Evaluation of the combinatorial effect of Tinospora cordifolia and Zingiber officinale on human breast cancer cells.

The present study was aimed to investigate the anticancer potential of the combination treatment of Tinospora cordifolia (TC) and Zingiber officinale (ZO) using network pharmacology approach. In silico analysis of the anticancer activity of TC + ZO was carried out using Cytoscape 3.2.0 software to elucidate the mechanism. The MTT assay confirms the combination of TC and ZO is more active (IC₅₀; 2 μg ml^-1) as compared to TC (509 μg ml^-1) and ZO (1 mg ml^-1) alone in MCF-7 cells. The TC + ZO combination treatment inhibits DNA synthesis, migration, and induces apoptosis in MCF-7 cells as compared to TC and ZO alone at a concentration of 1 µg ml^-1. TC + ZO combination treatment arrested cell cycle significantly at the G₀/G₁ phase. The proposed synergistic activity of the two herbs in the treatment of several cancers was correlated with an appropriate associated target/s, based on the pharmacological network. Interestingly, when both the plants used in combination, were found to regulate a total of 16 genes in 27 types of cancers. Further, ALOX5, MMP2, and MMP9 genes were identified as major targets which are responsible for the TC + ZO anticancer activity. According to merged and sub-networks of source-bioactive, bioactive-target, target-disease of TC, ZO alone and their combination; MMP9 was selected for validation purpose. The real-time PCR analysis confirmed that the TC + ZO combination treatment significantly down-regulated MMP9 mRNA expression by fivefold via up-regulation of its downstream target ER-α by 3.5-fold. In conclusion, the network analysis and in vitro validation confirmed the potent synergistic activity of TC + ZO combination treatment in breast cancer.