Identification And Validation Of Compound G-4 Acting On Triple-Negative Breast Cancer Targets

Triple-negative breast cancer(TNBC)has the characteristics of high malignancy and easy metastasis.Due to the common targets for estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(Her-2)are all negative.Currently,there is no clear targeted drug for TNBC,so finding therapeutic targets for TNBC is still a hot research topic in the world.Our research group designed and synthesized a series of purine derivatives in the early stage,and found that compound G-4 has a good effect on inhibiting the proliferation and metastasis of TNBC in vitro and in vivo,but its target remains unclear.In this study,the possible targets of purine derivative G-4 in TNBC cells were analyzed and preliminarily verified by highthroughput transcriptome sequencing,in order to provide new ideas for clarifying the mechanism of G-4 in the prevention and treatment of TNBC and developing drugs for TNBC treatment.Firstly,high-throughput transcriptome sequencing analysis was used to screen the differentially expressed genes of compound G-4 on MDA-MB-231 cells under different concentration and time.The Venn diagram was used to screen out the significantly different genes that overlapped in the DMSO-A0248 comparison group,the DMSOB0424 comparison group and the DMSO-C0448 comparison group,and these genes were subjected to GO enrichment analysis and KEGG analysis,and found that lipocalin2(LCN2)gene was enriched in the three pathways of iron ion transport,iron ion chelation and ferroptosis,and its expression was down-regulated in the experimental group.Therefore,it is speculated that compound G-4 may target LCN2 gene and cause down-regulation of LCN2 gene expression,thereby causing ferroptosis(Ferroptosis)of MDA-MB-231 cells.Ferroptosis is a novel cell death method proposed in 2012,which is an irondependent cel-mediated death caused by lipid peroxidation.In this experiment,the ferroptosis agonist Erastin,RSL3 and inhibitor Fer-1 were compared with compound G-4,and it was found that the level of cellular reactive oxygen species increased by 25%and lipid reactive oxygen species increased by 30% after the action of G-4.The iron content increased nearly 50 times,the MDA content increased 8 times,the SOD content decreased by 66%,and the GSH content decreased by 90%.These results suggest that LCN2 may be a potential target of G-4,as Erastin and RSL3 have the same effect on TNBC cells and can be inhibited by Fer-1.Finally,the LCN2 knockdown cell model was constructed by si RNA transfection for further verification,and it was found that G-4 could not cause the increase of lipid reactive oxygen species and MDA content in LCN2 knockdown cels.It can be speculated that LCN2 may be the target of compound G-4 on MDA-MB-231 cells.In conclusion,this study found that the purine derivative G-4 may inhibit the proliferation and metastasis of TNBC cells through LCN2-mediated ferroptosis,which provides a new idea for the prevention and treatment of triple-negative breast cancer.