Indirubin suppresses 4T1 murine breast cancer in vitro and in vivo by induction of ferroptosis

Objective Indirubin,isolated from Indigo Naturals,has been reported to have the inhibitory activity of MCF-7 human breast cancer cells in vitro.However,studies on its anti-breast cancer activity in vivo and the underlying mechanism are insufficient.We explored whether indirubin could trigger ferroptosis of breast cancer cells to exert anti-tumor activity.Methods Bioinformatical analysis was performed to detect the expression of prostaglandin-endoperoxide synthase 2(Ptgs2)in breast cancer tissues and Ptgs2-related prognosis for patients with breast cancer.The inhibitory effects of indirubin on 4T1 cells were assessed using MTT assay and LDH activity detection.The levels of 4-HNE,GPX4,PTGS2 and GSK-3βproteins were detected by Western blotting,and the mRNA of Ptgs2 was tested by qPCR.The contents of GSH and MDA were determined by commercial kits.Molecular docking was employed to study the interaction between indirubin and GSK-3β.A 4T1 murine breast cancer was adopted to evaluate the in vivo antitumor activity of indirubin.Results Indirubin promoted ferroptosis of 4T1 breast cancer cells with depleting of GSH,increased MDA and 4-HNE levels,as well as decreased GPX4 expression.Indirubin suppressed 4T1 breast tumor in vivo.The mechanism study showed indirubin up-regulated Ptgs2 expression by promoting phosphorylation(Ser 9)of GSK-3β.Conclusion Indirubin suppresses 4T1 murine breast cancer in vitro and in vivo by induction of ferroptosis.