| Objective:Cervical cancer is one of the most common malignancy of the female lower genital tract,which characterized by high malignancy,high morbidity and high postoperative recurrence rate.Tanshinone I(Tan I)is one of the crucial lipid-soluble components of red sage(Salvia miltiorrhiza).Our previous research showed that Tan I inhibited the proliferation and migration of cervical cancer HeLa cells and directly affected the ultrastructure of mitochondria.While its mode of action against cervical cancer is unclear.Therefore,our study focuses on mitochondrial function to explore the effects of Tan I on cell proliferation,migration and mitochondrial energy metabolism,to elucidate the mechanism of Tan I by inducing mitophagy and affecting metabolic reprogramming,and finally inhibiting the metastasis of cervical cancer cells.Contents and Methods:CCK-8,cell counting,colony formation and Transwell assay were used to investigate the effects of Tan I on the proliferation,cell number,colony formation ability,cell migration and invasion ability of cervical cancer SiHa cells.Effect of Tan I on apoptosis and cell cycle of HeLa cells was detected by Annexin V-FITC/PI and PI/RNase staining assay respectively.Flow cytometry and laser confocal microscopy assay were performed to observe the effects of Tan I on autophagy markers and autophagic flux of HeLa cells.Flow cytometry,digital inverted fluorescence microscope and laser confocal microscopy were used to detect effects of Tan I on mitochondrial membrane potential,F-actin cytoskeleton structure,total mitochondrial content and colocalization of mitochondria with lysosomes of cervical cancer HeLa cells,respectively.The mode of actions between Tan I and BNIP3/NIX was detected by molecular docking.Effects of Tan I on differentially expressed genes and metabolites of cervical cancer HeLa cells were detected by RNA-seq and 1HNMR metabolomics experiments,respectively.Finally,Western Blot assay was performed to explore the effects on the BNIP3/NIX mitochondrial autophagy pathway of HeLa cells influenced by Tan I.Results:Tan I significantly inhibited the SiHa cells proliferation,reduced the cell numbers,inhibited the colony-forming ability as well as the migration and invasion ability in a time-and dose-dependent manner.Tan I induced apoptosis and S phase of cell cycle blocked at concentrations of 12.5μM and 25μM,the effect was pronounced after 48h.However,the highest concentration of Tan I used(50μM)did not show obvious effect on the apoptosis and cell cycle arrest.Interestingly,50μM of Tan I significantly induced the expression of autophagy marker(LC3B),the autophagic flux of HeLa cell and CQ blocked this effect.In addition,50μM of Tan I siginificantly decreased mitochondrial membrane potential,disrupted the F-actin structure,increased the total mitochondrial content and induced colocalization of mitochondria with lysosomes of HeLa cells.Analysis of the molecular docking suggested that Tan I interacted with BNIP3 and NIX through hydrogen bond.Further mechanism studies showed:50μM of Tan I significantly altered the RNA profile and signal processing in cervical cancer cells.KEGG analysis revealed that Tan I significantly influenced "central carbon metabolism in cancer" and "mitophagy-animal"processes.Metabonomics profiling identified 25 metabolites affected by Tan I treatment in cancer cells such as Glutamate,Valine,ATP and NDA,etc.KEGG pathway enrichment results suggested its affected cell metabolism process including protein digestion and absorption,as well as energy,amino acid metabolism in cervical cancer cells.Moreover,Tan I significantly induced the expression of mitophagy-related proteins BNIP3,NIX and Optineurin and the conversion of LC3-Ⅰ to LC3-Ⅱ,inhibited the expression of NDP52 and P62.While CQ further increased the conversion of LC3-Ⅰ to LC3-Ⅱ and the expression of P62.Conclusion:(1)Tan I significantly inhibited the proliferation,invasion and migration abilities of cervical cancer SiHa cells in a concentration-and time-dependent manner;(2)Tan I induced the apoptosis and cell cycle S phase arrest of cervical cancer HeLa cells;(3)Tan I induced mitophagy and mitochondria dysfunction of cervical cancer HeLa cells;(4)Tan I interacted with BNIP3 and NIX through hydrogen bond and significantly influenced the differentially expressed gene profiles and reprogramming mitochondrial metabolism of the cervical cancer HeLa cell;(5)Tan I significantly increased the level of BNIP3,NIX and Optineurin,increased the conversion of LC3-Ⅰ to LC3-Ⅱ and decreased the expression of NDP52 and P62;... |
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