Study On The Anti-Breast Cancer Effect And Mechanism Of Indirubin Obtained From Indigo Naturalis

Objective:Breast cancer is a malignant tumor caused by the uncontrolled proliferation of breast epithelial cells.It has been the most prevalent cancer in women.Radiation and chemotherapy are the mainly treatments.However,both radiation and chemotherapy can lead to drug resistance and serious side effects.Therefore,new effective drug and treatment are needed.Indirubin,obtained from Indigo naturalis,has anti-tumor effects.Ferroptosis is a newly discovered way of cell death.Studies have shown that ferroptosis is closely related to the occurrence and development of tumor cells.Therefore,this study aimed to investigate whether indirubin by triggering ferroptosis on breast cancer cells.Methods:1.The gene expression profiles of breast cancer and normal breast tissue provided by NCBIGEO database were analyzed by bioinformatics.Differentially expressed genes(DEGs)were identified using the GEO2 R online tool.Kaplan-Meier plotter and GEPIA online tools were used to analyze the prognostic relationship between PTGS2 gene and breast cancer.2.MTT assay was used to determine the effect of indirubin and indigo on the proliferation of 4T1 cells.Cell scratch assay was used to determine the migration of 4T1 cells.Ferroptosis inhibitor Fer-1,necrosis inhibitor z-VAD,and apoptosis inhibitor Nec-1 were used to explore the effect of indirubin on breast cancer.MDA and GSH were measured with test kits.Western blotting was used to measure the effect of indirubin and indigo on the expression of 4-HNE,GPX4,PTGS2.The expression of PTGS2 was determined by q-PCR.3.The Discovery Studio 3.0 Docking Program software was used to dock indirubin and indigo with GSK-3β molecules.Western blotting assay was utilized to determine the effect of indirubin and indigo on the expression of GSK-3β and P-GSK-3β in 4T1 cells.4.The subcutaneously inoculated mouse tumor model was established,and H&E staining method was used on pathological evaluation of mouse breast tumor.MDA and GSH levels in mouse breast tumors were measured with test kits.The protein expressions of 4-HNE,GPX4,PTGS2 were determined by Western blotting.To evaluate the anti-breast cancer activity of indirubin and indigo in vivo.Results:1.Bioinformatics analysis showed that there were 16 co-down-regulated genes in the differentially expressed genes between breast cancer and normal breast tissue.Among them,PTGS2 gene was low expressed in breast cancer tissues,and the low expression of PTGS2 gene was associated with poor prognosis of breast cancer patients.PTGS2 gene is a marker of ferroptosis,suggesting that breast cancer may be related to ferroptosis.2.The experimental results show that in vivo and in vitro,indirubin has inhibitory effect on breast cancer.With the addition of ferroptosis inhibitor Fer-1,necrosis inhibitor Z-VAD,apoptosis inhibitor Nec-1,Fer-1 can reverse indirubin-induced cell death,suggesting that the mode of indirubin-induced breast cancer cell death may be ferroptosis.Indirubin can promote lipid peroxidation in tumor cells,accompanied by decreasing GSH and increasing MDA and 4-HNE,GPX4 expression was reduced.These effects suggested that indirubin could inhibit proliferation by enhancing ferroptosis.3.Mechanism studies suggested that the anti-breast cancer and induction ferroptosis of indirubin was related to promote the phosphorylation(Ser9)of GSK-3β and up-regulate PTGS2 expression.The molecular docking results showed that the difference in molecular structure between indirubin and indigo may be the reason for the difference in activity.Conclusion:Indirubin could inhibit breast cancer by promoting phosphorylation at Ser9 of GSK-3β,up-regulating the expression of PTGS2,thereby inducing ferroptosis.