Proteomic-based Study On Mechanism Of Natural Product Tryptanthrin Inhibiting Breast Cancer In Vitro And In Vivo

Objective:To study the effect of tryptanthrin on MAPK signaling pathway,EMT cell invasion and metastasis,and inflammatory tumor microenvironment breast cancer in mice,and to explore the mechanism of tryptanthrin in anti-breast cancer by non-standard quantitative proteomics.Methods: In vitro experiment: MCF-7 cells were treated with different concentrations of tryptanthrin and ERK,p38 MAPK and JNK pathway inhibitors,and the cell proliferation activity was detected by MTT method.Cell plate clone formation experiment was used to detect the effect of tryptanthrin on MCF-7 clone formation ability.H&E staining was used to observe the morphological changes of cells.TGF-?1 induced epithelial-mesenchymal transition(EMT)in MCF-7 cells.Cell scratch and Transwell cell experiment were used to evaluate the effect of tryptanthrin on cell migration and invasion after EMT.Western-Blot examined the expression levels of ERK,p-ERK,p38,p-p38,JNK and p-JNK proteins,and the expression of EMT-related marker proteins E-cadherin,Snail and MMP-2.In vivo experiment: A 4T1 mouse breast cancer model was established.Enzyme-linked immunosorbent assay(ELISA)was used to determine the effect of tryptanthrin on the expression levels of IL-2,IL-6,IL-10,IL-12 and TNF-? in mouse serum.H&E staining was used to evaluate the pathological changes of tumor tissues and organs in mice.Immunohistochemistry was used to detect the expression of NOS1 and COX-2 proteins in tumor tissues of mice.Western-Blot was used to detect the expression of NF-?B and STAT3 proteins in tumor tissues of mice.Proteomic experimental study: Nano-Flow UPLC/tims TOF was used to detect the expression protein of tryptanthrin in anti-breast cancer of mice,and proteomic study of tryptanthrin in anti-breast cancer was carried out.Results: Tryptanthrin significantly inhibited MCF-7 cell proliferation activity in a time-concentration dependent manner.The cell proliferation activity of tryptanthrin combined ERK inhibitor group,p38 MAPK inhibitor group and JNK inhibitor group was significantly different from that of tryptanthrin group alone.Cell plate clone formation experiment found that tryptanthrin can significantly inhibit the formation of MCF-7 cell clone.Under H&E staining,the cell morphology of tryptanthrin administration group was significantly different.Cell scratch test and Transwell cell test show that tryptanthrin can significantly inhibit cell migration and invasion after EMT.Western-Blot experiments showed that the expression of p-ERK and p-JNK in MCF-7 cells treated with tryptanthrin increased significantly,while the expression of p-p38 MAPK,ERK,p38 MAPK and JNK did not show any obvious changes.The expression of E-cadherin protein decreased in TFG-?1 model group,while the expression of tryptanthrin group increased.Snail and MMP-2 protein increased in TFG-?1 model group,while the expression of tryptanthrin group decreased significantly.Different doses of tryptanthrin group have certain inhibitory effect on tumor growth in mice,but have no obvious effect on body weight and spleen index of mice.ELISA results showed that there was no significant difference in the expression levels of IL-6 and IL-12 in serum of tryptanthrin administration group.The expression levels of IL-10,IL-2 and TNF-? were significantly increased.H&E staining showed that the 0.5% CMC-Na group had more atypical cells,pathological nuclear division,imbalance of nuclear-plasma ratio,more vacuolization and fewer apoptotic cells.In the low,medium and high dose tryptanthrin group,the cellular atypia and pathological mitosis were reduced,and some cells showed nuclear consolidation and apoptosis.Immunohistochemical results showed that COX-2 and NOS1 protein expressions in tumor tissues of tryptanthrin group were significantly decreased.Western-Blot analysis showed that the STAT3 protein expression in tryptanthrin group had no significant change,while the NF-?B protein expression was significantly decreased.Proteomic studies have identified 3997 proteins,2911 of which can be quantified,750 differentially expressed proteins between model group and tryptanthrin group,286 of which are up-regulated and 464 are down-regulated.GO analysis shows that these differentially expressed proteins are mainly involved in biological processes such as proliferation,cell migration,apoptosis,immunity,angiogenesis,and inflammatory regulation.KEGG pathway analysis further showed that differentially expressed proteins were mainly concentrated in T cell receptor,B cell receptor,Toll-like receptor,NF-?B,Ras,IL-17,TNF,PI3K-Akt,MAPK and other signal pathways,and there were differential proteins closely related to tumor proliferation,apoptosis,inflammatory regulation and prognosis.Conclusion: Tryptanthrin can inhibit the proliferation activity of MCF-7 cells,and its mechanism of action may be related to activation of ERK and JNK signaling pathways.Tryptanthrin can significantly inhibit migration and invasion of breast cancer MCF-7 cells after EMT induced by TGF-?1,and affect EMT effect by regulating EMT markers.Tryptanthrin has certain inhibitory effect on the growth of breast cancer in vivo,and can regulate the expression level changes of tumor microenvironment related inflammatory factors.Non-quantitative proteomics technology can effectively screen differentially expressed proteins after tryptanthrin acts on breast cancer mice,and it is found that proteins closely related to tryptanthrin 's anti-breast cancer mice effect may be found.