Targeted Metabolic Profiling and PRM Analysis of Proteins Revealed Impaired Polyunsaturated Fatty Acid Metabolism and GTP Metabolism in the Brainstem of Spontaneously Hypertensive Rats.

An untargeted multi-omics study implicated the potential dysregulation of fatty acid, nucleotide, and energy metabolism in the brainstems of spontaneously hypertensive rats (SHRs). A further quantitative exploration of the alterations in the metabolic pathways is necessary for a deep understanding of the central nervous system in SHRs. Targeted metabolic profiling of 40 fatty acids (PeptideAtlas: PASS01671) and 32 metabolites of nucleotides and energy metabolism (PeptideAtlas: PASS01672) and parallel reaction monitoring analysis of 5 proteins (PeptideAtlas: PASS01673) were performed on the brainstems of SHRs (n = 8, 11 weeks old) and normotensive Wistar rats (n = 8, age-matched) using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem MS. The targeted profiling results of metabolites and proteins revealed decreased polyunsaturated fatty acid (PUFA) synthesis with a significant downregulation of cis-11,14-eicosadienoic acid, cis-13,16-docosadienoic acid, and docosatetraenoate and impaired PUFA oxidation with the accumulation of γ-linolenate induced by the significantly downregulated expression of 2,4-dienoyl-CoA reductase (p < 0.05). Dysregulated GTP and ATP metabolism was observed, with significantly decreased GDP and ADP (p < 0.05) correlated with reduced GTPases of guanine nucleotide-binding protein subunit beta-1 (GNB1), transforming protein RhoA (RHOA), and Rho-related GTP-binding protein RhoB (RHOB) in the brainstem of SHRs. In addition, protein-arginine deiminase type-2 was significantly reduced in the brainstems of SHRs (p < 0.05). The aberrant PUFA and energy metabolism might help to explain the alterations in the brainstem of SHRs. The findings on both metabolites and proteins could provide systemic insights into the pathology basis of altered PUFA and energy metabolism in hypertension, especially in the central nervous system.

Targeted metabolomics in the cell culture media reveals increased uptake of branched amino acids by breast cancer cells.

In addition to the altered amino acids in many cancer cells for their uncontrolled growth, targeted metabolomics in cell culture media could display a dynamic interaction between cancer cells and their micro-environments. Methodology for cell culture medium samples is different from that of cell lysates on sampling points, calculation and statistical analysis. Targeted profiling method of 40 amino acid and derivatives was validated and performed on cell culture medium samples from cell lines of HCC 1806 (breast cancer cell) and MCF-10A (normal breast epithelial cell). Different from the common up-regulation of amino acids in cancer cell lysates, significantly increased uptake (>2.5-fold, VIP>1 and p < 0.001) of branched amino acids was observed in the cell culture media from the breast cancer cells while acetylmethionine, cysteine-glutathione, glutathione, cysteine and glutamic acid were excreted significantly more by the cancer cells to their media. The characteristic metabolic changes of amino acid and derivatives in the cell culture media provide a dynamic portrayal for the interaction of the breast cancer cells, normal breast cells with their micro-environments, which helps to understand the underlying proliferation mechanism of breast cancer cells.

Targeted metabolomics reveals dynamic portrayal of amino acids and derivatives in triple-negative breast cancer cells and culture media.

Triple-negative breast cancer (TNBC) is well-known for its metastatic aggressiveness and poor survival prognosis, accounting for nearly a quarter of cases in breast cancer. We performed intra- and extra-cellular profiling of 40 amino acids and derivatives on three cell lines and their culture media, including TNBC, non-TNBC and normal breast epithelial cells, using HILIC-MS/MS. Characteristic metabolic alteration of amino acids and derivatives was observed in TNBC cells, compared to non-TNBC cells, especially in correlated intra- and extra-cellular metabolic pathways. Intra-cellularly, quantified glutamic acid, ß-alanine, aspartic acid, glutathione, N-acetyl-serine and N-acetyl-methionine were most significantly increased (>2-fold, p < 0.01 and VIP > 1) in TNBC cells. Extra-cellularly, significantly increased uptake of glutamine, serine, ß-alanine, and lysine and elevated excretion of glutamic acid and l-cysteine-glutathione (p < 0.01 and VIP > 1) were observed by TNBC cells from or to their cell culture media. This study depicted a novel dynamic portrayal of metabolic dysregulation between TNBC and non-TNBC cells, correlated in both intra- and extra-cellular amino acid profiles. Quantification of these distinctive metabolites of TNBC cells might offer advanced understanding and new treatment targets for TNBC.

Integrative Metabolic and Proteomic Profiling of the Brainstem in Spontaneously Hypertensive Rats.

The brainstem, the core of the central nervous system, plays a vital role in controlling arterial blood pressure and its elevation of hypertension subtypes, especially essential hypertension. Integrative metabolic and proteomic profiling was performed on the brainstem samples of 11 week old spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar rats, using hydrophilic interaction liquid chromatography quadrupole/time-of-flight mass spectrometry (HILIC-Q/TOFMS) (PeptideAtlas: PASS01621) and nano-liquid chromatography-high-resolution-MS (nano-LC-high-resolution) combined with quantitative tandem mass tags (ProteomeXchange: PXD021210). The results showed a potentially significant measure of metabolic disorders in the brainstem of SHRs, including purine and pyrimidine metabolism and carnitine and acylcarnitine deficiency. By integrating the differential metabolites (VIP > 1 and p < 0.1) with the differentially expressed proteins (>1.2-fold and p < 0.05), the results revealed aberrant insulin signaling in the brainstem of SHRs, including reduced carnitine and acetylcarnitine; increased arginine; and increased flotillin-1 (FLOT1), hemoglobin subunit alpha-1/2, and hemoglobin subunit beta-2 proteins verified by the parallel reaction monitoring analysis (PeptideAtlas: PASS01622). The aberrant insulin signaling pathway in the brainstem of SHRs might help explain the correlation between essential hypertension and insulin resistance. These findings on the brainstem of SHRs could provide new insights into the dysregulation of the central nervous system in hypertension, especially as it relates to metabolite and protein levels.

Nitidine chloride inhibits proliferation, induces apoptosis via the Akt pathway and exhibits a synergistic effect with doxorubicin in ovarian cancer cells.

Nitidine chloride (NC) exhibits anti-tumor properties in various types of tumor. However, to the best of our knowledge there is no previous evidence of NC involvement in the apoptosis or proliferation of ovarian cancer cells and the underlying molecular mechanisms. The present study aimed to investigate the influence of NC on the viability and apoptosis of ovarian cancer cells and the synergistic effect NC and doxorubicin (DOX) may have on ovarian cancer cells. The viability and proliferation of ovarian cancer cells were examined using a methyl thiazolyl tetrazolium assay and 3H-thymidine incorporation assay. The apoptotic rate of ovarian cancer cells was detected by flow cytometry. The expression of apoptosis­associated proteins and Akt serine/threonine kinase 1 (Akt) were determined by western blot analysis following NC treatment. The inhibitory effect of NC on the proliferation of ovarian cancer cells was demonstrated in a time and dose­dependent manner. The pro-apoptotic effect of NC on ovarian cancer cells was also observed. It was determined that NC significantly downregulated the protein expression levels of B­cell CLL/lymphoma 2 (Bcl-2) and upregulated the expression of Bcl­2­associated X protein, p53, caspase­3 and ­9. NC suppressed Akt phosphorylation. Additionally, the present study demonstrated that the effect of NC on the proliferation and apoptosis of ovarian cancer cells was Akt­dependent by using the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt signaling pathway inhibitor, LY294002. NC exhibited a synergistic inhibitory effect on the viability of ovarian cancer cells when combined with DOX. The current study demonstrated that NC inhibited the proliferation and induced the apoptosis of ovarian cancer cells via the Akt signaling pathway and highlighted its potential clinical application for the treatment of ovarian cancer.

Simultaneous determination of nineteen major components in Qi She Pill by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Qi She Pill (QSP) is a traditional Chinese medicine (TCM) prescription that has been used in treating cervical spondylosis radiculopathy for many years. In this study, a simple and sensitive method using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) on a reverse-phase C18 column was developed for the simultaneous determination of the 19 major components in QSP. We found that the optimum mobile phase for gradient elution was 0.1% formic acid and methanol. The correlation coefficients of all calibration curves were greater than 0.99. Recoveries measured at three concentration levels varied from 95.43% to 102.35%. Relative standard deviations of intra- and inter-day precisions were less than 4.45%. After successfully validating our method, we then applied it to the quantification of 19 components in QSP products to show that this method provides a new standard in quality assessment of TCM prescriptions containing multiple bioactive components.