Background and objectives: Mushrooms that have medicinal properties are part of many traditional diets. The aim of the present study was to use the human breast cancer cell line MCF-7 to investigate the anticancer activity of Pleurotus highking mushroom purified extract fraction-III (PEF-III) and to elucidate the possible mechanism of that activity. Materials and Methods: The effects of PEF-III on cell proliferation and viability were evaluated by a colony formation assay and an MTT assay, respectively. Cell morphological changes, annexin-V phycoerythrin and propidium iodide (PI) staining, DNA fragmentation, and caspase 3/7 activity assays were performed to determine the induction of apoptosis by PEF-III. The genes responsible for regulation of apoptosis were analyzed by means of Western blot analysis. In vitro tumor sphere formation assay was performed using a 3D sphere culture system. Results: PEF-III significantly reduced the proliferation and viability of MCF-7 cells. Cell shrinkage and rounding, and annexin-V phycoerythrin and PI staining followed by flow cytometry indicated that the cell death was due to apoptosis. Additionally, a laddering DNA pattern and increased levels of caspase-3/7 enzyme also corroborated the notion of apoptosis-mediated cell death. This incidence was further confirmed by upregulation of proapoptotic genes (p53 and its target gene, Bax) and downregulation of the expression of an antiapoptotic gene (Bcl-2). PEF-III also reduced the size and number of the tumor spheres in 3D culture conditions. Conclusions: The anticancer activity of PEF-III is due to induction of apoptosis by a shift in the balance of proapoptotic and antiapoptotic genes. Therefore, the findings of the present study may open a path to exploring potential drug candidates from the P. highking mushroom for combating breast cancer.
Keywords: 3D culture medium; Bax/Bcl-2 ratio; MCF-7 cells; apoptosis; breast cancer; caspase 3/7 activity; flow cytometry; tumor sphere.