OPTIMIZATION OF HARVESTING, EXTRACTION, AND ANALYSTICAL PROTOCOLS FOR UPLC-ESI-MS BASED METABOLOMIC ANALYSIS OF ADHERENT MAMMALIAN CANCER CELLS There is an urgent need for methods be developed and standardized for the extraction of small metabolites from solid tissues. a liquid chromatography mass spectrometry-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. EDTA treatment and cell scraping in water were examines for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared, and the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was found to be the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties. ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) ALPHA IN TRICHLOROETHYLENE-INDUCED TOXICITIES: Trichloroethylene (TCE) exposure can lead to liver toxicity and contribute to hepatocarcinogenesis. TCE-induced liver toxicity and carcinogenesis is mediated in part by activation of the PPARalpha. However, the contribution of the two TCE metabolites, dichloroacetate (DCA) and trichloroacetate (TCA) to the toxicity of TCE has not been established. To determine the metabolic response to TCE exposure and the contribution of DCA and TCA to TCE toxicity, metabolic profiling was carries out in serum and urine after TCE exposure. C57BL/6N mice were administered TCE, TCA, or DCA, and urine and serum subjected to ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based global metabolomics analysis. The ions were identified through searching metabolomics databases and by comparison with authentic standards, and quantitated using multiple reactions monitoring. Quantitative polymerase chain reaction of mRNA, biochemical analysis, and liver histology were also performed. TCE exposure resulted in a decrease in urine of metabolites involved in fatty acid metabolism, resulting from altered expression of PPARalpha target genes. TCE treatment also induced altered phospholipid homeostasis in serum, as revealed by increased serum lysophosphatidylcholine 18:0 and 18:1, and phosphatidylcholine metabolites. TCA administration revealed similar metabolite profiles in urine and serum upon TCE exposure, which correlated with a more robust induction of PPARalpha target gene expression associated with TCA than DCA treatment. These data show the metabolic response to TCE exposure and demonstrate that TCA is the major contributor to TCE-induced metabolite alterations observed in urine and serum. BIOMARKERS OF COORDINATE METABOLIC REPROGRAMMING IN COLORECTAL TUMORS IN MICE AND HUMANS: Frequently, colorectal cancer is diagnosed after symptoms appear, as early colorectal cancer is usually asymptomatic with symptoms usually only appear with more advanced disease. Early colon cancer is usually diagnosed through the use colonoscopies There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis. Mass spectrometry-based metabolomic analysis of urine and tissues from wild-type C57BL/6J and Apc(Min/+) mice, and mice with azoxymethane (AOM)-induced tumors, was employed in tandem with gene expression analysis. Metabolic profiling was also performed on colon tumor and adjacent nontumor tissues from cancer patients. The effects of beta-catenin activity on metabolic profiles were also assessed in mice with colon-specific disruption of the Apc gene. Thirteen markers were found in urine associated with development of colorectal tumors in Apc(Min/+) mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice revealed that derangement of metabolites were a reflection of coordinate metabolic reprogramming in tumor tissue. Similar changes in urinary metabolites were observed in mice with AOM-induced tumors and in mice with colon-specific activation of beta-catenin. The metabolic alterations indicated by markers in urine, therefore, appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 17 metabolites associated with the same metabolic pathways identified in mice;ten metabolites that were increased in tumor tissues, compared with non-tumor tissues, were also increased in urine from tumor-bearing mice. Gene expression and metabolomic profiles of urine and tissue samples from mice with colorectal tumors and of tumor samples from patients revealed pathways associated with derangement of specific metabolic pathways that are indicative of early-stage tumor development. These urine and tissue markers might be used for the early detection of colorectal cancer. Any patients showing alteration in the urine metabolites could then be subjected to colonoscopies and biopsies.
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