The mechanism of formononetin/calycosin compound optimizing the effects of temozolomide on C6 malignant glioma based on metabolomics and network pharmacology

The complex of formononetin and calycosin (FMN/CAL) shows a synergistic effect on temozolomide in the treatment of malignant glioma, however the mechanism is unclear. We investigated the mechanism through means of metabolomics, network pharmacology and molecular biology. FMN/CAL enhanced the inhibition of TMZ on the growth and infiltration of C6 glioma. The metabolomic results showed that the TMZ sensitization of FMN/CAL mainly involved 5 metabolic pathways and 4 metabolites in cells, 1 metabolic pathway and 2 me-tabolites in tumor tissues, and 7 metabolic pathways and 8 metabolites in serum. Further network pharmaco-logical analysis revealed that NOS2 was a potential target for FMN/CAL to regulate the metabolism in TMZ-treated C6 glioma cells, serums and tissues, and TNF-alpha was another potential target identified in tissues. FMN/CAL down-regulated the expression of NOS2 in tumor cells and tissues, and reduced the secretion of TNF-alpha in tumor region. FMN/CAL promoted TMZ-induced C6 cell apoptosis by inhibiting NOS2, but the inhibition of cell vitality and migration was not through NOS2. Our work revealed that FMN/CAL can increase the sensitivity of malignant glioma to TMZ by inhibiting NOS2-dependent cell survival, which provides a basis for the appli-cation of this combination in adjuvant treatment of glioma.

Automated summary

The combination of formononetin and calycosin enhances the sensitivity of malignant glioma to temozolomide. The mechanisms involved include metabolic pathway regulation and inhibition of specific targets.