*3.6. B Induces Apoptosis and Autophagy in Caco2 Cells by Activating ROS/JNK Signaling Pathways*

The involvement of ROS and JNK activation for the induction of apoptosis and autophagy by **B** and EL000327 was further investigated. Pretreatment of Caco2 with NAC or the JNK inhibitor SP600125 significantly reduced the sensitivity to **B** and EL000327 compared to the cells treated with **B** or EL000327 in the absence of inhibitors (Figure 6a). Flow cytometric analysis detected very low numbers of apoptotic cells in the presence of NAC or SP600125, confirming that ROS and JNK inhibitors rescued **B**-induced cell death in the Caco2 cells (Figures 6b,c and S4a). Moreover, the application of SP600125 significantly decreased the expression of apoptotic and autophagy markers as well as JNK pathway related proteins, as analyzed by Western blotting after treatment with **B** for 24 or 48 h (Figure 6d,e). Taken together, these results reveal that triggering the generation of ROS by

**B** activates JNK/c-jun signaling and leads to the induction of apoptosis and autophagy in Caco2 cells.

#### *3.7. B Exhibits Synergy with 5-FU and Compound D on CRC Cells*

To investigate the potential of **B** as an anticancer therapeutic drug in the clinic, we analyzed its effect when used in combination with 5-FU, a well-known chemotherapeutic that is used to treat several types of cancers including colorectal cancers, and D (IC50 = 10 μg/mL), another cytotoxic compound isolated from EL000327 (Figure 1c). The chemical structure of D is yet to be determined. Caco2 were treated with **B**, 5-FU, and D singly or 5-FU combined with **B** or D, and the HCT116 cells were treated with **B**, and 5-FU singly or 5-FU combined with **B** at various concentrations for 48 h; cell survival was analyzed using a MTT assay, The Chou–Talalay method was used to calculate the combination index (CI) of synergy using compuSyn software. Anti-cancer agents with synergism have a CI value of <1, and those with the smallest CI value are considered to be more suitable for cancer therapy. The combination of 2 μg/mL of **B** with 4 μg/mL (**1**; CI = 0.4) or 6 μg/mL of 5-FU (**2**; CI = 0.6), the combination of 4 μg/mL of **B** with 4 μg/mL of 5-FU (**3**; CI = 0.41), and the combination of 6 μg/mL of **B** with 2 μg/mL of 5-FU (**3**; CI = 0.23) all produced CI values of less than 1 on Caco2 cells. Furthermore, **B** exhibited good synergism with 5FU on HCT116 cells as well as when combined with 2 μg/mL of **B** with 4 μg/mL of 5-FU (**1**; CI = 0.96), 4 μg/mL of **B** with 2 μg/mL (**2**; CI = 0.52), or 4 μg/mL of 5-FU (**3**; CI = 0.54) and 6 μg/mL of **B** with 2 μg/mL of 5-FU (**4**; CI = 0.45) (Figure 7a). Western blot analysis demonstrated that the combination of **B** and 5-FU at the concentrations that provided a CI value of <1 considerably increased the cleaved PARP and Caspase-3expressions and decreased the level of anti-apoptotic protein Bcl-XL compared to treatment with **B** and 5-FU individually. Decreased level of p62 was observed after the combination treatments (Figure 7b,c). Furthermore, 2 μg/mL of **B** combined with 5 μg/mL (**1**; CI = 0.27), 5 μg/mL of **B** with 5 μg/mL of D (**2**; CI = 0.47), and 10 μg/mL of **B** with 1 μg/mL of D (**3**; CI = 0.19) (Figure 7d). Expression of cleaved PARP, caspase-3, and LC3BI/II was elevated when the cells were subjected to combined **B** and D treatments, as demonstrated by Western blotting. Furthermore, the levels of anti-apoptotic protein Bcl-XL and autophagy related protein p62 were downregulated by the combination treatment with **B** and D (Figure 7e,f). These results suggest that **B** acts synergistically with both 5-FU and compound D to significantly increase its cytotoxic effects on CRC cells at low concentrations. Therefore, **B** shows a high potential to act synergistically with other anticancer therapeutics to enhance their effect by increasing the cytotoxicity.

**Figure 7. B** shows synergy with 5-FU and with D, a compound isolated from the crude extract of EL000327 on the CRC cells. (**a**) Fa-CI plot of combination treatment with 5-FU (2, 4 or 6 μg/mL) and **B** (2, 4 or 6 μg/mL) on the Caco2 cells and the Fa-CI plot of combination treatment with 5-FU (2 or 4 μg/mL) and **B** (2, 4 or 6 μg/mL) on the HCT116 cells. (**b**) Expression of apoptosis and autophagy related protein in Caco2 cells after combination treatment with 5-FU (4 or 6 μg/mL) and **B** (2 or 4 μg/mL) or **B** (2 or 4 μg/mL) or 5FU (4 or 6 μg/mL) individually for 48 h, as detected by Western blotting. (**c**) Quantification of Bcl-XL and p62 protein expression after the treatment with **B**+5FU or **B** or 5FU at the indicated concentrations. (**d**) Fa-CI plot of combination treatment with D (5 or 1 μg/mL) and **B** (2, 5 or 10 μg/mL) on the Caco2 cells. (**e**) Western blot analysis of apoptosis and autophagy related protein expression in the Caco2 cells after combination treatment with D (5 or 1 μg/mL) and **B** (2 or10 μg/mL) or **B** (10 μg/mL) or D (1 or 5 μg/mL) individually for 48 h. (**e**) Quantification of Bcl-XL and p62 protein expression after the treatment with **B** + D or **B** or D at the indicated concentrations. 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 NAC-treated group or the DMSO-treated control.

#### **4. Discussion**

Natural products have been highly recognized as rich reservoirs of bioactive compounds with the potential to lead to the discovery of novel anticancer therapeutics. As a result of the tremendous efforts by scientists, many naturally derived anticancer agents have been discovered and successfully developed into drugs within the last 30 years. Such compounds account for approximately 25% of newly approved anticancer drugs [25]. Lichen substances and the secondary metabolites of endolichenic fungi have also been proven to have a wide range of anticancer activities against various types of cancers [5,26–31]. Therefore, it is essential to carry out a thorough investigation of bioactive compounds derived from the rich bioresources of lichens. Libertellenone T **(B)** is a novel cytotoxic compound, isolated as a pink oil from a secondary metabolite extract of the endolichenic fungi EL000327. Its molecular formula was determined to be C20H26O4, based on HR-FAB-MS, coupled with the analysis of the NMR data. As the crude extract of EL000327 exerted comparatively high cytotoxicity on CRC, which is considered to be the second most lethal cancer type in the world [32], the cytotoxicity of **B** on CRC was assessed. Interestingly, the effect of **B** on the CRC cells was much stronger than EL000327 and the Caco2 cells were highly sensitive to the treatment of **B** compared to the HCT116, DLD1, and HT29 cells. Furthermore, the effect of **B** on the non-cancerous cell line Madin–Darby canine kidney epithelial cells (MDCK) was very low and the human non-cancerous cell line HaCaT was similar to the Caco2 cells. In the current study, the mechanisms underlying **B**-induced cell death were comprehensively investigated. We found that **B** induced mitotic arrest in CRC cells by stabilizing microtubules and preventing their depolymerization. Moreover, **B** activated apoptosis and autophagy via the ROS/JNK signaling pathway. **B**-induced autophagy had a protective effect on Caco2 cells. Most importantly, **B** showed synergy with the well-known chemotherapeutic 5-FU and another novel compound D isolated from the same crude extract of EL000327. Here, we mainly used Caco2 cells to study the mechanism of action of **B**, but our results revealed that **B** has an effect on other CRC cells such as HCT116, DLD1, and HT29. The induction of apoptosis in these cells upon the treatment of **B** was confirmed by the results of Hoechst staining and flow cytometry.

Check points prevent cells with damaged DNA from entering the next phase of the cell cycle. This phenomenon is highly regulated by a series of proteins, and the G2/M transition is mainly regulated by the Cyclin B/Cdc2 (Cdk1) complex. Phosphorylation of Cdc2 negatively regulates cell cycle progression from the G2 phase to M phase [11]. Furthermore, the Cyclin B/Cdk1 complex is highly activated in the metaphase as it supports the assembly of the mitotic apparatus and chromosome alignment. Once chromosomes are properly attached to spindles, the APC/C is activated and promotes progression to anaphase.

Degradation of cyclin B by the activation of the APC/C complex leads to Cdk1 inactivation [33]. Flow cytometric analysis of cell cycle progression in our study indicated a clear accumulation of Caco2 cells in the G2/M phase than other CRC cell lines HCT116, DLD1, and HT29 after treatment with **B** for 24 h. However, increasing G2/M cell populations compared to the control indicated that HCT116, DLD1, and HT29 cells require longer treatment to induce G2/M phase arrest. Furthermore, treatment with **B** resulted in the upregulation of Cyclin B1 and p-Cdc-2 expression. Therefore, we continued our studies to investigate whether **B** affected the microtubule dynamics in cells. According to the results of our in vitro tubulin polymerization assay, **B** stabilized microtubules in a manner similar to the clinically approved microtubule stabilizer, paclitaxel. These results were further confirmed by the immunofluorescence staining of cells treated with **B** and paclitaxel. Microtubule targeting agents in anticancer therapy can be classified into two groups based on their mode of action. Microtubule destabilizers prevent microtubule formation by inhibiting tubulin dimerization. In contrast, microtubule stabilizers promote tubulin dimerization and stabilize microtubules [34]. Both the stabilization and destabilization of microtubules lead to mitotic catastrophe followed by cell death due to failure to form the spindle required for chromosome segregation in the M phase of the cell cycle. Similarly, our compound, **B**, induced cell death after prolonged mitotic arrest, as shown by the appearance of a sub G1 population in cell cycle analysis.

Apoptosis is the process of programmed cell death and the most popular target of many anticancer therapies. Dysregulation of apoptosis signals in cancers promotes abnormal cell growth and tumorigenesis [9]. Restoring the lost apoptotic function in cancer cells is the main objective of much of the research into cancer treatments. The initiation of apoptosis in cells can be identified by morphological changes such as nuclear fragmentation, chromatin condensation, cell shrinkage, and membrane blebbing [35,36]. Hoechst, Caspase 3/7, and Annexin V staining demonstrated the initiation of apoptosis in CRC cells upon treatment with **B**. Apoptosis can be classed as either caspase-dependent or caspase-independent. Many anticancer agents mainly activate the caspase-dependent mitochondrial pathway. In addition, the death receptor mediated, and endoplasmic reticulum pathways also activate caspases at the final phase of apoptosis. Disruption of the mitochondrial membrane potential followed by translocation of AIF and endonuclease G to the nucleus induces caspase-independent apoptosis [37]. Data from the IncuCyte apoptosis assay and flow cytometric analysis of apoptosis demonstrated that treatment with the caspase inhibitor Z-VAD-FMK significantly reduced the extent to which **B** induced apoptosis in cells, indicating that apoptosis induced by **B** is caspase-dependent. Furthermore, Western blot analysis showed the activation of the proapoptotic protein BAX, the anti-apoptotic protein Bcl-xL, and eventually, Caspase-3and PARP cleavage in response to **B** treatment. Caspase-3 is the executioner caspase in all caspase dependent apoptosis pathways. Caspase-3 can be cleaved and activated by the upstream caspase, caspase-8, in the extrinsic pathway as well as by caspase-9, an initiating caspase, in the intrinsic pathway [38]. Cleavage of PARP by caspases is considered as a hallmark of apoptosis and an indicator that caspase dependent apoptosis has been accomplished [39]. Here, as predicted, Z-VAD-FMK treatment significantly impaired the expression of apoptotic markers in cells treated with **B**. Interestingly, the expression of the autophagic proteins Beclin 1 and LC3BI/II slightly decreased while p62 expression slightly increased upon treatment with **B** in the presence of Z-VAD-FMK. However, this result is not sufficient to assess the action of **B** toward autophagy in the presence of Z-VAD-FMK.

Autophagy plays a dual role in cancer treatment by either supporting or preventing cancer cell survival. In the current study, **B** induced autophagy in the Caco2 cells. Moreover, treatment with the autophagy inhibitors 3MA and CQ significantly increased **B**-induced cell death, suggesting that the activation of autophagy promotes CRC cell survival. Western blot analysis further confirmed that the inhibition of autophagy enhanced the activation of apoptosis, as demonstrated by elevated cleaved caspase-3 and PARP levels in the presence of CQ. Beclin 1, P62, and LC3BI/II are key regulators of the autophagic process. Beclin 1 regulates autophagosome formation at the beginning of autophagy. During autophagosome formation, cytosolic LC3B-I is converted to the membrane-bound LC3B-II form. Binding of LC3B to the adapter protein p62/SQSTM1 facilitates autophagic degradation [40]. In the present study, we observed increased the expressions of Beclin 1 and, LC3B-II and decreased p62 upon treatment with **B**. While elevated expression of Beclin 1 and, LC3B is indicative of the activation of autophagy in CRC cells, generally, activation of autophagy results in reduced expression of p62. However, under some circumstances, p62 expression can be elevated by upregulation of p62 transcription during starvation of cells, regardless of the effect of autophagy. Under prolonged starvation, p62 expression is restored to basal levels by transcriptional regulation, even, when its expression has been decreased by autophagic activities at earlier time points [41,42]. Furthermore, p62 transcription is modulated by oxidative stress (Nrf2), the Ras/MAPK pathway, and the JNK/c-Jun pathway as well as some chemicals, including autophagy inducers [43]. Following treatment with **B**, cell viability was lower in the presence of CQ than 3MA. Furthermore, the level of LC3B-II was significantly increased upon treatment with CQ in western blot analysis. CQ inhibits autophagy by inhibiting fusion of the autophagosome and lysosome, and by degradation of the autophagolysosome [44]. These results suggest that **B** may induce autophagy by

promoting late phase autophagy, fusion, and degradation. Moreover, the expression of autophagy related proteins was decreased when apoptosis was suppressed by Z-VAD-FMK. In contrast, expression of apoptotic markers was increased when autophagy was blocked by CQ. This leads to the hypothesis that **B** predominantly induces apoptosis in CRC cells, and that autophagy is activated as a counter mechanism to protect the cells from apoptosis. However, this potential interconnection between activation of apoptosis and autophagy by **B** needs further, detailed investigation.

Our study revealed that ROS generation is highly induced upon the treatment with **B**. Excess levels of ROS can be deleterious to cells due to the induction of oxidative stress within cells. Blocking ROS generation using the antioxidant NAC decreased expression of both apoptotic and autophagy markers in response to treatment with **B**, as demonstrated by western blot analysis. Furthermore, NAC significantly decreased the phosphorylation of JNK and expression of c-JUN. Given that ROS generation began after 12 h of treatment with **B**, while apoptotic markers were detected after 48 h, it seems likely that **B** induced apoptosis is initiated by ROS. ROS activate many signaling pathways (PI3K/Akt, MAPK, Nrf2) and transcription factors (NF-κB, p53) that eventually induce apoptosis, autophagy, or necrosis. Oxidants like OH•, ONOO−, and H2O induce apoptosis and/or necrosis, while O2 •− and H2O2 induce autophagy and mostly trigger cell survival.

Cell viability upon treatment with **B** was markedly increased in the presence of the NAC and JNK inhibitor, SP600125, while apoptosis was significantly decreased, according to flow cytometric and western blot analysis. Furthermore, levels of LC3B were also reduced in the presence of SP600125, indicating that autophagy was inhibited. Taken together, this suggests that JNK plays a significant role in the apoptosis and autophagy activated by treatment with **B**. Induction of c-jun/JNK signaling by ROS blocks the antiapoptotic protein, Bcl-2, and activates the proapoptotic proteins in the Bcl-2 family, which are critical for the release of cytochrome c to the cytosol. Activation of caspase-9 and the effector caspase-3 by cytochrome c eventually leads to cell death via the mitochondrial apoptotic pathway. ROS can also activate the extrinsic apoptotic pathway by directly causing damage to DNA [45]. Taken together, the above results suggest that **B** induces apoptosis and autophagy in CRC via activation of ROS/JNK signaling (Figure 8).

EL000327 is the crude extract from which **B** was isolated. This crude extract contained six compounds, including **B** and another highly cytotoxic compound, D. In our study, we compared the effect of our isolated compound on CRC cells with that of the crude extract, EL000327. Initiation of apoptosis upon treatment with EL000327 was detected by Hoechst staining, the incucyte apoptosis assay, and flow cytometric analysis of apoptosis. However, apoptotic markers were not clearly detected by western blot analysis at the same time points as **B**. Induction of cell cycle arrest, activation of autophagy, or the ROS/JNK pathway related proteins in Caco2 cells were also not observed upon treatment with EL000327. These results suggest that interactions between different compounds in the crude extract may alter the activity of EL000327. Alternatively, the effects of EL000327 and **B** may have been detected at different time points because EL000327 was either faster or slower to act than **B**.

*5-Fluorouracil* (*5-FU*) is a first-line treatment for many cancers, including CRC. 5-FU leads cells to death by preventing DNA replication and RNA synthesis through inhibition of cellular thymidylate synthase (TS) [46,47]. Combination treatments in which 5-FU is combined with different anticancer agents, enhance the anticancer effect and response rate of these treatments. Similarly, combination of **B** with 5-FU enhanced the cytotoxicity of **B** toward Caco2 and HCT116 cells at significantly lower treatment concentrations [48,49]. Combination of **B** and 5-FU induced apoptosis in Caco2 cells by enhancing the expression of cleaved caspase-3and PARP. Further investigations are required to understand the mechanism by which cytotoxicity is increased in the synergy between **B** and 5-FU. Furthermore, **B** exhibited excellent synergic effects with compound D. A combination of **B** and D induced apoptosis and autophagy in the Caco2 cells at comparatively low concentrations. These results provide evidence that **B** may act synergistically with a range of different anticancer agents to enhance their cytotoxic effect.

**Figure 8.** Schematic representation of the proposed mechanism for **B**-induced apoptosis and autophagy in the Caco2 cells. **B** induces apoptosis in Caco2 cells through the induction of G2/M phase arrest caused by tubulin stabilization. Simultaneously, **B** induces apoptosis and autophagy in Caco2 cells via the ROS/JNK signaling pathway. Inhibition of ROS, JNK, and caspases by NAC, SP600125, and Z-VAD-FMK, respectively, decreases **B** induced caspase-dependent apoptosis in Caco2 cells. 3MA and CQ inhibit autophagy in the Caco2 cells. The inhibition of autophagy by preventing the fusion of lysosomes with autophagosomes by CQ significantly increases the induction of caspase-dependent apoptosis by **B** in the Caco2 cells.

**B** is a novel naturally-derived compound, and this is the first study of the activity of **B** on CRC cells. We thoroughly investigated the mechanisms by which **B** induces apoptotic cell death and have laid the groundwork for a detailed analysis of the clinical usefulness of **B.** Moreover, we have demonstrated that the combination of **B** with known and novel anticancer agents may provide effective new treatment options for patients with CRC, which warrants further investigation.
