| Objective:To investigate the effect of Baicalin on the 5-FU resistance of colorectal cancer(CRC)cells,and to further explore the molecular mechanism of Baicalin reversing colorectal cancer drug resistance through metabolic reprogramming.Methods:1.Network Pharmacology:(1)Predict potential targets of Baicalin based on PubChem database website(https://pubchem.ncbi.nlm.nih.gov/)and pharmmapper prediction database(http://lilab.ecust.edu.cn/pharmmapper/).(2)Build target interaction network based on string database(https://www.string-db.org/).(3)Build drug and gene network through Cytoscape3.10.0 software.(4)Based on the bioconductor package,the Genecard database(https://www.genecards.org/),and the online version of Venny2.1(http://www.liuxiaoyuyuan.cn/),the predicted target and key genes are intersected.(5)Molecular docking of Baicalin and intersection genes by PMV-1.5.6 and Chem3D software.2.In vitro experiments:Construct colorectal cancer 5-FU drug-resistant cell lines and treat them with Baicalin.(1)Cell viability was detected by CCK-8 assay,and drug-resistant proteins expression were detected by western blot(WB)and qRT-PCR.EdU assay and flow cytometry was used to detect cell proliferation and apoptosis.(2)Transwell assay for cell invasion and migration.(3)Sphere formation and WB was performed to detect cell stemness characteristics.(4)Several kinds of kits were used to detect and Glucose uptake,lactate production,LDH activity,and pyruvate kinase M2(PKM2)activity in the parental cells(WT)and drug-resistant cells(5-FU-R).(5)extracellular acidification rates(ECAR)and oxygen consumption rates(OCR)were detected by Seahorse Bioscience XF-96 extracellular flux analyzer.(6)CO-IP assay was used to detect the combination of PKM2 and HIF1α.(7)WB and qRT-PCR were used to detect the expression of metabolism-related genes and proteins in cells.3.In vivo experiments:(1)Inject HCT116/5-FU-R cells to construct a subcutaneous tumor-bearing model,and monitor the body weight and tumor volume changes of the mice.(2)Immunohistochemical staining to detect the expression of Ki67 and cleaved caspase3 in tumor tissues.(3)The expressions of PKM2,HIF1α,GLUT1,HK2,and LDHA in tumor tissues were detected by WB and qRT-PCR.Results:1.Network pharmacology:(1)A total of 293 potential targets of Baicalin were predicted,and 64 effective targets remained after removing invalid targets.(2)63 nodes were obtained in the protein interaction network,and 34 nodes were effective node,the expected number of edges is 29,the actual number of passes is 35,and the PPI enrichment value is 1.14×10-9.(3)The results of drug and gene network showed the interaction relationship between Baicalin and target gene.(4)GO function enrichment Set analysis showed that Baicalin was involved in the regulation of cytokine receptor binding and other functions.(5)KEGG pathway enrichment analysis showed that Baicalin was involved in the regulation of colorectal cancer,glycolysis/gluconeogenesis and other pathways.(6)There are 24 common genes in Baicalin and drug-resistant colorectal cancer,of which only PKM2 can be docked with Baicalin.2.In vitro experiments:(1)The results of CCK-8 experiments showed that after 48 hours of treatment with 5-FU(10-9 mol/L to 10-2 mol/L),the viability of HCT116 and HCT15 parental cells(WT group)were significantly lower than 5-FU resistant cell lines(5-FU-R group).IC50 value of HCT116/WT cell was 3.24×10-6 mol/L,IC50 of HCT116/5-FU-R cell was 7.51×10-4 mol/L,RI was 231.79.IC50 value of HCT15/WT cell was 1.16×10-6 mol/L,the IC50 value of HCT15/5-FU-R cells was 2.28×10-4 mol/L,and the RI was 196.5.(2)Results of qRT-PCR and WB showed that the mRNAs and proteins levels of PGP,MRP1 and BCRP in 5-FU-R cells was upregulated.(3)The results of EdU experiment showed that the proliferation of 5-FU-R cells was higher than that of WT cells.After 5-FU(10 μmol/L)treatment,the proliferation of WT cells decreased but 5-FU-R cells did not change significantly.(4)Flow cytometry results showed that after 5-FU(10 μmol/L)treatment,the apoptosis rates of WT cells increased but 5-FU-R cells did not change significantly.After treatment of different concentrations of Baicalin on WT and 5-FU-R cells,(5)CCK-8 results showed that Baicalin had a significant inhibitory effect on the viability of both WT cells and 5-FU-R cells,and the IC50 values were similar.(6)All concentrations of Baicalin(2.5,5 and 10 μmol/L)reduced the IC50-5-FU of 5-FU-R cells.(7)The results of qRT-PCR showed that Baicalin could significantly reduce the mRNA expression of PGP,MRP1 and BCRP in 5-FU-R cells.5-FU-R cells were treated with 10μmol/L Baicalin and 10 μmol/L 5-FU alone or in combination.(8)The results of TUNEL experiment showed that the apoptosis rate of the combined treatment group was significantly increased.(8)The results of WB experiment showed that the protein expressions of cleaved caspase3 and cleaved PARP in the combined treatment group were significantly increased.(9)Transwell results showed that the invasion and migration ability of 5-FU-R cells in the combined treatment group was significantly reduced.(10)The results of sphere formation experiments showed that The size and number of spheres in the combined treatment group were significantly reduced.(11)WB results showed that the rotein expression levels of stemness-related proteins SRY-Box Transcription Factor 9(SRY-Box Transcription Factor 9,SOX9)and Lysine Demethylasel(Lysine Demethylase 1A,LSD1)in the combined treatment group pwas significantly reduced.(12)The results of flow cytometry and kits showed that compared with WT cells,5-FU-R cells had significantly increased glucose uptake,lactate production,LDH activity,ECAR levels,and decreased OCR levels and OCR/ECAR value.Baicalin reversed the above metabolic index levels in 5-FU-R cells.(13)CCK-8 results showed that inhibition of glycolysis can reduce the activity of 5-FU-R cells.The 5-FU IC50 value of 5-FU-R cells in the high glucose combined with Baicalin group was significantly increased compared with that of Baicalin group.(14)The results of qRT-PCR and WB experiments showed that Baicalin reduced the mRNA and protein levels of PKM2,HIF1A,GLUT1,HK2,LDHA,and decreased the phosphorylation level of PKM2 Y105.(15)The kit and immunofluorescence results showed that Baicalin significantly increased the activity of PKM2 and inhibited nuclear transfer in 5-FU-R cells.5-FU-R cells were treated with PKM2 activity activator DASA-58 and HIF-1α inhibitor BAY87-2243 alone or in combination with Baicalin.(16)WB results showed that treatment with DASA-58 alone or in combination with Baicalin inhibited the expressions of p-PKM2 Y105,PKM2,HIF1α,GLUT1,HK2 and LDHA.Treatment with BAY87-2243 alone or in combination with Baicalin inhibited the expression of HIF-lα,GLUT1,HK2 and LDHA,but had no significant effect on the expression of p-PKM2 Y105.PKM2-silenced 5-FU-R cells(sh-PKM2)were treated with Baicalin.(17)The results of flow cytometry,kits and energy meters showed that after silencing PKM2,The levels of glucose uptake,lactate production and ECAR were significantly decreased,and the OCR level was increased in 5-FU-R cells.after Baicalin treatment,there was no significant change in the metabolism-related indicators of sh-PKM2 cells.3.In vivo experiments:(1)The tumor growth rate,volume and weight of the Baicalin treatment group and the combination treatment group were lower than those of the Control group,and the inhibition effect of the combination group was more obvious.(2)There was no significant change in the weight of the mice in single treatment group and the combination treatment group compared with the Control group.(3)The results of immunohistochemistry showed that the Baicalin alone or combined with 5-FU group significantly reduced the positive cells proportion of Ki67 and increased the expression of cleaved caspase3.(4)The results of qRT-PCR and WB experiments showed that the mRNA and protein levels of metabolic key enzymes were signifiicantly reduced,and the phosphorylation level of PKM2 Y105 was down-regulated in tumor tissue of the Baicalin alone or combined with 5-FU group.Conclusion:Baicalin reversed the 5-FU resistance of CRC cells through PKM2/HIF1α mediated metabolic reprogramming. |
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