Global metabolomics revealed deviations from the metabolic aging clock in colorectal cancer patients.

Background: Markers of aging hold promise in the context of colorectal cancer (CRC) care. Utilizing high-resolution metabolomic profiling, we can unveil distinctive age-related patterns that have the potential to predict early CRC development. Our study aims to unearth a panel of aging markers and delve into the metabolomic alterations associated with aging and CRC. Methods: We assembled a serum cohort comprising 5,649 individuals, consisting of 3,002 healthy volunteers, 715 patients diagnosed with colorectal advanced precancerous lesions (APL), and 1,932 CRC patients, to perform a comprehensive metabolomic analysis. Results: We successfully identified unique age-associated patterns across 42 metabolic pathways. Moreover, we established a metabolic aging clock, comprising 9 key metabolites, using an elastic net regularized regression model that accurately estimates chronological age. Notably, we observed significant chronological disparities among the healthy population, APL patients, and CRC patients. By combining the analysis of circulative carcinoembryonic antigen levels with the categorization of individuals into the "hypo" metabolic aging subgroup, our blood test demonstrates the ability to detect APL and CRC with positive predictive values of 68.4% (64.3%, 72.2%) and 21.4% (17.8%, 25.9%), respectively. Conclusions: This innovative approach utilizing our metabolic aging clock holds significant promise for accurately assessing biological age and enhancing our capacity to detect APL and CRC.

A proteomic clock for malignant gliomas: The role of the environment in tumorigenesis at the presymptomatic stage.

Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, "clocked", adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies.

Cerebrospinal fluid protein dynamic driver network: At the crossroads of brain tumorigenesis.

To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150 days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.

Alterations in cerebrospinal fluid proteins in a presymptomatic primary glioma model.

BACKGROUND: Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period. MATERIALS AND METHODS: Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified. RESULTS: Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin. CONCLUSIONS: The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.

Capture of phosphopeptides using alpha-zirconium phosphate nanoplatelets.

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.

Intracellular mediators of granulysin-induced cell death.

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.

Selenomethionine does not affect PSA secretion independent of its effect on LNCaP cell growth.

BACKGROUND: Individuals supplemented with selenium have reduced incidence of prostate cancer. This study determines whether selenomethionine specifically affects the secretion of prostate specific antigen (PSA) in vitro. METHODS: LNCaP cells were supplemented with selenomethionine for 7 days. PSA secretion was determined by ELISA. Cell proliferation was assessed by enumeration of trypan blue excluding cells. Colony formation was determined in soft agar. Cell cycle distribution was determined by FACS analysis of propidium iodide stained cells. RESULTS: Selenomethionine at > or = 70 microM inhibited LNCaP cell growth and colony formation. 0-100 microM selenomethionine did not affect the secretion of PSA by LNCaP cells in cell culture supernatants when normalized to the number of cells in culture. At supra-nutritional concentrations of selenomethionine, LNCaP cells had longer G(0)/G(1) phase in agreement with the inhibitory effects on cell growth. CONCLUSIONS: PSA secretion is not specifically inhibited by concentrations of selenomethionine corresponding to plasma selenium concentrations found in individuals supplemented with chemopreventive concentrations of selenized yeast. These data suggest that changes in serum PSA levels in individual patients during selenium supplementation is not an effect specific for PSA secretion, but rather may be a useful indicator for changes in disease progression in individual patients.