Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/ß-Catenin and Akt/NF-κB signaling pathways.

BACKGROUND: Surgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PURPOSE: This study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. METHODS: The potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. RESULTS: Network pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/ß-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. CONCLUSION: Our study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/ß-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Xihuang Pill-destabilized CD133/EGFR/Akt/mTOR cascade reduces stemness enrichment of glioblastoma via the down-regulation of SOX2.

BACKGROUND: Our previous study found that XHP could induce GBM cells to undergo apoptosis. A lot of evidence suggests that glioma stem-like cells (GSCs) are key factors that contribute to disease progression and poor prognosis of glioblastoma multiforme (GBM). Traditional Chinese medicine has been applied in clinical practice as a complementary and alternative therapy for glioma. PURPOSE: To evaluate the effect and the potential molecular mechanism of Xihuang pill (XHP) on GSCs. METHODS: UPLC-QTOF-MS analysis was used for constituent analysis of XHP. Using network pharmacology and bioinformatics methods, a molecular network targeting GSCs by the active ingredients in XHP was constructed. Cell viability, self-renewal ability, apoptosis, and GSC markers were detected by CCK-8 assay, tumor sphere formation assay and flow cytometry, respectively. The interrelationship between GSC markers (CD133 and SOX2) and key proteins of the EGFR/Akt/mTOR signaling pathway was evaluated using GEPIA and verified by western blot. A GBM cell line stably overexpressing Akt was constructed using lentivirus to evaluate the role of Akt signaling in the regulation of glioma stemness. The effect of XHP on glioma growth was analyzed by a subcutaneously transplanted glioma cell model in nude mice, hematoxylin-eosin staining was used to examine pathological changes, TUNEL staining was used to detect apoptosis in tumor tissues, and the expression of GSC markers in tumor tissues was identified by western blot and immunofluorescence. RESULTS: Bioinformatics analysis showed that 55 matched targets were related to XHP targets and glioma stem cell targets. In addition to causing apoptosis, XHP could diminish the number of GBM 3D spheroids, the proportion of CD133-positive cells and the expression level of GSC markers (CD133 and SOX2) in vitro. Furthermore, XHP could attenuate the expression of CD133, EGFR, p-Akt, p-mTOR and SOX2 in GBM spheres. Overexpression of Akt significantly increased the expression level of SOX2, which was prohibited in the presence of XHP. XHP reduced GSC markers including CD133 and SOX2, and impeded the development of glioma growth in xenograft mouse models in vivo. CONCLUSION: We demonstrate for the first time that XHP down-regulates stemness, restrains self-renewal and induces apoptosis in GSCs and impedes glioma growth by down-regulating SOX2 through destabilizing the CD133/EGFR/Akt/mTOR cascade.

Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway.

BACKGROUND: Glioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear. PURPOSE: This study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells. METHODS: CCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting. RESULTS: Here we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. CONCLUSION: We first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT+ GBM patients.