The Study Of The Inhibition Of Luteolin On TNBC Through SGK1-FOXO3a-BNIP3 Signaling

PurposeThe purpose of this study was to clarify the effects of luteolin on the cell proliferation,apoptosis,migration,and invasion of Triple-negative breast cancer(TNBC)cells as well as tumor growth,and to explore the specific mechanism of inhibiting TNBC.It provides a theoretical basis for clinical treatment and drug research and development of TNBC,as well as a theoretical basis for finding new therapeutic targets for TNBC.MethodsThe effects of luteolin on the proliferation,apoptosis,migration,and invasion of MDA-MB-231 and 4T1 cells were observed by CCK-8 assay,AnnexinV-FITC/PI staining,Wound healing assay,and Transwell assay.The effects of luteolin at different concentrations on the growth of orthotopic triple-negative breast cancer mice models were observed.RNA transcriptome sequencing was performed to analyze the differentially expressed mRNAs in MDA-MB-231 cells after luteolin treatment and GO and KEGG pathway enrichment analyses were performed to find the target genes and pathways of luteolin.RT-qPCR was used to verify the enriched pathways and genes at the mRNA level.Molecular docking analysis was used to analyze the relationship between the selected target genes and luteolin.The changes in apoptosis and autophagy after luteolin treatment were observed by Transmission Electron Microscope(TME).Western Blot was used to detect the effects of luteolin on the expressions of SGK1-FOXO3a-BNIP3,and apoptosis and autophagy-related proteins.SGK1 overexpression vector was constructed and transfected into MDA-MB-231 cells.The effects of luteolin on the proliferation and apoptosis of breast cancer cells after SGK1 overexpression were detected.RT-qPCR and Western Blot were used to detect the changes of SGK1-FOXO3a-BNIP3 and apoptosis and autophagy-related proteins in luteolin overexpressing SGK1.Immunohistochemistry(IHC)and Western Blot were used to further verify the effects of SGK1 on cell apoptosis and autophagy through the FOXO3a-BNIP3 pathway in a mouse model.Results1.Luteolin inhibited the proliferation,migration,and invasion and significantly promoted cell apoptosis.of TNBC cells MDA-MB-231 and 4T1 and tumor growth in mice in a time-and concentration-dependent manner.2.GO analysis of differentially expressed genes showed that the most enriched genes were transcription,cell cycle,protein phosphorylation,and apoptotic process.KEGG pathway analysis showed that the pathways related to autophagy and apoptosis included PI3K-Akt,p53,Hioop,and FOXO signaling pathways.PI3K-Akt and FOXO3a shared 6 common different genes IL6,CDKN1A,SGK1,EGF,AKT3 and IL7R.RT-qPCR confirmed that the mRNA expression levels of these genes were consistent with the sequencing results.The autophagy and apoptosis of MDA-MB-231 cells after luteolin treatment was significantly correlated with PI3K-Akt and FOXO pathways.3.Both AKT3 and SGK1 belong to the AGC kinase family and share certain structural and functional similarities.Molecular docking results showed that luteolin had a better binding affinity with SGK1.Therefore,SGK1 was selected for the next study.Transcription also showed that in the FOXO3a pathway,the expression of BNIP3,a downstream protein of SGK1,was significantly increased.4.Obvious apoptotic characteristics and autophagosomes were observed in luteolin-treated MDA-MB-231 cells by TME.In cell and animal experiments,Western Blot results showed that with the increase of luteolin concentration,the expression of SGK1 decreased,and its downstream p-FOXO3a,FOXO3a,and BNIP3 changed in response to the increase of luteolin concentration.5.Further experiments showed that luteolin significantly promoted the up-regulation of autophagy and apoptosis-related proteins LC3Ⅱ/Ⅰ,Beclin-1,Bax,Bim,and the down-regulation of P62 and BCl-2 through SGK1-FOXO3a-BNIP3,which promoted the occurrence of autophagy and apoptosis and thus inhibited the occurrence and development of TNBC.6.Overexpression of SGK1 significantly inhibited luteolin-induced proliferation inhibition and apoptosis induction in TNBC cells,while SGK1 overexpression significantly promoted proliferation and inhibited apoptosis in TNBC cells.SGK1-FOXO3a-BNIP3 pathway p-FOXO3a expression was increased while FOXO3a and BNIP3 were down-regulated.The intervention of luteolin could inhibit the above trend,and the overexpression of SGK1 alleviated the up-regulation of autophagy-and apoptosis-related proteins LC3Ⅱ/Ⅰ,Beclin-1,Bax,Bim,and the down-regulation of P62 and BCl-2 in TNBC treated by luteolin.ConclusionsLuteolin significantly inhibited the proliferation,migration,and invasion of TNBC cells and promoted apoptosis in a time-and concentration-dependent manner.Luteolin significantly inhibited the expression of SGK1 and AKT3 in TNBC cells,and up-regulated its downstream gene BNIP3.However,the direct binding ability of luteolin to SGK1 was better than that of AKT3,Luteolin can promote FOXO3a nuclear translocation by inhibiting SGK1 and lead to the transcription of BNIP3 in vitro and in vivo and finally promote the interaction between BNIP3 and apoptosis and autophagy proteins.