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 metabolites in tumor tissues, and 7 metabolic pathways and 8 metabolites in serum. Further network pharmacological 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-α 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-α 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 application of this combination in adjuvant treatment of glioma.