Target pathway validation for Compound Kushen Injection active against cancer cells.

Traditional Chinese medicines are based on complex mixtures of natural products and their multi-target mechanism of action. Therefore the discovery and validation of targets and mechanisms have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and candidate target genes for validation, through which CKI exerts its pharmacological effects. This study aimed to demonstrate the involvement of multiple genetic targets in different pharmacological activities for natural products in Compound Kushen Injection. In this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity. Although the sensitivity of different cell lines to gene knockout varied, overall, it led to reductions in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI. These findings validate the results of our previous transcriptomic analysis and further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures.