000327, Exerts Cytotoxicity on the CRC Cells*

The endolichenic fungus EL000327 (Figure 1a) was isolated from lichen specimen Graphis, collected from Hallasan in Jeju Island, South Korea in 2009 using the surface sterilization method. EL000327 was identified as a *Pseudoplectania* sp. according to a BLAST search of the GeneBank Database (Table S1). The cytotoxicity of the crude extract of EL000327 was tested against several human cancer cell lines: HT29, HCT116, Caco2, DLD1, CSC221, AGS, TMK1, RV1, A549, a mouse colon cancer cell line CT26, and noncancerous cell lines HaCaT and MDCK. Among the cancer cell lines, EL000327 showed the highest cytotoxicity toward the human CRC cell line, Caco2 (IC50 = 52.2 μg/mL) and the mouse colon cancer cell line, CT26 (IC50 = 33.12 μg/mL). Cytotoxicity on the spontaneously transformed human keratinocyte cell (HaCaT) was similar to that of the Caco2 cells (IC50 = 48.7 μg/mL) (Figure 1b). The extract of EL000327 was subjected to a purification process to isolate and identify active compounds from the crude extract. First, the extract of EL000327 was separated into seven fractions, and Fr.2 (IC50 = 24.35 μg/mL) was identified as the fraction with the strongest cytotoxicity against Caco2 cells. In a further purification of Fr.2, six purified compounds (A', A, **B**, B', C, D) were isolated, as indicated in Figure 1c.

Compound **B** was isolated as a pink oil, and its molecular formula was deduced as C20H26O4, based on the HR-FAB-MS data. The chemical structure of **B** was determined to be a novel compound 7β-9α-dihydroxy-1,8(14),15-pimaratrien-3,11-dione (Libertellenone T), based on intensive interpretation of MS, UV, and NMR spectroscopic data (Figure 1d).

Furthermore, the stereo-configurations of compound **B** were determined by comparing the NMR spectroscopic data and the values of optical rotation with the literature [24].

**Figure 1.** Compound **B** isolated from the crude extract of EL000327 exhibited cytotoxicity toward the human CRC cell line, Caco2. (**a**) Image of the endolichenic fungus, EL000327, belonging to *Pseudoplectania* sp. isolated from the lichen Graphis. (**b**) IC50 values of EL000327 in HT29, HCT116, Caco2, DLD1, CSC221, TMK1, RV1, A549, HaCaT, CT26, and MDCK cells. (**c**) Schematic representation of the process for the purification of compound **B** from the crude extract, EL000327. (**d**) Chemical structure of the novel compound, **B**. (**e**) IC50 values of human CRC cells Caco2, HCT116, DLD1, and HT29 after treatment with **B** for 48 h. (**f**) Comparison of IC50 values of the CRC cells Caco2 and non-cancer cell lines HaCaT and MDCK treated with single compound **B**, fraction 2, or crude extract, EL000327 for 48 h. Results are representative of three independent experiments. Data represent the mean ± S.D. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, NS: no significant difference (*p* > 0.05) compared with the Caco2 cells.

The cytotoxicity of purified compound **B** on Caco2, HCT116, DLD1, HT29, HaCaT, and MDCK cell lines was evaluated. **B** was toxic to the Caco2 (IC50 = 17.5 μg/mL) cell line at much lower doses than the HCT116 (IC50 = 28 μg/mL), DLD1 (IC50 = 36.6 μg/mL), and HT29 (IC50 = 28 μg/mL) cell lines and the non-cancer cell lines, HaCaT (IC50 = 17.6 μg/mL) and MDCK (IC50 = 171.8 μg/mL) (Figure 1e). Furthermore, for MDCK cells, the IC50 of **B** was significantly higher than those of Fr.2 (IC50 = 81.35 μg/mL) or the crude extract EL000327 (IC50 = 102.5 μg/mL) (Figure 1f). Thus, **B** was identified as a novel chemical compound, which exerted the highest cytotoxicity toward Caco2 among the tested human CRC cell lines, and was suitable for further investigation of its mechanism of action.

#### *3.2. B Induces G2/M Phase Arrest in CRC Cells as a Result of Microtubule Stabilization*

To determine whether **B** inhibits CRC cell growth by regulating the cell cycle, the cell cycle distribution of Caco2, HCT116, DLD1, and HT29 cells was analyzed by flow cytometry after treatment with **B**. Caco2 cells were treated with cytotoxic concentrations of **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 24 h, 48 h, and 72 h. **B** markedly increased the proportion of cells at the G2/M phase in a dose-dependent manner after 24 h of treatment. At 48 h and 72 h after treatment, the proportion of cells at the G2/M phase decreased in a time-dependent manner, accompanied by an increase in the sub G1 population, indicating cell death after G2/M phase arrest. Treatment with EL000327 caused some accumulation of cells in the G2/M phase, but to a lesser extent than the treatment with **B** (Figures 2a and S1a). Analysis of the expression of known cell cycle regulatory proteins by Western blotting demonstrated that the expression of Cyclin B1, Cyclin D1, and p-Cdc2 was upregulated after treatment with **B** or EL000327 for 48 h (Figures 2b and S5a). To compare the effect of **B** on cell cycles of other CRC cell lines, the HCT116, DLD1, and HT29 cells were treated with 20 μg/mL of **B** for 24 h and the distribution of the cell cycle was analyzed. Cells accumulated in the G2/M phase was increased in all cell lines compared to the control (Figure S1b,c).

The dynamics of microtubules play a vital role in the progression of the cell cycle through the G2/M phase because microtubules form mitotic spindles, which provide structural support for chromosome segregation during mitosis. Therefore, tubulin polymerization assays were performed to evaluate the effect of **B** on tubulin polymerization in vitro. Treatment with the well-known microtubule stabilizer paclitaxel (10 μM) enhanced tubulin polymerization, whereas the microtubule destabilizer, vinblastine (10 μM) impaired tubulin polymerization. In untreated conditions, microtubules self-assembled to form tubulin polymers in a time dependent manner. Treatment with **B** (3.3, 20, 60 μg/mL) had a similar effect to paclitaxel and enhanced tubulin polymerization in a dose- and time-dependent manner (Figure 2c). qRT-PCR analysis demonstrated that the tubulin destabilizing gene *Satathmin* was significantly downregulated upon treatment with **B** or EL000327 (Figure 2d). The changes in mitotic spindle organization in Caco2 cells seen after treatment with **B** were visualized by immunofluorescence staining to confirm the results described above. Immunofluorescence microscopy demonstrated that **B** had a similar effect on the microtubules to paclitaxel (100 nM) by inducing multipolar mitotic spindles as a result of enhanced tubulin polymerization. Treatment with high concentrations of **B** (20, 60, 100 μg/mL) increased the bundling and stabilization of microtubules in Caco2 cells in a dose dependent manner. In contrast, the known microtubule destabilizers, vinblastine (50 nM) and DPT (25 nM), resulted in disrupted microtubule organization (Figure 2e). These data suggest that **B** induced G2/M phase arrest by regulating the cell cycle related protein and by stabilizing the microtubules. Furthermore, **B** was identified as a microtubule stabilizing agent, which has a functional effect that is similar to that of paclitaxel.

**Figure 2. B** induces G2/M phase arrest in Caco2 cells by inducing tubulin polymerization. (**a**) The cell-cycle distribution of Caco2 cells treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 24 h, 48 h, and 72 h as assessed by flow cytometry. (**b**) Western blot analysis of cell cycle regulating proteins, Cyclin B1, D1, and p-Cdc2 after treatment with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 24 h, 48 h, and 72 h. (**c**) Effect of **B** on tubulin polymerization in vitro, at concentrations of 20, 60, and 3.3 μg/mL. DMSO, paclitaxel, microtubule stabilizer (10 μM), and vinblastine microtubule destabilizer (10 μM) were used as the controls. (**d**) Relative mRNA levels of stathmin and MAP4, which are associated with microtubule destabilization and stabilization, respectively, after treatment with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h. (**e**) Immunofluorescence microscopy of the microtubule organization in the Caco2 cells after treatment with **B** (20, 60, 100 μg/mL), paclitaxel (100 nM), vinblastine (50 nM) or DPT microtubule destabilizer (25 nM) for 24 h. Actin was stained with Alexa Fluor 568 phalloidin (red), microtubules were stained with α-tubulin antibodies (green), and DNA was stained with DAPI (blue). Results are representative of three independent experiments. Data represent the mean ± S.D. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, NS: no significant difference (*p* > 0.05) compared with the DMSO-treated control group.

#### *3.3. B Induces Apoptotic Cell Death in CRC Cells*

We next wished to determine whether the cytotoxicity exerted by **B** on CRC cells was due to the induction of apoptosis. Thus, Caco2, HCT116, DLD1, and HT29 cells were stained with Hoechst 33258 after treatment with cytotoxic concentrations of **B** or EL000327 for 12 h or 24 h to examine the morphological changes in the nuclei of cells. Cells treated with both **B** and EL000327 exhibited condensed chromatin, indicative of the initiation of apoptosis (Figures 3a and S2a,b). The number of condensed nuclei significantly increased in all CRC cell lines (Figures 3b and S2c). Furthermore, cell death induced by treatment with **B** was analyzed using the IncuCyteTM apoptosis assay, which employs Caspase 3/7 (green) and Annexin V (red) dyes, in the presence or absence of the caspase inhibitor, Z-VAD-FMK. The level of Caspase-3/7 fluorescent green signal in Caco2 cells was markedly increased upon treatment with **B** or EL000327 for 48 h (Figure 3c,d). In this assay, the Annexin V dye emits a red fluorescent signal upon binding to the exposed phosphatidylserines (PS) of apoptotic cells. The number of red-labeled apoptotic cells increased after exposure to **B** in a dose-dependent manner. However, the suppression of caspases by Z-VAD-FMK significantly decreased the number of apoptotic cells detected by Annexin V staining (Figure 3e,f). To confirm the induction of caspase dependent apoptosis by **B** in Caco2 cells, flow cytometric analysis of apoptosis was also carried out. Cells were double stained with PI and Annexin V following treatment with **B** or EL000327 in the presence or absence of Z-VAD-FMK. Dose-dependently increasing numbers of apoptotic cells were observed after 48 h of treatment. Moreover, the inhibition of caspase significantly decreased apoptosis in the Caco2 cells (Figures 3g,h and S3a). In addition, the flow cytometric analysis revealed that **B** significantly induced the apoptosis of other CRC cell lines HCT116, DLD1, and HT29 as well as at the concentration of 20 μg/mL (Figure S3b,c). Taken together, both **B** and EL000327 induced apoptotic cell death in CRC cells.

**Figure 3.** *Cont*.

**Figure 3. B** induces caspase dependent apoptosis in Caco2 cells. (**a**) Nuclei condensation of Caco2 cells upon treatment with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 12 h, as determined by Hoechst staining. Arrowheads indicate nuclear condensation in cells. (**b**) Quantification of condensed nuclei in Caco2 cells treated with indicated concentrations of **B** or EL000327. (**c**) Caspase 3/7 (green) staining of Caco2 cells treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h. (**d**) Quantification of apoptotic cells stained with Caspase 3/7 after treatment with the indicated concentrations of **B** or EL000327. (**e**) Annexin V staining of Caco2 cells treated with **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h in the presence or absence of the caspase inhibitor Z-VAD-FMK (10 μM). (**f**) Quantification of apoptotic cells stained with Annexin V after treatment with the indicated concentrations of **B** or EL000327 in the presence or absence of Z-VAD-FMK (10 μM). (**g**) Flow cytometric analysis of dead cells stained by Annexin v-FITC (apoptotic cells) and PI (necrotic cells) upon the treatment of **B** (20, 60 μg/mL) or EL000327 (60 μg/mL) for 48 h in the presence or absence of Z-VAD-FMK (10 μM). (**h**) Quantification of the percentage of apoptotic cells treated with indicated concentrations of **B** and EL000327 and analyzed by flow cytometry in the presence or absence of Z-VAD-FMK (10 μM). Results are representative of three independent experiments. Data represent the mean ± S.D. \*\* *p* < 0.01, \*\*\* *p* < 0.001; compared with the DMSO-treated control or Z-VAD-FMK treated group.
