Two novel compounds, ergosterol and ergosta-5,8-dien-3-ol, from Termitomyces heimii Natarajan demonstrate promising anti-hepatocarcinoma activity

Mushroom-derived components have immense potential to become a safe alternative in identifying lead anti-cancer molecules. Termitomyces heimii Natarajan (T. heimii) is a traditionally used edible mushroom with no previous record of anti-hepatocarcinoma activity. The anti-proliferative efficacy of the mushroom ethyl acetate extract was screened against a panel of seven cancer cell lines, namely Hep G2, MCF-7, MDA-MB-231, MAD-MB-436, MOLT-4, Reh, and K-562, and against peripheral blood mononuclear cells isolated from normal healthy donors by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The impact of the extract on nuclear morphology was examined by 4′,6-diamidino-2-phenylindole staining and the apoptotic potential of the extract was evaluated through flow cytometry and Annexin V-PI dual staining, followed by an in vitro scratch assay to elucidate the anti-migratory potential of the extract. The apoptotic and anti-migratory effects were further validated using in silico molecular docking with four compounds, ergosterol, ergosta-5,8-dien-3-ol, lanosterol, and eburicol, against two anti-apoptotic proteins, Bcl-2 and Bcl-XL, and two angiogenic receptors, VEGFR-1 and VEGFR-2. The screening data revealed that ethyl acetate extract exhibited remarkable anti-proliferative efficacy against Hep G2 cells, with a half maximal inhibitory concentration (IC₅₀) value of 263.53 (8.09) μg/mL, followed by MCF-7 cell lines and showed a negligible effect on peripheral blood mononuclear cells. A clear alteration of the cellular and nuclear morphology was concentration-dependently observed in Hep G2 cells. The extract induced robust apoptosis and a significant concentration-dependent increase in the scratch area. The results of in silico docking revealed that compared to standard drug sunitinib, both ergosterol and ergosta-5,8-dien-3-ol displayed lower binding energy, and satisfactory drugability and absorption, distribution, metabolism, excretion, and toxicity properties. T. heimii is a potential source for isolating lead anticancer molecules in the future. Ergosterol and ergosta-5,8-dien-3-ol hold great promise as new drugs against hepatocarcinoma.