JSH⁃23 combined with Stattic inhibits proliferation and migration of glioma cells by targeting NF⁃κB and STAT3 signaling pathways

Abstract

Objective To investigate the effects of JSH⁃23 combined with Stattic targeting inhibition of nuclear factor⁃κB (NF⁃κB) and signal transducer and activator of transcription factor 3 (STAT3) signaling pathways on the proliferation and migration ability of mesenchymal glioblastoma cells and the expressions of NF⁃κB pathway and STAT3 pathway⁃related proteins. Methods The mRNA⁃seq results of 529 glioma patients were downloaded from The Cancer Genome Atlas (TCGA). Bioinformatics was used to analyze the correlation between RelA/P65 and STAT3 and the markers of mesenchymal glioblastoma, as well as the expression of NF⁃κB pathway and STAT3 pathway⁃related proteins in mesenchymal, classical and proneural glioblastoma. The human glioma cell lines U87MG and U251MG in vitro were treated with JSH⁃23, Stattic, JSH⁃23 and Stattic, respectively. The half⁃inhibitory concentration (IC₅₀) of the two drugs was calculated by cytotoxicity assay, and the synergistic effect of the two drugs was observed by drug synergistic assay. CCK⁃8 assay and colony⁃forming assay were used to detect the proliferation activity of tumor cells. Wound healing assay and Transwell assay were used to detect the migration ability of tumor cells. Western blotting was used to detect the relative expressions of NF⁃κB pathway and STAT3 pathway⁃related proteins P65, phosphorylated P65 (p⁃P65), STAT3, phosphorylated STAT3 (p⁃STAT3) and CD44. Results 1) Bioinformatics analysis showed that RelA/P65 mRNA and STAT3 mRNA were positively correlated with mesenchymal glioblastoma markers CD44, CXCR4, CHI3L1, IL⁃4R and TRADD (r=0.206-0.605; P＜0.01, for all); the expressions of NF⁃κB pathway and STAT3 pathway⁃related proteins in mesenchymal glioblastoma were the highest, followed by classical glioblastoma, and the lowest in proneural glioblastoma. 2) The cytotoxicity assay showed the IC₅₀ of JSH⁃23 on U87MG and U251MG cells were 59.39 and 56.21 μmol/L, and Stattic were 0.96 and 1.08 μmol/L. The drug synergistic assay showed that the synergistic effect of JSH⁃23 at 40-80 μmol/L and Stattic at 0.50-1 μmol/L on U87MG was the highest, and the synergistic effect of JSH⁃23 at 40 μmol/L and Stattic at 1 μmol/L on U251MG was the highest. The final concentration of JSH⁃23 was 60 μmol/L and Stattic was 1 μmol/L. 3) After the combined treatment of JSH⁃23, Stattic, JSH⁃23 and Stattic, the proliferation activity of U87MG and U251MG cells decreased (P=0.000, for all), the colony forming efficiency decreased (P=0.000, for all), the cell mobility decreased (P＜0.05, for all), and the positive cell numbers of crystal violet staining decreased (P=0.000, for all). The relative expressions of P65 (P=0.000, for all), p⁃P65 (P=0.000, for all), STAT3 (P=0.000, for all), p⁃STAT3 (P=0.000, for all) and CD44 (P=0.000, for all) were decreased, especially after JSH⁃23 combined with Stattic treatment (P＜0.01, for all). Conclusions JSH⁃23 combined with Stattic targeting inhibition of NF⁃κB and STAT3 signaling pathways can effectively inhibit the proliferation and migration ability of mesenchymal glioblastoma cells, and down⁃regulate the relative expressions of NF⁃κB pathway and STAT3 pathway⁃related proteins.

doi：10.3969/j.issn.1672⁃6731.2022.05.011