Targeted Metabolomics On Cancer Cell Metabolism Study

Endogenous metabolites are a kind of important substances in biosystems.The traditional non-targeted metabolomic studies provide a panoramic view of all the metabolites in different bio-samples,and the targeted metabolomics provide a further expanded view of some critical metabolites in the bio-samples.In this study,we developed rapid,sensitive,high-throughput and reliable targeted metabolomics platforms to simultaneously profile 20 endogenous nucleosides and nucleotides,40amino acids and their derivatives in cancer cell lines,based on ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry?UHPLC-MS/MS?,respectively.The two platforms were applied to the targeted metabolomics of three different cancer cells?A549,SW480 and MDA-MB-231?and breast epithelial cells?the breast cancer cell HCC1806 and the normal breast epithelial cell MCF10A?,respectively.In addition,the potential mechanism of endogenous metabolite 5-HT in the treatment of hypertension with Ketanserin was studied.This dissertation aims to provide new targeted metabolomics platforms and new ideas for the study of characteristic metabolite pathways,the mechanism of disease,the function of metabolites and the mechanism of drug actions in the future.This dissertation is divided into seven sections,Chapter 1 provides an introduction and Chapter 7 is the summary and prospect.The other five sections are as follows:In Chapter 2:Endogenous nucleosides and nucleotides are highly significant compounds,and play multiple,critical roles in biosystems.They are frequently highlighted as the most differential metabolites in recent metabolomics studies.We developed a rapid,sensitive,high-throughput and reliable quantitative platform to simultaneously profile 20 endogenous nucleosides and nucleotides in cancer cell lines based on ultra-high performance liquid chromatography-electrospray tandem mass spectrometry?UHPLC-MS/MS?by using a porous graphitic carbon column and basic mobile phase.In this study,we evaluated the effectiveness of two different extraction methods to cancer cells and optimized the MS parameters and LC conditions?columns,mobile phase composition,pH,etc.?.The results indicated that high pH value of mobile phase containing 0.12%diethylamine?DEA?and 5 mM NH₄OAC?pH 11.5?was the critical factor to prevent the adsorption of multi-phosphorylated species,and significantly improved peak shape and sensitivity.The optimized method was successfully validated with satisfactory linearity,sensitivity,accuracy,precision,matrix effects,recovery and stability for all analytes.The limit of quantification?LOQ?was in the range of 0.6⁶ nmol/L?6⁶0 fmol on column?.The validated method was applied to the extract of HCC1806 cancer cell lines,and 18 nucleosides and nucleotides can be detected.This quantified analytical platform of 20 endogenous nucleosides and nucleotides in cancer cell lines meets the requirement of quantification in specific expanded metabolomics studies,with good selectivity and sensitivity.In Chapter 3:Cancer is one of the most life-threatening diseases and still has a relatively high mortality rate.Cancer cells reprogram various metabolic pathways to satisfy their unique bioenergetic requirements.The validated target metabolomics platform to simultaneously profile 20 endogenous nucleosides and nucleotides in cancer cell lines based on UHPLC-MS/MS was applied to the extract of three epithelial cancer cell lines?A549,SW480 and MDA-MB-231?.The PCA and OPLS-DA models derived from the metabonomic analysis showed clear separation among MDA-MB-231,A549 and SW480 cell lines.Significant difference?p<0.01?of most of nucleosides and nucleotides were found between MDA-MB-231 and A549cells,which indicated a clear discrimination of the breast cancer cell line from the lung cancer cell line.On the basis of VIP>1 of the OPLS-DA model,p<0.01 of t-test and fold change>2.0,a total of 12 metabolites?CMP,CTP,Uridine,UMP,UDP,UTP,Adenosine,ADP,GMP,GDP,cAMP and IMP?were differentially expressed between MDA-MB-231 and A549 cells.The obtained metabolite levels of nucleosides and nucleotides are correlated with tumor characteristics of three cancer cells,and the result could serve as potential biomarkers for future investigation.In Chapter 4:Amino acids and their derivatives are biologically important metabolites which play an essential role in energy metabolism,neurotransmission,the synthesis of protein and lipid transport and so on.Endogenous metabolites of amino acids and their derivatives in bio-samples are frequently highlighted as the most differential metabolites in recent metabolomics studies.We developed a rapid,high-throughput,sensitive and reliable quantitative target metabolomics platform to simultaneously profile 40 underivatized amino acids and their derivatives including essential amino acids,non-essential amino acids and amino acid derivatives?N-acetyl amino acids and oligopeptides?in cell lines,based on ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry?UHPLC-MS/MS?by using a hydrophilic interaction liquid chromatography?HILIC?column.In this study,we evaluated the effectiveness of different extraction methods to breast cancer cells and optimized the MS parameters and LC conditions?columns,mobile phase composition,pH,etc.?.The optimized method was successfully validated with satisfactory linearity,sensitivity,accuracy,precision,matrix effects,recovery and stability for all analytes.The limit of detection?LOD?and the limit of quantifcation?LOQ?for most compounds were in the range of 0.2³.0 ng/mL?2³0 pg on column?and 0.6¹0.0 ng/mL?6¹00 pg on column?,respectively.The intra-day and inter-day precision were 1.65⁸.97%and 3.02⁹.94%,respectively,and the accuracy was-14.18¹5.13%and-13.13¹5.17%,respectively.The matrix effect¹ of samples to treated matrix was 80.0%¹14%,and the matrix effect² of treated matrix to pure solution was 80.2%¹19%.The recoveries of most compounds ranged from 89.6%to 114%.The targeted metabolomics platform has the advantages of high sensitivity,accuracy and high-throughput,and can be used for the study of disease metabolic pathway,disease progression prediction,pathogenesis,potential marker verification,metabolite function and drug action mechanism in the future.In Chapter 5:Breast cancer is a common female malignant cancer.Target metabolomics on amino acids and their derivatives are helpful to reveal the potential metabolite mechanisms of breast cancer metabolism at the molecular level.The established accurate targeted metabolomics platform was successfully applied to the analysis of amino acids and their derivatives in the breast cancer cell HCC1806 and the normal breast epithelial cell MCF10A.Principal component analysis?PCA?and the orthogonal projections to latent structures?OPLS?showed a clear discrimination of the non-tumorigenic breast epithelial cell line MCF-10A from the breast cancer cell line HCC 1806.Characteristic metabolic changes in amino acid metabolism were observed in the breast cancer cell line.Significant difference?p<0.001?of amino acids and their derivatives were found between MCF-10A and HCC 1806 cells,which indicated a clear discrimination of the breast cancer cell line from the non-tumorigenic breast epithelial cell line.On the basis of VIP>1 of the OPLS model,p<0.001 of t-test,a total of 24 analytes were increased more than 1.5-fold in breast cancer cell line of HCC1806 in comparison with the ones in the breast epithelial cell line MCF-10A.Among the 24 potential marker metabolites,it was observed that the levels of seven target compounds including L-proline,L-citrulline,Lcysteine,glutathione,N-acetyl-L-methionine,glycyl-glycine and Nacetyl-L-serine were significantly higher?>2.5 fold?in breast cancer cell line HCC1806 than those in the epithelial cell line MCF-10A.Characteristic metabolic changes in amino acid metabolism were observed in the breast cancer cell line and the details of amino acid metabolism disorder provided us more information on understanding the underlying biological mechanism.In Chapter 6:Hypertension,a common chronic disease,is also the key risk factors of cardio-cerebro-vascular diseases.Serotonin and its receptors in the peripheral circulatory system were reported to play an important role in the formation of hypertension in previous literature.We developed a rapid,sensitive,and reliable quantitative method to detect serotonin in whole blood of rats,based on HILIC-MS/MS.Various types and concentrations of anticoagulants,which might affect platelet deposition and the release of serotonin from dense granules in plasma,were systematically investigated.The comparisons of various kinds and concentrations of anticoagulant revealed that platelet-poor plasma with 10 mmol/L EDTA-K2 could minimize the release of serotonin from platelet activation.The developed fast method?only 2.5 min?was successfully validated with satisfactory linearity,sensitivity,accuracy,precision,matrix effects,recovery,and stability for quantification of serotonin in whole blood.The mechanism of Ketanserin in the treatment of hypertension was studied by detection of serotonin in the plate-poor plasma and serum of the normal group,the treatment group and the model group.The serotonin receptor 5-HT_2A on peripheral blood vessels and platelets can be inhibited by the receptor antagonist Ketanserin,and the peripheral vascular resistance and the platelet aggregation will be reduced.