Cinobufotalin reduces glioblastoma resistance to temozolomide by inhibiting the CCL5-mediated PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is a key factor that restricts the therapeutic effects of glioblastoma (GBM). The pharmacological properties of cinobufotalin (CB) indicated that CB promotes the cell death of GBM cells, while the underlying mechanism remains not fully elucidated. This study aims to elucidate the mechanism by which CB reduces TMZ resistance during GBM progression. METHODS: BALB/c nude mice were used to construct in situ models, and were intraperitoneally injected with CB and TMZ to evaluate their effects on tumor growth. Live imaging, hematoxylin-eosin (HE), and immunohistochemical (IHC) were employed to detect tumor growth in BALB/c nude mice. T98G cells were treated with CB, TMZ and the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway inhibitor or activator, or transfected with C-C motif chemokine ligand 5 (CCL5) up/down-regulation plasmids to evaluate whether CB could reverse TMZ resistance and further explore whether the PI3K/Akt/mTOR pathway was involved in this mechanism. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EDU) staining, transwell assay, flow cytometry, and Western blot were performed to detect cell proliferation after different treatments. RESULTS: In BALB/c nude mice, the combination therapy of CB and TMZ reduced TMZ resistance and inhibited tumor growth, as evidenced by suppressing tumor proliferation, decreasing MGMT expression levels, and increasing tumor cell death. In T98G cells, CB also reduced TMZ resistance, as indicated by decreased cell proliferation and invasion capacity, reduced MGMT expression, and increased cell death. The PI3K/Akt/mTOR pathway is essential for CB's function, as its activation not only promotes cell growth but also enhances TMZ resistance. Further investigation demonstrated that CB inhibits CCL5 transcription, thereby blocking the PI3K/Akt/mTOR pathway and ultimately inhibiting cell growth. CONCLUSIONS: Our findings indicate that CB enhanced the sensitivity of GBM to TMZ by blocking the CCL5-mediated PI3K/Akt/mTOR pathway. It provides a promising therapeutic strategy to reduce TMZ resistance in GBM treatment.