NMR Methods for Determining Lipid Turnover via Stable Isotope Resolved Metabolomics

Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex. Furthermore, although stable isotope tracers enable the determination of synthesis and degradation of complex lipids, the numbers of distinguishable molecules increase enormously, which exacerbates the problem. Although LC-MS-MS (Liquid Chromatography-Tandem Mass Spectrometry) is the standard for lipidomics, NMR can add value in global lipid analysis and isotopomer distributions of intact lipids. Here, we describe new developments in NMR analysis for assessing global lipid content and isotopic enrichment of mixtures of complex lipids for two cell lines (PC3 and UMUC3) using both 13C6 glucose and 13C5 glutamine tracers

A Systematic Protocol for the Characterization of Hsp90 Modulators

This is the author's accepted manuscript. Made available by the permission of the publisher.Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy

Objective: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival. Methods: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor. Results: HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses. Conclusions: HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC

Quantification of a new anti-cancer molecule MJC13 using a rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometry method

MJC13 is a novel molecule that has potential use for the treatment of hormone refractory prostate cancer (HRPC). The purpose of this work was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of MJC13. Itraconazole was used as the internal standard (IS). Acetonitrile was used to extract MJC13 from rat plasma and urine samples. A LC system equipped with a Waters XTerra MS C18 column (125Å, 3.5 µm, 4.6×150 mm) was used for chromatographic separation with acetonitrile-water as mobile phase. The API 3200 QTRAP triple quadrupole mass spectrometer was used for chromatographic analysis by multiple reaction monitoring (MRM) at positive mode with the transitions m/z 272→m/z 162 for MJC13, and m/z 705→m/z 392 for IS. The retention times for MJC13 and IS were 4.98 min and 4.42 min, respectively. The standard curves of MJC13 in solution, rat plasma, and rat urine were linear in the concentration range of 1 – 1000, 1 – 1000 and 1 – 200 ng/mL, respectively. The intra- and inter-day accuracy (relative error) ranged from 1.99 – 4.20% and 1.83 – 4.39%, respectively. The intra- and inter-day precision (coefficient of variation) ranged from 2.27 – 3.88% and 2.80 – 4.79%, respectively. The extraction recovery rates of rat plasma and urine samples were 95.3% and 96.2%, respectively. No measurable matrix effect interfered with MJC13 identification and quantification in rat plasma and urine. In summary, a rapid, specific, sensitive, and reproducible LC-MS/MS method was developed and validated to quantify MJC13 in solution, rat plasma, and rat urine