Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.
ABSTRACT Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.
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ABSTRACT: Urinary biomarker tests for the diagnosis of prostate cancer (PCa) have gained considerable interest. Urine is a complex mixture that can be subfractionated. In this study we evaluated two urinary fractions that contain nucleic acids, i.e. cell pellet and exosomes. The influence of a digital rectal examination (DRE) before urine collection was also studied and PCa specific biomarkers PCA3 and TMPRSS2-ERG were assayed. Urine samples before and after DRE were prospectively obtained from 30 subjects scheduled for prostate biopsy. Cell pellet and exosomes were isolated and used for biomarker analysis. Analytical and diagnostic performance was tested using Student's t-test and ROC curves. Unlike the exosome fraction, urinary sediment gene expression analysis is compromised by amorphous precipitation in 10% of all specimens. The DRE results in increased mRNA levels in both fractions. This was particularly relevant for the exosomal fraction, since after DRE the number of samples, in which the cancer specific markers were below the analytical detection limit, decreased. The diagnostic performance of the biomarkers was comparable to that in large clinical studies. In exosomes, the biomarkers needed to be normalized for PSA mRNA, whereas the cell pellets absolute PCA3 levels had diagnostic value. Exosomes contain characteristics to serve as a stable substrate for biomarker analysis. Thereby, DRE enhances analytical performance of biomarker analysis in exosomes and cell pellet. Diagnostic performance of biomarkers in exosomes is different from the cell pellet. Its clinical utility needs to be prospectively assessed in larger clinical cohorts.The Journal of urology 11/2013; · 3.75 Impact Factor
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ABSTRACT: A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. Whereas these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib) and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes and signaling pathways that are simultaneously engaged by multi-targeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments.Molecular Cancer Therapeutics 09/2014; · 5.60 Impact Factor
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ABSTRACT: The toxicokinetics of trenbolone was characterized during 500 ng/L water exposures in female rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas). Related experiments measured various toxicodynamic effects of exposure. In both species, trenbolone was rapidly absorbed from the water and reached peak plasma levels within 8 hrs of exposure. Afterwards, trenbolone concentrations in trout (66-95 ng/ml) were 2-6 times higher compared to minnows (15-29 ng/ml), which was attributable to greater plasma binding in trout. During water exposures, circulating levels of estradiol (E2) rapidly decreased in both species to a concentration that was 25-40% of control values by 8-24 hrs of exposure and then remained relatively unchanged for the subsequent six days of exposure. In trout, changes in circulating levels of follicle stimulating hormone were also significantly greater after trenbolone exposure, relative to controls. In both species, the pharmacokinetics of injected E2-d3 was altered by trenbolone exposure with increased total body clearance and a corresponding decrease in elimination half-life. The unbound percentage of E2 in trout plasma was 0.25%, which was similar in pre- or post-vitellogenic female trout. Subsequent incubation with trenbolone caused the unbound percentage to significantly increase to 2.4 % in the pre-vitellogenic trout plasma. iTRAQ based toxicoproteomic studies in minnows exposed to 5, 50 and 500 ng/L trenbolone identified a total of 148 proteins with 19 down-regulated including vitellogenin and 18 up-regulated. Other down-regulated proteins were fibrinogens, α-2-macroglobulin and transferrin. Up-regulated proteins included amine oxidase, apolipoproteins, parvalbumin, complement system proteins and several uncharacterized proteins. The results indicate trenbolone exposure is a highly dynamic process in female fish with uptake and tissue equilibrium quickly established leading to both rapid and delayed toxicodynamic effects.Toxicological Sciences 09/2013; · 4.48 Impact Factor