FOMES INZENGAE EXTRACT LIMITS PROLIFERATION AND MIGRATION OF HEPATOCELLULAR CARCINOMA CELLS THROUGH MODULATION OF THE PTEN/PI3K/AKT PATHWAY

Fomes inzengae, a sister species of F. fomentarius, is a lignicolous fungus parasitic on deciduous trees and widely distributed across Europe. While other species within the genus Fomes are well-documented for their production of bioactive compounds, including polysaccharides, triterpenes and their derivatives, lipids, and secondary metabolites, the properties of F. inzengae remain largely unexplored, highlighting the need for further research. These compounds are known for their diverse pharmacological activities, including anticancer, immunomodulatory, anti-inflammatory, and antiviral effects [1]. This study investigates the bioactive properties and biological effects of Fomes inzengae ethanol extract on two hepatocarcinoma (HCC) cell lines, HepG2 and Huh7. Firstly, we assessed its cytotoxic potential and antiproliferative activity using various methods, including the Trypan Blue Exclusion Test, Lactate Dehydrogenase (LDH) test, and Live/Dead assays. The results demonstrated that F. inzengae extract significantly reduced the number of both HepG2 and Huh7 cells in a time- and dose-dependent manner. Based on these preliminary experiments, we identified 100 μg/mL as the optimal concentration for evaluating both cytotoxic and biological effects. At this concentration, treatment with F. inzengae extract led to an increase in the levels of tumor suppressor proteins p53, p21, and p27, suggesting a slowdown of the cell cycle. Additionally, we observed a decrease in the expression of the anti-apoptotic protein Bcl-2, alongside an increase in pro-apoptotic proteins Bax, cleaved caspase-9, and caspase-3. Furthermore, the extract impaired the wound-healing ability of Huh7 cells, which are highly migratory, suggesting its potential role in limiting cancer cell dissemination. This was further supported by the upregulation of E-cadherin and downregulation of Twist, as confirmed by Western blot analysis. Additionally, to explore the molecular mechanisms underlying these effects, we investigated potential signaling pathways that could be involved. Given its well-established role in regulating cell survival, proliferation, migration, and metabolism, we focused on the PTEN/PI3K/AKT pathway [2]. PTEN, a tumor suppressor, dephosphorylates PIP3, thereby inhibiting the activity of Phosphoinositide 3-Kinase (PI3K) and subsequently Protein Kinase B (AKT), which are key mediators of cell growth and survival. Western blot analysis of Huh7 cells revealed an increase in active (dephosphorylated) PTEN levels and a decrease in the phosphorylated (active) forms of PI3K and AKT, aligning with the observed biological effects. Although further research is needed to fully characterize the bioactive components and precise molecular targets of F. inzengae ethanol extract, our findings indicate that this polypore fungus exerts antiproliferative, proapoptotic, and antimigratory effects, at least in part, through modulation of the PTEN/PI3K/AKT signaling pathway. [1] Diego Morales, Fomes fomentarius: An underexplored mushroom as source of bioactive compounds, Food Bioscience, Volume 61,2024,104781,ISSN 2212-4292. [2] Papa A, The PTEN-PI3K Axis in Cancer. Biomolecules. 2019 Apr 17;9(4):153. doi: 10.3390/biom9040153. PMID: 30999672; PMCID: PMC6523724.