Methionine-Homocysteine Pathway in African-American Prostate Cancer.

African American (AA) men have a 60% higher incidence and two times greater risk of dying of prostate cancer (PCa) than European American men, yet there is limited insight into the molecular mechanisms driving this difference. To our knowledge, metabolic alterations, a cancer-associated hallmark, have not been reported in AA PCa, despite their importance in tumor biology. Therefore, we measured 190 metabolites across ancestry-verified AA PCa/benign adjacent tissue pairs (n = 33 each) and identified alterations in the methionine-homocysteine pathway utilizing two-sided statistical tests for all comparisons. Consistent with this finding, methionine and homocysteine were elevated in plasma from AA PCa patients using case-control (AA PCa vs AA control, methionine: P = .0007 and homocysteine: P < .0001), biopsy cohorts (AA biopsy positive vs AA biopsy negative, methionine: P = .0002 and homocysteine: P < .0001), and race assignments based on either self-report (AA PCa vs European American PCa, methionine: P = .001, homocysteine: P < .0001) or West African ancestry (upper tertile vs middle tertile, homocysteine: P < .0001; upper tertile vs low tertile, homocysteine: P = .002). These findings demonstrate reprogrammed metabolism in AA PCa patients and provide a potential biological basis for PCa disparities.

Tobacco-Specific Carcinogens Induce Hypermethylation, DNA Adducts, and DNA Damage in Bladder Cancer.

Smoking is a major risk factor for the development of bladder cancer; however, the functional consequences of the carcinogens in tobacco smoke and bladder cancer-associated metabolic alterations remain poorly defined. We assessed the metabolic profiles in bladder cancer smokers and non-smokers and identified the key alterations in their metabolism. LC/MS and bioinformatic analysis were performed to determine the metabolome associated with bladder cancer smokers and were further validated in cell line models. Smokers with bladder cancer were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons, DNA adducts, and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with bladder cancer. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade bladder cancer. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in bladder cancer cells. Cotreatment of bladder cancer cells with these carcinogens and the methylation inhibitor 5-aza-2'-deoxycytidine rewired the methylated metabolites, DNA adducts, and DNA damage. These findings were confirmed through the isotopic-labeled metabolic flux analysis. Screens using smoke-associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in bladder cancer smokers. Noninvasive predictive biomarkers that can stratify the risk of developing bladder cancer in smokers could aid in early detection and treatment. Cancer Prev Res; 10(10); 588-97. ©2017 AACR.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer.

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid ß oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

EMT-induced metabolite signature identifies poor clinical outcome.

Metabolic reprogramming is a hallmark of cancer. Epithelial-mesenchymal transition (EMT) induces cancer stem cell (CSC) characteristics and promotes tumor invasiveness; however relatively little is known about the metabolic reprogramming in EMT. Here we show that breast epithelial cells undergo metabolic reprogramming following EMT. Relative to control, cell lines expressing EMT transcription factors show ≥1.5-fold accumulation of glutamine, glutamate, beta-alanine and glycylleucine as well as ≥1.5-fold reduction of phosphoenolpyruvate, urate, and deoxycarnitine. Moreover, these metabolic alterations were found to be predictive of overall survival (hazard ratio = 2.3 (95% confidence interval: 1.31-4.2), logrank p-value = 0.03) and define breast cancer molecular subtypes. EMT-associated metabolites are primarily composed of anapleurotic precursors, suggesting that cells undergoing EMT have a shift in energy production. In summary, we describe a unique panel of metabolites associated with EMT and demonstrate that these metabolites have the potential for predicting clinical and biological characteristics associated with patient survival.

A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases.

The growth of cancer cells relies more on increased proliferation and autonomy compared to non-malignant cells. The rate of de novo nucleotide biosynthesis correlates with cell proliferation rates. In part, glutamine is needed to sustain high rates of cellular proliferation as a key nitrogen donor in purine and pyrimidine nucleotide biosynthesis. In addition, glutamine serves as an essential substrate for key enzymes involved in the de novo synthesis of purine and pyrimidine nucleotides. Here, we developed a novel liquid chromatography (LC-MS) to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines). For this, DNA from 5637 bladder cancer cell line cultured in 15N labelled glutamine and then enzymatically hydrolyzed by sequential digestion. Subsequently, DNA hydrolysates were separated by LC-MS and Selected Reaction Monitoring (SRM) was employed to identify the nucleobases and nucleosides. Thus, high sensitivity and reproducibility of the method make it a valuable tool to identify the nitrogen flux primarily derived from glutamine and can be further adaptable for high throughput analysis of large set of DNA in a clinical setting.

Pathway-centric integrative analysis identifies RRM2 as a prognostic marker in breast cancer associated with poor survival and tamoxifen resistance.

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2-enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies. We measured the metabolome of luminal and basal-like BCa cell lines using mass spectrometry, linked metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in disease progression. Pyrimidine metabolism was altered in luminal versus basal BCa, whereas the combined expression of its associated genes or expression of one key gene, ribonucleotide reductase subunit M2 (RRM2) alone, associated significantly with decreased survival across all BCa subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. Our study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in BCa.

Application of 13C isotope labeling using liquid chromatography mass spectrometry (LC-MS) to determining phosphate-containing metabolic incorporation.

Here, we describe an approach wherein negative electrospray ionization mass spectrometry has used to understand the relative flux through phosphate containing metabolic intermediates associated with central carbon metabolism after administering cells with 13C-labeled substrates. The method was applied to examine the 13C incorporation through glycolysis in T47D breast cancer cells and showed reduction of glycolytic relative flux upon treatment with 2-Deoxyglucose.

Alteration of bile acid metabolism in pseudo germ-free rats [corrected].

To characterize the impact of gut microbiota on host bile acid metabolism, we investigated the metabolic profiles of oxysterols and bile acids (BAs) in a conventional rat model (SD) (n=5) and its pseudo germ-free (GF) equivalent (n=5). GF rats were developed by the oral administration of bacitracin, neomycin and streptomycin (200 mg/kg, each) twice a day for 6 days. Urinary levels of oxysterols and bile acid metabolites were quantified using gas chromatography-mass spectrometry (GC-MS). The activity levels of enzymes involved in the bile acid metabolic pathway were determined through urinary concentration ratio between product to precursor. Cholic acid (CA) and α-/ß-muricholic acid (α-/ß-MCA) were significantly elevated at pseudo germ-free condition. An increase of hydroxylase (cholesterol 7α-hydroxylase, oxysterol 7α-hydroxylase and cytochrome P450 scc) and a significant decrease of 7α-dehydroxylase were observed. The urinary concentration ratio of primary bile acids, a marker for hepatotoxicity, increased in pseudo germfree conditions. Therefore, it was found that gut microbiota could play a significant role in the bile acids homeostasis and metabolism.

Analysis of plasma nucleotides in rat by micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry.

RATIONALE: The aim of this study was to establish a simultaneous quantitative analysis method of nine endogenous nucleotides in rat plasma using micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry (MEKC/ESI-MS). METHODS: To select the optimum conditions for separation of the nucleotides, various pH, concentrations of running buffers and surfactants were tested. Ammonium acetate (20 mM) containing the surfactant dodecyltrimethylammonium bromide (2 mM, pH 3.5) was selected as the micellar running buffer. The plasma samples were prepared by precipitating the proteins with 2 mM EDTA in 60% ethanol. The samples were analyzed using capillary electrophoresis (CE)/MS and selected ion monitoring (SIM) mode with positive ionization. CE was performed using a silica capillary column in reversed polarity mode. RESULTS: The limits of detection (LODs) and limits of quantification (LOQs) of the nucleotides ranged from 0.05-5 and 2.0-20 µM, respectively. The calibration curves were linear (R(2) >0.99) for all analytes, and the accuracy and precision were within ±15%. The developed method was applied to the analysis of nucleotides in rat plasma that was collected after oral administration of acetaminophen (1000 mg/kg/day) to evaluate the changes in plasma nucleotide levels under hepatotoxic conditions. CONCLUSIONS: Decreased level of GTP and increased level of cytosine nucleotides were found to be associated with liver toxicity, which led to the conclusion that liver toxicity is closely related to changes in nucleotide levels in plasma.

Identification of salt tolerant rice cultivars via phenotypic and marker-assisted procedures.

Eleven genotypes, including the salt tolerant cultivar Pokkali as check, were used to evaluate salinity tolerance phenotypically and genotypically. Three selected SSR primers viz., RM7075, RM336 and RM253 were used to evaluate rice genotypes for salt tolerance. Two setups were maintained for this study viz., the seedling and reproductive stages. Phenotyping at the seedling stage was done in hydroponic system using salinized (EC 12 dS m(-1)) nutrient solution and at the reproductive stage using salinized tap water (EC 6 dS m(-1)). IRRI standard protocol was followed to evaluate salinity tolerance in rice. The genotypes having similar banding pattern with Pokkali were considered as tolerant. Phenotypically, three genotypes Pokkali, THDB and TNDB-100 and five genotypes RD-2586, TNDB-100, Dhol Kochuri, PNR-519 and Pokkali were identified as salt tolerant at the seedling and reproductive stages, respectively. These genotypes were also identified as salt tolerant genotypically (with markers). Through phenotypic and genotypic study, five genotypes viz., Pokkali, Dhol Kochuri, RD-2586, TNDB-100 and PNR-519 were identified as salt tolerant. Therefore, these microsatellite markers used in this study could be efficiently used for identification of salt tolerant rice varieties in marker-assisted breeding and quantitative trait loci analysis.

Control of endogenous bacterial contamination and micropropagation of a traditional table banana (Musa spp. cv. Kanthali) of Bangladesh.

Shoot tips of a traditional table banana (Musa spp. cv. Kanthali) of Bangladesh were evaluated for in vitro propagation. Initial surface sterilization (with 0.1% HgCl2 for 12 minutes) of shoot tips was successful but microbial contamination (mostly bacteria) at the rhizomatous base of the explants was observed within 6-15 days after inoculation which eventually killed 85% of inoculated explants. So, for contamination free culture establishment explants were soaked in two broad spectrum antibiotics namely ampicillin and gentamicin. Cent percent contamination free cultures were established by soaking the explants in 400 mg/L ampicillin or 200 mg/L gentamicin for 1h. Antibiotic treated explants were found to be full contamination free but failed to regenerate after 3 weeks of culture. But some of them absorbed media for up to 2nd subculture and showed swelling of explants and some color changes from pale white to light/deep green. Finally, a few days after 3rd subculture, no growth of explants was observed and all treated explants eventually started to die. Among the untreated alive explants the best medium for single shoot development was MS + 4.0 mg/L BA + 0.5 mg/L KT + 15% CW and average time required for shoot development was 18-21 days. But the regeneration percentage was very low (30%). The best medium for shoot multiplication was MS + 4.0 mg/L BA + 2.0 mg/L IAA + 15% CW and only average 3-4 shoots were formed per shoot. Finally, in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 0.5 mg/L IBA.