Pharmacologically inhibiting phosphoglycerate kinase 1 for glioma with NG52.

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.

Osimertinib successfully combats EGFR-negative glioblastoma cells by inhibiting the MAPK pathway.

Glioblastoma (GBM) patients have extremely poor prognoses, and currently no effective treatment available including surgery, radiation, and chemotherapy. MAPK-interacting kinases (MNK1/2) as the downstream of the MAPK-signaling pathway regulate protein synthesis in normal and tumor cells. Research has shown that targeting MNKs may be an effective strategy to treat GBM. In this study we investigated the antitumor activity of osimertinib, an FDA-approved epidermal growth factor receptor (EGFR) inhibitor, against patient-derived primary GBM cells. Using high-throughput screening approach, we screened the entire panel of FDA-approved drugs against primary cancer cells derived from glioblastoma patients, found that osimertinib (3 µM) suppressed the proliferation of a subset (10/22) of EGFR-negative GBM cells (>50% growth inhibition). We detected the gene expression difference between osimertinib-sensitive and -resistant cells, found that osimertinib-sensitive GBM cells displayed activated MAPK-signaling pathway. We further showed that osimertinib potently inhibited the MNK kinase activities with IC50 values of 324 nM and 48.6 nM, respectively, against MNK1 and MNK2 kinases; osimertinib (0.3-3 µM) dose-dependently suppressed the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). In GBM patient-derived xenografts mice, oral administration of osimertinib (40 mg· kg-1 ·d-1, for 18 days) significantly suppressed the tumor growth (TGI = 74.5%) and inhibited eIF4E phosphorylation in tumor cells. Given the fact that osimertinib could cross the blood-brain barrier and its toxicity was well tolerated in patients, our results suggest that osimertinib could be a new and effective drug candidate for the EGFR-negative GBM patients.

[Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18].

OBJECTIVE: To construct a transgenic strain of Toxoplasma gondii high-expressing ROP18. METHODS: The gene sequence of encoding ROP18 was amplified by RT-PCR with RNA of T. gondii RH strain. The purified PCR product was subcloned into pCR-Blunt II-Top vector to construct pROP18. The gene sequence of encoding ROP18 was amplified from pROP18, and subcloned into pTUB8-mycGFPPftail-Ty1. The recombinant plasmid pTUB8-ROP18-Ty1 was electroporated into T. gondii RH strain. Stable transgenic cells were selected in the presence of 25 µg/ml mycophenolic acid and 50 µg/ml xanthine, and parasite clones were isolated by limiting dilution after drug selection. The expression of ROP18 in transgenic parasites was detected by immunofluorescence analysis and Western blotting. Giemsa assay was used to detect the proliferation rate of RH strain and the transgenic strain of T. gondii high-expressing ROP18. Twenty mice were divided into two groups. Each mouse in RH strain group was intraperitoneally injected with 1 x 10(3) RH strain, and that of transgenic strain group received 1 x 10(3) transgenic high-expressing ROP18 strain. RESULTS: The full-length sequence of ROP18 gene (1,665 bp) was amplified by RT-PCR. The recombinant plasmid pTUB8-ROP18-Ty1 was identified by restriction enzyme digestion and sequencing methods. Western blotting analysis showed that the transgenic high-expressing ROP18 strain expressed ROP18-Ty1 (Mr 56,000). Immunofluorescence assay showed that ROP18-Ty1 was localized in the rhoptry of T. gondii. Giemsa assay confirmed that ROP18 protein enhanced the proliferation of T. gondii. On the 6th, 12th, and 24th hour after HFF cells infected with T. gondii, the number of tachyzoites in transgenic strain group was 100.0 ± 16.9, 476.0 ± 31.1, and 860.0 ± 52.3, respectively, higher than that of RH strain group (88.0 ± 16.9, 300.0 ± 11.3, 675.0 ± 35.4) (P < 0.05). In RH strain group, all the mice were survival on the 8th day post-infection, the survival rate on the 14th day was 30% (3/10), and all died on the 16th day. In transgenic strain group, all the mice were survival on the 5th day post-infection, the survival rate on the 8th day was 30% (3/10), and all died on the 9th day. CONCLUSION: The transgenic high-expressing ROP18 strain of T. gondii is constructed.