Synergistic apoptotic and epithelial–mesenchymal transition-inhibitory effects of Hericium erinaceus extract in canine mammary cancer cell lines

Background and Aim: Canine mammary cancer (CMC) is one of the most common malignant neoplasms in female dogs, with high metastatic potential and limited therapeutic options. Natural bioactive compounds derived from medicinal mushrooms have gained increasing attention because of their anticancer properties and low toxicity. Hericium erinaceus (HE) is a medicinal mushroom known for its antioxidant and antitumor activities; however, its anticancer effects in CMC remain poorly understood. Therefore, this study investigated the in vitro antiproliferative, pro-apoptotic, and epithelial–mesenchymal transition (EMT)-inhibitory effects of HE methanolic extract in two CMC cell lines, CHMp-13a and CHMp-5b. Materials and Methods: The anticancer activity of HE methanolic extract was evaluated using CHMp-13a and CHMp-5b CMC cell lines, while Madin-Darby canine kidney cells were used as normal controls. Cell viability was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration and invasion were evaluated using wound healing and Transwell assays, respectively. Apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry. Relative mRNA and protein expression levels of apoptosis- and EMT-related markers were determined using quantitative real-time polymerase chain reaction and western blotting. The phytochemical profile of the extract was characterized using liquid chromatography quadrupole time-of-flight mass spectrometry. Results: HE extract significantly inhibited proliferation of both CMC cell lines in a dose- and time-dependent manner, with greater selectivity toward CHMp-13a cells and minimal cytotoxicity in normal cells. Morphological analysis revealed apoptotic features, including cell shrinkage, detachment, and cytoplasmic vacuolization. The extract significantly suppressed migration and invasion capacities of both CMC cell lines. Flow cytometric analysis demonstrated increased apoptotic cell populations following treatment. Molecular analyses showed upregulation of the pro-apoptotic marker BAX and downregulation of the anti-apoptotic marker BCL-2. Furthermore, HE extract suppressed EMT progression by increasing E-cadherin expression while reducing N-cadherin expression. Phytochemical screening identified 17 bioactive compounds, including erinacines, hericenones, hericene derivatives, and phenolic compounds, which may contribute to the observed anticancer activities. Conclusion: HE extract demonstrated potent in vitro anticancer activity against CMC cells through synergistic induction of apoptosis and suppression of EMT-associated metastatic behavior. These findings suggest that HE extract may serve as a promising natural adjuvant candidate for the management of CMC and warrants further in vivo and mechanistic investigations. Keywords: apoptosis, canine mammary cancer, epithelial–mesenchymal transition, Hericium erinaceus, medicinal mushroom, metastasis inhibition, phytochemicals, selective cytotoxicity.