Natural small molecule thymoquinone increases the chemosensitivity of glioblastoma to temozolomide through inhibiting Wnt/β-catenin signaling pathway to downregulate MGMT expression: In vitro and in vivo validation

Temozolomide (TMZ) is the only one oral first-line chemotherapeutic drug for glioblastoma treatment. However, O⁶-methylguanine-DNA methyltransferase (MGMT) can repair the lethal O⁶-methylguaine (O⁶-MeG) lesion produced by TMZ, thus imparting resistance to TMZ. Currently, the clinical utility of small molecule covalent MGMT inhibitors is limited by the occurrence of severe hematological toxicity. Therefore, developing new strategies for overcoming MGMT-mediated resistance is highly urgent. Here, we explored the feasibility that modulating Wnt/β-catenin signaling pathway in glioblastoma to inhibit MGMT expression to overcome TMZ resistance. From eight natural products or approved drugs with inhibitory effects on Wnt/β-catenin pathway, we found thymoquinone (TQ) completely suppressed MGMT expression in glioblastoma SF763 and SF767 cell lines within 24 h. As expected, TQ exhibited synergistic killing effects with TMZ in SF763 and SF767 cells, while in MGMT-negative SF126 cells only additive effect observed. Moreover, TQ remarkably enhanced the inhibition of TMZ on cell proliferation, clone formation, invasion and migration, and promoted cell apoptosis. In resistant SF763 mice tumor xenograft model, TQ significantly increased the suppression of TMZ on tumor growth, meanwhile maintaining good biosafety. Western blotting analysis indicated that TQ significantly inhibited the nuclear translocation of β-catenin and the expression of downstream proteins Cyclin D1 and MGMT. The addition of Wnt activator LiCl reversed the nuclear translocation of β-catenin and the expression of Cyclin D1 and MGMT induced by TQ. For the first time, our findings indicate that TQ can considerably increase the sensitivity of glioblastoma to TMZ by interfering Wnt/β-catenin pathway to downregulate MGMT expression.