Our study shows that Ganoderma lucidum/reishi/lingzhi shows antitumor properties on osteosarcoma cells in vitro. It was found that Ganoderma lucidum inhibits breast cancer cell growth and migration by suppressing Wnt/β-catenin signaling. It suppresses lung cancer through disruption of focal adhesions and induction of MDM2-mediated Slug degradation. Ganoderma lucidum inhibits breast cancer via downregulating the PI3K/AKT/mTOR pathway, Ganoderma lucidum plays an antitumor role in acute leukemia cells by blocking the MAPK pathway.

CCK-8 and colony formation assays, for assessing the effect of Ganoderma lucidum on osteosarcoma cell line viability and proliferation, showed that Ganoderma lucidum suppresses the proliferation of MG63 and U2-OS cells in a time- and concentration-dependent manner, and reduces the ability of cells to colonize.

Ganoderma lucidum upregulates the expression of proapoptotic genes, and flow cytometry analysis showed that apoptosis of MG63 and U2-OS cells is increased after treatment with Ganoderma lucidum. Cell migration is the basis of a variety of biological behaviors, including angiogenesis, wound healing, inflammation, and cancer metastasis. Ganoderma lucidum decreases the migration and invasion of both cell lines and inhibits the proliferation, migration, and invasion, and induces apoptosis of osteosarcoma cells.

Aberrant Wnt/β-catenin signaling is closely related to the formation, metastasis, and apoptosis of numerous types of cancers, with upregulation of Wnt/β-catenin signaling being observed in osteosarcoma.

In this study, dual-luciferase reporter assays showed that Ganoderma lucidum treatment blocks CHIR-99021-activated Wnt/β-catenin signaling. This is further proved by our demonstration that transcription of Wnt target genes, such as LRP5, β-catenin, cyclin D1, and MMP-9, is inhibited when osteosarcoma cells are treated with Ganoderma lucidum.

Previous studies have shown in clinical samples that LRP5 is upregulated in osteosarcoma relative to normal tissue, and expression of LRP5 correlates with metastatic disease and poor disease-free survival, making LRP5 a potential therapeutic target for osteosarcoma.

β-catenin itself is a key target in the Wnt/β-catenin signaling pathway, and the expression of β-catenin in osteosarcoma is significantly increased. When β-catenin translocates into the nucleus from the cytoplasm, it activates expression of its downstream target genes, which include cyclin D1, C-Myc, and MMPs.

Myc is one of the major proto-oncogenes and plays an important role in regulating the activation, transcription, and inhibition of gene expression.It has been reported that suppression of the C-Myc oncogene induces aging and apoptosis of several tumor cell types, including osteosarcoma.

Cyclin D1 is an important cell cycle G1 phase regulator and accelerates G1/S phase transition. Excessive expression of cyclin D1 can shorten the cell cycle and promote rapid cell proliferation in diverse tumor types.

MMP-2 and MMP-9 are stromelysins that have the ability to degrade extracellular matrix components, a crucial feature for tumor angiogenesis and invasion.

This suggests that Wnt/β-catenin target genes play a key role in the progression of osteosarcoma, and that blocking these signal nodes may have a dramatic therapeutic effect.

Subsequently, we detected the expression of mRNA and protein of Wnt/β-catenin signaling-related target genes by PCR and western blotting. In both cell lines, Ganoderma lucidum inhibited the expression of these proteins and genes. These results further demonstrate that Ganoderma lucidum inhibits Wnt/β-catenin signaling by targeting LRP5, β-catenin, C-Myc, cyclin D1, MMP-2, and MMP-9.

E-cadherin is a transmembrane glycoprotein widely expressed in epithelial cells and mediates adhesion between epithelial cells and stromal cells. Deletion or loss of E-cadherin expression leads to loss or weakening of adhesion between tumor cells, enabling tumor cells to move more easily, and then make the tumor infiltrate, diffuse, and metastasize. In this study, we found that Ganoderma lucidum can upregulate E-cadherin, thereby countering the Wnt/β-catenin–mediated phenotype of osteosarcoma cells.

In conclusion, our results indicate that Ganoderma lucidum blocks osteosarcoma Wnt/β-catenin signaling and ultimately leads to the decrease of osteosarcoma cell activity. These findings suggest that Ganoderma lucidum may be a useful and effective therapeutic agent for the treatment of osteosarcoma, the related products include ganoderma lucidum spore oil softgels/reishi spore oil softgels, ganoderma lucidum spore powder/reishi spore powder.