Pyrvinium pamoate regulates MGMT expression through suppressing the Wnt/β-catenin signaling pathway to enhance the glioblastoma sensitivity to temozolomide

Abstract

Temozolomide (TMZ) is the mainstream chemotherapeutic drug for treating glioblastoma multiforme (GBM), but the intrinsic or acquired chemoresistance to TMZ has become the leading clinical concern, which is related to the repair of DNA alkylation sites by O⁶-methylguanine-DNA methyltransferase (MGMT). Pyrvinium pamoate (PP), the FDA-approved anthelminthic drug, has been reported to inhibit the Wnt/β-catenin pathway within numerous cancer types, and Wnt/β-catenin signaling pathway can modulate the expression of MGMT gene. However, whether PP affects the expression of MGMT and enhances TMZ sensitivity in GBM cells remains unclear. In the present study, we found that PP and TMZ had synergistic effect on inhibiting the viability of GBM cells, and PP induced inhibition of MGMT and enhanced the TMZ chemosensitivity of GBM cells through down-regulating Wnt/β-catenin pathway. Moreover, the overexpression of MGMT or β-catenin weakened the synergy between PP and TMZ. The mechanism of PP in inhibiting the Wnt pathway was indicated that PP resulted in the degradation of β-catenin via the AKT/GSK3β/β-catenin signaling axis. Moreover, Ser552 phosphorylation in β-catenin, which promotes its nuclear accumulation and transcriptional activity, is blocked by PP that also inhibits the Wnt pathway to some extent. The intracranial GBM mouse model also demonstrated that the synergy between PP and TMZ could be achieved through down-regulating β-catenin and MGMT, which prolonged the survival time of tumor-bearing mice. Taken together, our data suggest that PP may serve as the prospect medicine to improve the chemotherapeutic effect on GBM, especially for chemoresistant to TMZ induced by MGMT overexpression.

Fig. 1. PP inhibited the viability and colony forming capacity in GBM cells.

A PP chemical structure. B PP (0–8 μM) was used to treat the LN18, T98G, LN229, and U87MG GBM cell lines for 24, 48, and 72 h; then, MTT assay was conducted to detect cell viability (n = 3). C, D LN18 (C) and T98G (D) cells were subjected to PP treatment for 10 days. Later, the colonies formed were photographed and counted, as displayed in the histogram (n = 3). The results were presented in the manner of mean ± SD. Compared with control group, **p < 0.01; ****p < 0.001.

Fig. 2. PP induced GBM cells apoptosis and the arrest of cell cycle.

A LN18 and T98G cells were subjected to PP treatment at various concentrations for 48 h, and then flow cytometry was carried out along with annexin V-FITC and PI staining (left panel). The elevated apoptotic cells were quantified and presented in the histogram (right panel) (n = 3). B The protein levels of PARP-1, Bcl-2, Bax, caspase-9, cleaved caspase-3, and survivin were evaluated through western blotting following PP treatment at different contents. C PI staining combined with flow cytometry showed cell cycle distribution in the LN18 and T98G cell lines following PP treatment at different contents. D Histograms showed LN18 and T98G cell percentages at G0/G1, S as well as G2/M phase, respectively. E The cyclin B1 and cyclin D1 protein expression within LN18 and T98G cell lines following PP treatment for 48 h. The results were presented in the manner of mean ± SD. Compared with control group, *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.

Fig. 3. PP down-regulated MGMT expression and enhanced the TMZ chemotherapeutic effect on GBM cell lines.

A The PP role in MGMT protein level was measured through western blotting. B The PP role in MGMT mRNA level was measured through quantitative RT-PCR (n = 3). C LN18 and T98G cell viability under PP and TMZ treatment at different contents (n = 3). CalcuSyn 2.0 was adopted to generate the curves. The Fa-CI plots showed the value of combination index (CI) in every fractional effect, and PP was synergized with TMZ (CI < 1). D Surface plots of LN18 and T98G cells treated with TMZ, PP, or combined TMZ + PP shown in 2D and 3D. Plots were generated using Combenefit program by applying HSA model. E MGMT protein levels in over-expressing MGMT and control cells were examined within LN18 and T98G cells (top panel). Densitometric analysis of MGMT/β-actin expression fold change (bottom panel) (n = 3). F Effect of MGMT overexpression on cell viability under TMZ, PP, or TMZ combined PP treatment (n = 3). Results were presented in the manner of mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.

Fig. 4. PP inhibited the Wnt/β-catenin signaling pathway within the GBM cells.

A The PP role in β-catenin protein level was measured through western blotting. B Expression of β-catenin in the cytoplasm or nucleus was examined by western blotting following PP treatment at different contents. C β-catenin expression within LN18 and T98G cells following 2.0 μM PP treatment examined by immunofluorescence. The scale bar is 20 µm. D TCF/LEF-dependent luciferase activity was measured following PP treatment for 24 h (n = 3). The results were presented in the manner of mean ± SD. ***p < 0.005; ****p < 0.001.

Fig. 5. PP-induced MGMT suppression and weakened-resistance to TMZ of GBM cells mediated by β-catenin.

A MGMT and β-catenin protein levels in β-catenin knockdown and control cells were examined within LN18 and T98G cells (left panel). Densitometric analysis of β-catenin and MGMT expression (right panel) (n = 3). B Cell viability in β-catenin knockdown and control cells under TMZ treatment within LN18 and T98G cells (n = 3). C Effect of MGMT overexpression on cell viability in β-catenin knockdown and TMZ treatment groups within LN18 and T98G cells (n = 3). D MGMT and β-catenin protein levels in β-catenin overexpression and control cells under PP treatment. E Densitometric analysis of β-catenin/β-actin and MGMT/β-actin expression relative fold change in LN18 (top panel) and T98G cells (bottom panel) (n = 3). F Effect of β-catenin overexpression on cell viability under PP or PP combined TMZ treatment within LN18 and T98G cells (n = 3). G Schematic diagram for the MGMT promoter luciferase reporter plasmids (P-755/−39 ML, P-1346/−39 ML, and P-1989/−39 ML) possessed desired candidate Tcf/Lef-binding site numbers. H MGMT promoter luciferase reporter activity was measured following PP treatment at different contents in LN18 cells (n = 3). Data were presented in the manner of mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig. 6. The underlying mechanism of PP in regulating β-catenin.

A The protein levels of AKT, p-AKT, GSK3β, p-GSK3β (S9), p-GSK3β (Y216), p-β-catenin (S552), and p-β-catenin (S33/37/T41) were evaluated through western blotting following PP treatment at different contents. B Effect of pretreatment with GSK3β inhibitor CHIR-99021 on PP-induced β-catenin down-regulation (top panel). Densitometric analysis of β-catenin/β-actin expression fold change (bottom panel). C Schematic diagram for the underlying mechanism by which PP regulated β-catenin. PP inhibits the activation of AKT, thereby increasing the activity of GSK3β, leading to increased degradation of β-catenin by multi-protein destruction complex, which mainly includes Axin, APC, and GSK-3β. β-catenin has at least two distinct pools. One is located in cytoplasm which binds with E-cadherin, and the other one is involved in the Wnt signaling pathway so that shuttles between cytoplasm and nucleus [45]. AKT mediates the phosphorylation of β-catenin associated with plasma membrane, and then induces the dissociation of β-catenin from E-cadherin, which accumulates in cytoplasm and nucleus [30]. In the case of PP addition, PP reduces the p-AKT protein level and further down-regulates p-β-catenin (S552) expression in the cells. Consequently, dissociation of β-catenin from E-cadherin within cell membrane is reduced, so that the combination of β-catenin with the LEF/TCF transcription factors in cell nucleus is reduced, either, which hinders the activation of MGMT gene. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig. 7. PP suppressed GBM xenograft growth and was synergized with TMZ in vivo.

A Typical mouse bioluminescence images at 13, 22, 28 and 34 days following GBM cell implantation. B The bioluminescence images were quantitatively analyzed for four experimental groups (n = 5). *p < 0.05; ****p < 0.001. C Representative images of HE-stained brain sections in PP and control group. D Kaplan−Meier survival curves for the four experimental groups (n = 5). E Representative images of TUNEL staining in PP and control group. The scale bar is 10 µm. F The protein levels of p-AKT, p-GSK3β (S9), MGMT, and β-catenin were evaluated through western blotting within orthotopic GBM xenograft (n = 3). The results were displayed in the manner of mean ± SD. *p < 0.05 vs. control group; ^#p < 0.05 vs. single group.