Dataset Information

The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

ABSTRACT:

Background

P7C3 is a neurogenic compound that exhibits neuroprotective properties in neural cells. However, its target proteins and effects in glioma are unknown.

Methods

The candidate P7C3 target proteins were analyzed using a human protein microarray containing 23136 human proteins. A streptavidin agarose affinity assay was used to verify the direct interaction between P7C3 and phosphoglycerate kinase 1 (PGK1). Mass spectrometry was used to identify the binding sites of PGK1 for P7C3 binding. Seahorse XF96 extracellular flux analyzer was used to measure the cell oxygen consumption rate and extracellular acidification rate. Glycolytic metabolites were measured using the related kits. Protein level was detected by western blotting and immunohistochemical staining. Autophagy was analyzed using a transmission electron microscope and western blotting. The malignancy of tumor progression in vitro and in vivo was analyzed based on cell viability, apoptosis and proliferation, migration and invasion, and xenograft model. Glial cells were marked by antibodies via immunohistochemical staining.

Results

The human protein microarray identified 577 candidate P7C3 target proteins. The global profile of P7C3 target proteins indicated that P7C3 regulates glycolysis. Metabolic experiments confirmed that P7C3 regulates aerobic glycolysis in glioma cells. The underlying mechanism of P7C3 was found to be direct targeting PGK1 at lysine residues and asparagine residues, and the specific P7C3-PGK1 interaction led to decreased protein level and total intracellular kinase activity of PGK1. The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases indicated that the mRNA level of PGK1 is significantly increased in high-grade glioma, and the abnormally high mRNA level of PGK1 is associated with a poor prognosis in patients with glioma, suggesting that PGK1 is a promising target for glioma therapy. The inhibition of PGK1 and the subsequent suppression of aerobic glycolysis caused by P7C3 inhibited the malignant growth of glioma in vitro and in vivo. Furthermore, P7C3 did not damage normal glial cells under concentration, which exhibit an inhibitory effect on gliomas.

Conclusions

This study revealed that P7C3 suppresses glioma by regulating aerobic glycolysis via directly targeting PGK1. Furthermore, we identified the P7C3 target proteins for the first time which is expected to provide scientific clues for future studies.

SUBMITTER: Chen W

PROVIDER: S-EPMC8252887 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inhibition of glycolysis process has been an attractive approach for cancer treatment due to the evidence that tumor cells are more dependent on glycolysis rather than oxidative phosphorylation pathway. Preliminary evidence shows that inhibition of phosphoglycerate kinase 1 (PGK1) kinase activity would reverse the Warburg effect and make tumor cells lose the metabolic advantage for fueling the proliferation through restoration of the pyruvate dehydrogenase (PDH) activity and subsequently promotion of pyruvic acid to enter the Krebs cycle in glioma. However, due to the lack of small molecule inhibitors of PGK1 kinase activity to treat glioma, whether PGK1 could be a therapeutic target of glioma has not been pharmacologically verified yet. In this study we developed a high-throughput screening and discovered that NG52, previously known as a yeast cell cycle-regulating kinase inhibitor, could inhibit the kinase activity of PGK1 (the IC50 = 2.5 ± 0.2 μM). We showed that NG52 dose-dependently inhibited the proliferation of glioma U87 and U251 cell lines with IC50 values of 7.8 ± 1.1 and 5.2 ± 0.2 μM, respectively, meanwhile it potently inhibited the proliferation of primary glioma cells. We further revealed that NG52 (12.5-50 μM) effectively inhibited the phosphorylation of PDHK1 at Thr338 site and the phosphorylation of PDH at Ser293 site in U87 and U251 cells, resulting in more pyruvic acid entering the Krebs cycle with increased production of ATP and ROS. Therefore, NG52 could reverse the Warburg effect by inhibiting PGK1 kinase activity, and switched cellular glucose metabolism from anaerobic mode to aerobic mode. In nude mice bearing patient-derived glioma xenograft, oral administration of NG52 (50, 100, 150 mg· kg-1·d-1, for 13 days) dose-dependently suppressed the growth of glioma xenograft. Together, our results demonstrate that targeting PGK1 kinase activity might be a potential strategy for glioma treatment.