**1. Introduction**

An abnormal cell growth that occurs in the colon or rectum is simply recognized as colorectal cancer (CRC). A noncancerous cell growth, known as a polyp, can develop in the mucosal layer of the colon or rectum, and may develop into CRC. Less than 10% of polyps have a high potential to progress into invasive cancer over 10–20 years. CRC occurs predominantly in adults aged 50 and older [1]. In 2020, CRC was recognized as

**Citation:** Gamage, C.D.B.; Kim, J.-H.; Yang, Y.; Ta¸s, ˙ I.; Park, S.-Y.; Zhou, R.; Pulat, S.; Varlı, M.; Hur, J.-S.; Nam, S.-J.; et al. 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. *Cancers* **2023**, *15*, 489. https://doi.org/10.3390/ cancers15020489

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 28 November 2022 Revised: 10 January 2023 Accepted: 11 January 2023 Published: 12 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the third most common cancer type worldwide and the second most common cause of cancer death. Geographically, the occurrence of CRC is highest in Asian regions. The International Agency for Research on Cancer (IARC) estimated that the global burden of CRC will increase by 56%, and CRC related mortality will increase by 69% between 2020 and 2040 [2]. Therefore, a strong research focus on the discovery of novel anticancer therapeutics is urgently required.

Many scientists have focused their attention on the development of innovative drugs derived from natural sources such as plants, lichens, and micro-organisms over a few decades. A lichen is a symbiotic living form associated with a fungus (mycobiont) and a cyanobacterium or green alga (the photobiont), or both [2]. Some endolichenic fungi (ELF) reside inside the lichen thalli and produce bioactive secondary metabolites with medicinal and economic potential. These secondary metabolites show cytotoxic, antioxidant, antifungal, and antibacterial bioactivities, which are crucial in drug development in the pharmaceutical industry [3,4]. Therefore, the ability of ELF to produce unique secondary metabolites with anti-cancer properties provides a novel opportunity to identify effective cancer therapeutics [5–7]. Libertellenone T (**B**) is a novel cytotoxic compound isolated from secondary metabolites extracted from the endolichenic fungus, EL000327, from *Pseudoplectania* sp. found in the lichen *Graphis*, collected from Hallasan in Jeju Island, South Korea in 2009.

Many chemotherapeutics exert cytotoxicity and trigger cancer cell death via inducing apoptosis in cells. Apoptosis, or programmed cell death, is a cell suicide process that activates when their continuous survival is blocked [8]. Morphological and biochemical changes such as chromatin condensation, nuclear fragmentation, cell shrinkage, membrane blebbing, DNA, protein breakdown, and caspase activation can be observed in apoptotic cells. Cancer cells evade natural cell death as a result of genetic mutations acquired during transformation from normal cells to malignant cells. Disruption of the balance between pro-apoptotic and anti-apoptotic proteins, reduction in caspase function, and impairment of death receptor signaling all cause apoptosis resistance in cancer cells [9]. Therefore, restoration of these functions by therapeutics can successfully prevent cancer progression. Furthermore, alterations that occur in cell cycle regulators at check points cause abnormal cell proliferation in cancers [10]. Thus, the induction of G2/M phase arrest is another target of many anti-cancer agents. Controlling cell progression through the cell cycle by regulating related proteins or disrupting tubulin organization eventually leads cells to G2/M phase arrest and subsequent cell death [11,12].

Autophagy (macroautophagy) mainly involves the formation of an autophagosome by engulfing damaged organelles, fusion with lysosome, and degradation of cellular debris by lysosomal hydrolase [13,14]. Autophagy plays a paradoxical role in cancer progression. In the early stage of many cancers, autophagy acts as a tumor suppressor by protecting cell homeostasis. Conversely, autophagy promotes tumor growth in more advanced stages of cancer by increasing stress tolerance [15]. Many anticancer therapeutics are known to activate autophagy concurrently with apoptosis. This activation of autophagy can either promote or suppress cancer cell survival. Therefore, the role of autophagy is crucial in anticancer drug development [16]. Furthermore, the relationship between autophagy and apoptosis is unclear and yet to be investigated.

Reactive oxygen species (ROS) are highly reactive and short-lived small molecules in the form of free radicals [17]. ROS support many physiological functions in cells under optimum conditions. However, the over production of ROS can have a deleterious effect on cells and trigger oxidative stress. The generation of ROS can be triggered by both endogenous and exogenous sources such as mitochondrial transport chain leakage, high metabolic rate, environmental pollutants, radiation, and drugs [18]. ROS are responsible for activating many cellular signaling pathways that lead to cell survival or cell death including autophagy, apoptosis, and necrosis. c-Jun N-terminal kinase (JNK) signaling is activated in response to ER stress or activation of the mitochondrial pathway of apoptosis by ROS [19].

In this study, we investigated the possible mechanisms underlying the impact of **B** on CRC cells. We selected four CRC cell lines, HCT116, DLD1, HT29, and Caco2, harboring different genetic mutations and status of microsatellite instability, to evaluate the effect of **B** on them. HCT116 is a microsatellite instable (MSI) cell line with KRAS and PIK3CA mutations. DLD1 is the MSI cell line, harboring KRAS, PIK3CA, and TP53 mutations. HT29 is microsatellite stable (MSS), and BRAF and PIK3CA are muted cell lines, while Caco2 is MSS and the wild type of KRAS PIK3CA, TP53 [20]. Furthermore, we demonstrated that **B** induced mitotic arrest, apoptosis, and autophagy via activating the ROS/JNK signaling pathway. In addition, we found that **B**-induced autophagy promotes cell survival. Furthermore, **B** exhibited synergy not only with the known anticancer drug *5-Fluorouracil* (5-FU), but also with a novel compound D. Collectively, our data suggest that **B** is a potential candidate as an anticancer therapeutic with clinical application.
