*Article* **Libertellenone T, a Novel Compound Isolated from Endolichenic Fungus, Induces G2/M Phase Arrest, Apoptosis, and Autophagy by Activating the ROS/JNK Pathway in Colorectal Cancer Cells**


**Simple Summary:** Libertellenone T (**B**) is a natural product derived from the secondary metabolites of the endolichenic fungus, *Pseudoplectania* sp. In this study, we investigated the underlying molecular mechanisms that induce the apoptotic cell death of the colorectal cancer cell line, Caco2, in response to **B**. Our findings demonstrate that **B** induces Caco2 cell apoptosis via G2/M phase arrest and activation of ROS/JNK signaling. Moreover, **B** exhibited excellent synergistic effects when combined with the known and novel anticancer agents 5-FU and compound D, respectively. In light of this investigation, we propose that **B** is a promising potential chemotherapeutic agent against colorectal cancer.

**Abstract:** Colorectal cancer (CRC) is the third most deadly type of cancer in the world and continuous investigations are required to discover novel therapeutics for CRC. Induction of apoptosis is one of the promising strategies to inhibit cancers. Here, we have identified a novel compound, Libertellenone T (**B**), isolated from crude extracts of the endolichenic fungus from *Pseudoplectania* sp. (EL000327) and investigated the mechanism of action. CRC cells treated by **B** were subjected to apoptosis detection assays, immunofluorescence imaging, and molecular analyses such as immunoblotting and QRT-PCR. Our findings revealed that **B** induced CRC cell death via multiple mechanisms including G2/M phase arrest caused by microtubule stabilization and caspase-dependent apoptosis. Further studies revealed that **B** induced the generation of reactive oxygen species (ROS) attributed to activating the JNK signaling pathway by which apoptosis and autophagy was induced in Caco2 cells. Moreover, **B** exhibited good synergistic effects when combined with the well-known anticancer drug, 5-FU, and another cytotoxic novel compound D, which was isolated from the same crude extract of EL000327. Overall, Libertellenone T induces G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK pathway in CRC. Thus, **B** may be a potential anticancer therapeutic against CRC that is suitable for clinical applications.

**Keywords:** CRC; Libertellenone T; G2/M phase arrest; apoptosis; autophagy; ROS/JNK signaling
