| Background:Colon cancer is the most common gastrointestinal malignant tumor in the world.At present,surgical resection is the main method.For advanced stages with metastasis,chemotherapy is the main treatment,while multidrug resistance(MDR)often leads to unsatisfactory results.P-glycoprotein(P-gp)is one of the main factors that produce MDR.Classic P-gp inhibitors include verapamil and cyclosporin A,and verapamil is an antiarrhythmic drugs,cyclosporin A is immunosuppressants,and they can produce toxic and side effects while enhancing the sensitivity of chemotherapy.Therefore,it is urgent to find a class of drugs that can enhance the chemo-sensitivity of a chemotherapeutic drug without obvious toxic and side effects after being combined with a chemotherapeutic drug.Based on the low toxicity of the medicinal and food homologous components,some researchers pay more attention to natural organic small molecule compounds.Quercetin(Que)is a polyhydroxy flavonoid compound that can be extracted from a variety of plants.It has a wide range of pharmacological effects,such as anti-tumor,anti-inflammatory,prevention and treatment of cardiovascular and cerebrovascular diseases.Que has achieved some results in the reversal of multidrug resistance in recent years,while the pharmacological effect of Que on multidrug resistant colon cancer cells SW620/Ad300 and its reversal mechanism have not been reported.Objective:This article aims to explore the pharmacological effects of Que on regulating the proliferation of colon cancer multi drug resistant cells SW620/Ad300 by doxorubicin(Dox),and to study the molecular mechanism of Que to enhance the sensitivity of colon cancer cells SW620/Ad300 to Dox from the perspective of systems biology.Methods:(1)MTT experiment:To determine the concentration of Que and Dox;the effect of Que on the chemotherapy effect of Dox.(2)Apoptosis experiment:To determine the effect of Que on the apoptosis of Dox-induced colon cancer cells SW620 and its drug-resistant cells SW620/Ad300.(3)Dox accumulation experiment:To determine the effect of Que on the intracellular Dox accumulation of colon cancer cells SW620 and SW620/Ad300.(4)Rhodamine 123(Rho 123)accumulation experiment:To measure the accumulation of Rho123 in colon cancer cells SW620 and SW620/Ad300,and evaluate the effect of Que on P-gp transport activity.(5)Metabolomics:UPLC-MS/MS is used to analyze cell samples;OSI-SMMS software is used to characterize metabolites,MZcloud and HMDB are used for confirmation;SIMCA software is used to screen differential metabolites;MetaboAnalyst and KEGG are used to analyze related pathways.(6)Human solute carrier family 1(Solute carrier family 1,mrmber 5,SLC1A5)enzyme-linked immunoassay(ELISA)and glutaminase(GLS)activity analysis:To determine the effect of Que on the expression of SLC1A5 and glutaminase activity in colon cancer cells SW620 and SW620/Ad300.(7)Analysis of ATP(Adenosine triphosphate)and Reactive oxygen(ROS)content:The effects of Que,Dox,and the combination of the two drugs on the levels of ATP and ROS in colon cancer cells SW620 and SW620/Ad300 were determined.Results:(1)MTT experiment results:After a series of gradient concentrations of Dox were applied to colon cancer cells SW620 and SW620/Ad300 for 48 h,their IC50 values were 0.52±0.0018 μM and 13.02 ± 0.0035 μM.The resistance index(RI)was 24.95.After the Que of gradient series concentration was applied to colon cancer cells SW620 and SW620/Ad300 for 48 h,their IC50 values were 594.37±4.32 μM and 652.32 ± 3.86 μM,repectively,there is no significant difference in the toxicity of Que on parental and resistant cells.The IC50 values of 33.00 μM Que combined with Dox for SW620 and SW620/Ad300 cells were 0.52±0.0030μM and 3.66±0.0024μM,respectively.It indicated that Que had no significant effect on Dox for parent cells,while Que could significantly increase the sensitivity of colon cancer resistant cells to Dox for drug-resistant cells.The resistance fold(RF)was 3.55.(2)Apoptosis results:0.52 μM Dox induced apoptosis in 45.36%of SW620 cells,while there is no significant effect on the apoptosis of drug-resistant cells;33.00μM Que in combination with Dox was used to SW620 cells for 48 hours,compared with the effect of Dox alone on SW620 cells,the apoptosis rate did not change significantly,while the apoptosis of colon cancer resistant cells SW620/Ad300 increased significantly.(3)Dox accumulation experiment results:Dox was significantly lower in P-gp-expressing drug-resistant cells than parental cells;33.00 μM Que can significantly increase the intracellular Dox accumulation in colon cancer resistant cells SW620/Ad300,but there is no significant effect on the intracellular Dox content of colon cancer parental cells SW620.(4)Rho 123 accumulation experiment results:Under the action of Que,the accumulation of Rho 123 in colon cancer resistant cells SW620/Ad300 was increased,while the content of Rho 123 in colon cancer parental cells SW620 did not change significantly.(5)UPLC-MS/MS-based metabolomics research results:Compared with colon cancer parent cells SW620 and drug-resistant cells SW300/Ad300,13 differential metabolites were identified,including isoleucine,citric acid,and glutamine acid and glutamine,etc.,the metabolic pathways involved are alanine,aspartic acid and glutamic acid metabolism,aminoacyl-tRNA biosynthesis,D-glutamine and D-glutamic acid metabolism,etc.;Que acted on drug-resistant cells SW620/Ad300,7 metabolites were significantly changed,including isoleucine,tryptophan,glutamic acid and glutamine,proline,citric acid and N-acetylaspartic acid.Pathway analysis showed that D-glutamine and D-glutamic acid metabolism was the most significant(P=3.94E-04,Impact=0.2834).Que may reverse the multidrug resistance caused by high expression P-gp by affecting this metabolism.(6)SLC1A5 enzyme-linked immunosorbent assay and GLS activity analysis results:The expression of SLC1A5 in drug-resistant cells SW620/Ad300 is higher than that of parental cells SW620,Dox has no effect on the expression of SLC1A5,and Que inhibits the expression of SLC1A5,which is highly expressed in drug-resistant cells.There was no significant difference in GLS activity between SW620 cells and SW620/Ad300 cells,and Que had no significant effect on the enzyme activity.(7)ATP and ROS analysis results:ATP levels in SW620/Ad300 cells were significantly higher than that in SW620 cells.Dox inhibited ATP production in SW620 cells,but stimulated a weak up-regulation of ATP in drug-resistant cells.Combination of Que and Dox reduces ATP production in drug-resistant cells.0.52μM of Dox induces ROS production in SW620 cells without significant effect on drug-resistant cells.After combined with 33.00 μM Que,ROS in drug-resistant cells increased significantly.Conclusions:(1)Que increases the intracellular Dox accumulation in colon cancer resistant cells SW620/Ad300 by inhibiting P-gp transport activity,thereby increasing the apoptosis of SW620/Ad300 cells induced by Dox.(2)Que inhibits the glutamine metabolism by inhibiting the expression of the glutamine transporter SLC1A5,and then down-regulates the ATP level to inhibit the P-gp transport activity,thereby increasing the intracellular Dox and increasing the level of ROS,these could enhance the toxic effect on SW620/Ad300 cells and reverse the resistance of SW620/Ad300 cells. |
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