The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

Abstract

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor with a poor median survival of less than 15 months. However, clinical strategies and effective therapies are limited. Here, we found that the second-generation small molecule multi-CDK inhibitor AT7519 is a potential drug for GBM treatment according to high-throughput screening via the Approved Drug Library and Clinical Compound Library (2718 compounds). We found that AT7519 significantly inhibited the cell viability and proliferation of U87MG, U251, and patient-derived primary GBM cells in a dose-dependent manner. Furthermore, AT7519 also inhibited the phosphorylation of CDK1/2 and arrested the cell cycle at the G1-S and G2-M phases. More importantly, AT7519 induced intrinsic apoptosis and pyroptosis via caspase-3-mediated cleavage of gasdermin E (GSDME). In the glioblastoma intracranial and subcutaneous xenograft assays, tumor volume was significantly reduced after treatment with AT7519. In summary, AT7519 induces cell death through multiple pathways and inhibits glioblastoma growth, indicating that AT7519 is a potential chemical available for GBM treatment.

Fig. 1. AT7519 inhibits glioblastoma cell proliferation and viability in vitro.

A Process for high-throughput drug screening. B, C U87MG, U251, GBM60, and GBM38 cell viability was determined using a CCK-8 assay after treatment with various concentrations of AT7519. *P < 0.05, ***P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. D U87MG and U251 cell viability was determined using a CCK-8 assay after treatment with 0.4 µM AT7519 for 6, 12, 24, 48, and 60 h. *P < 0.05, ****P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. E Colony formation assays to verify the effect of AT7519 on glioblastoma cell proliferation. *P < 0.05, ***P < 0.001, and ****P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. F, G The level of DNA synthesis of U87MG and U251 cells was determined using an EdU assay after treatment with increasing concentrations of AT7519. The nuclei were stained with Hoechst (blue), and the proliferating cells were stained with EdU (yellow). *P < 0.05, ***P < 0.001 and ****P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. The results are presented as the mean ± SD from three independent experiments.

Fig. 2. Differential gene expression and pathway enrichment analysis of glioblastoma cells treated with AT7519.

A, B U87MG cells were treated with AT7519 for 48 h for RNA sequencing, and a volcano plot and heatmap of differential expression were obtained by analysis (upregulated genes are in red; downregulated genes are in blue; nonregulated genes are in gray (|log2FC | ≥ 1 and q-value ≤ 0.05). C KEGG pathway analysis of differentially expressed genes. The volcano map was drawn based on R (https://www.r-project.org/) on the OmicStudio platform (https://www.omicstudio.cn/tool). KEGG pathway analyses were performed using the OmicStudio tools at https://www.omicstudio.cn/tool.

Fig. 3. AT7519 arrests glioblastoma cells at the G1/S and G2/M phases of the cell cycle.

A, B After U87MG and U251 cells were treated with different concentrations of AT7519 for 48 h, the cell cycle distribution was detected by flow cytometry. C, D Western blot analysis of cell lysates treated with different concentrations of AT7519 for 48 h. Detection of G1/S and G2/M arrest-related proteins.

Fig. 4. AT7519 induces apoptosis in glioblastoma cells.

A, B After treatment of U87MG, U251, and GBM-derived primary cells with AT7519 for 48 h, the percentage of cells undergoing apoptosis was detected by flow cytometry using PI/Annexin V-FITC double staining. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as determined by one-way ANOVA followed by Dunnett’s multiple test. C, D Western blotting detection of proteins associated with apoptosis in U87MG and U251 cells treated with 0.1, 0.2, or 0.4 μM AT7519 for 48 h. E Mitochondrial membrane potential was displayed by change in the ratio between red (aggregated JC-1) and green (monomeric JC-1) fluorescence intensity measured by spectrofluorimeter. *P < 0.05 by Student’s t-test. F, G U87MG and U251 cells were treated with AT7519 following 2-h pretreatment with Z-VAD-FMK or DMSO, and the apoptosis ratio was detected by flow cytometry using PI/Annexin V-FITC double stain. ****P < 0.0001 as determined by Student’s t-test.

Fig. 5. AT7519 induces pyroptosis through caspase-3 cleavage of gasdermin E.

A U87MG and U251 cells were treated with AT7519 and morphological features of pyroptosis in SEM (red arrows, membrane pore-forming). B After U87MG and U251 cells were treated with AT7519 for 48 h, cytotoxicity was detected by lactate dehydrogenase (LDH) released into the cell culture medium. ****P < 0.0001 by Student’s t-test. C, D Full-length GSDME (GSDME-FL) and N-terminal GSDME (GSDME-N) were detected in glioblastoma cells after treatment with AT7519 for 48 h by western blot analysis. E, F U87MG and U251 cells were pretreated with Z-VAD-FMK for 2 h and then treated with AT7519 for 48 h. Cytotoxicity was detected by LDH release assay. ***P < 0.001 and ****P < 0.0001 as assessed by Student’s t-test. The apoptosis marker cleaved PARP and pyroptosis marker GSDME-N were detected by western blot. G U87MG and U251 cells were treated with Z-DEVD-FMK combined with AT7519 for 48 h, and western blot analysis of cleaved caspase-3, GSDME-FL and GSDME-N proteins was performed.

Fig. 6. AT7519 inhibits tumor growth in glioblastoma xenograft mice.

A Image of the subcutaneous xenograft tumors formed in nude mouse models. B Tumor volumes were measured and calculated every week. ****P < 0.0001 as assessed by two-way ANOVA followed by Sidak’s multiple test. C Tumors were excised and weighed at the end of the experiment. ***P < 0.001 by Student’s t-test. D Body weight of nude mice during administration of AT7519. E Representative H&E-stained images of the intracranial xenograft model in the AT7519 treatment group and control group. *p < 0.05 as assessed by Student’s t-test. F Western blot assay of the apoptosis, pyroptosis, and cell cycle-related key protein expression levels in tumor tissue. G Schematic model of the antitumor mechanism of AT7519 in glioblastoma cells.