Magnetic Resonance Spectroscopy to Study Glycolytic Metabolism During Autophagy.

Cancer cells undergoing starvation- and treatment-induced autophagy were found to exhibit reduced intracellular lactate, reduced rates of steady-state lactate excretion and reduced real-time pyruvate-lactate exchange rates, indicating that glycolytic metabolism was altered in autophagic cells. In this chapter, we describe the technical details of the use of 1H-magnetic resonance spectroscopy (MRS) to measure endogenous cellular concentrations of lactate and glucose in autophagic cells and tissues, how to measure the rate of steady-state lactate excretion and glucose uptake by 1H-MRS in autophagic cells, and details of the real-time measurement of [1-13C] pyruvate to lactate exchange in autophagic cells by 13C-MRS-DNP (dynamic nuclear polarization).

Angiosuppressive properties of marine-derived compounds-a mini review.

Angiogenesis, formation of new blood vessels from preexisting one, is a critical step of tumorgenesis of solid tumors. Therefore, antiangiogenic therapy is one of the promising approaches to control tumor growth. In the past 20 years, a lot of compounds have been tested for their antiangiogenic properties. Bevacizumab, Avastin®, the first antiangiogenic drug approved by the US FDA, has been widely used in clinic for treating cancer. Indeed, many synthetic compounds are highly toxic and exert side effects even though they are effective in inhibiting neovessel formation and cancer cell growth. Using natural compounds or their derivatives is one of the ways to solve these problems. Sinomenine and ginsenosides are common antiangiogenic and anticancer compounds that are extracted from herbal medicines. Recent findings suggested that marine algae-derived natural pigments also possess similar activities. It has been reported that fucoxanthin from Undaria pinnatifida, Siphonaxanthin from Codium fragile, can inhibit angiogenesis and cancer growth effectively. In conclusion, natural compounds derived from marine algae could provide a novel and safe source for new drug development in anticancer and antiangiogenic properties in the future.

Dichloroacetate induces autophagy in colorectal cancer cells and tumours.

BACKGROUND: Dichloroacetate (DCA) has been found to have antitumour properties. METHODS: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, (1)H and hyperpolarised (13)C-magnetic resonance spectroscopy. RESULTS: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-(13)C] pyruvate-to-lactate exchange rate (k(PL)) were decreased in DCA-treated cells, along with increased NAD(+)/NADH ratios and NAD(+). Steady-state lactate excretion and k(PL) returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD(+) and NADH. Reduced k(PL) with DCA treatment was found in HT29 tumour xenografts in vivo. CONCLUSIONS: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k(PL) and increased NAD(+)/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

The HIF-pathway inhibitor NSC-134754 induces metabolic changes and anti-tumour activity while maintaining vascular function.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) mediates the transcriptional response to hypoxic stress, promoting tumour progression and survival. This study investigated the acute effects of the small-molecule HIF-pathway inhibitor NSC-134754. METHODS: Human PC-3LN5 prostate cancer cells were treated with NSC-134754 for 24 h in hypoxia. Orthotopic prostate tumour-bearing mice were treated with a single dose of NSC-134754 for 6, 24 or 48 h. Treatment response was measured using magnetic resonance spectroscopy and imaging. Ex-vivo histological validation of imaging findings was also sought. RESULTS: In vitro, NSC-134754 significantly reduced lactate production and glucose uptake (P<0.05), while significantly increasing intracellular glucose (P<0.01) and glutamine uptake/metabolism (P<0.05). Increased glutamine metabolism was independent of c-Myc, a factor also downregulated by NSC-134754. In vivo, a significantly higher tumour apparent diffusion coefficient was determined 24 h post-treatment (P<0.05), with significantly higher tumour necrosis after 48 h (P<0.05). NSC-134754-treated tumours revealed lower expression of HIF-1α and glucose transporter-1, at 6 and 24 h respectively, while a transient increase in tumour hypoxia was observed after 24 h. Vessel perfusion/flow and vascular endothelial growth factor levels were unchanged with treatment. CONCLUSION: NSC-134754 induces metabolic alterations in vitro and early anti-tumour activity in vivo, independent of changes in vascular function. Our data support the further evaluation of NSC-134754 as an anti-cancer agent.

Metabolic assessment of the action of targeted cancer therapeutics using magnetic resonance spectroscopy.

Developing rational targeted cancer drugs requires the implementation of pharmacodynamic (PD), preferably non-invasive, biomarkers to aid response assessment and patient follow-up. Magnetic resonance spectroscopy (MRS) allows the non-invasive study of tumour metabolism. We describe the MRS-detectable PD biomarkers resulting from the action of targeted therapeutics, and discuss their biological significance and future translation into clinical use.

Using metabolomics to monitor anticancer drugs.

The metabolome of a cancer cell is likely to show changes after responding to an anticancer drug. These changes could be used to decide whether to continue treatment or, in the context of a drug trial, to indicate whether the drug is working and perhaps its mechanism of action. (Nuclear) magnetic resonance spectroscopy (NMR/MRS) methods can offer important insights into novel anticancer agents in order to accelerate the drug development process including time-course studies on the effect of a drug on its site of action (termed pharmacodynamics), in this case the cancer. In addition, some classes of anticancer agents currently under development (e.g. antiangiogenics) are designed to be used in combination with other drugs and will not cause tumour shrinkage when used as single agents in Phase 1 clinical trials. Thus NMR/MRS may have a special role in monitoring the pharmacodynamic actions of such drugs in early-phase clinical trials. This review focuses on the use of ex vivo NMR and in vivo MRS methods for monitoring the effect of some novel anticancer drugs on the cancer metabolome. Ex vivo NMR methods are complementary to in vivo measurements, as they can provide additional information and help in the interpretation of the in vivo data.

A prognostic scoring system for locoregional control in nasopharyngeal carcinoma following conformal radiotherapy.

PURPOSE: This study established a prognostic scoring system for nasopharyngeal carcinoma (NPC), which estimates the probability of locoregional (LR) control following definitive conformal radiotherapy. METHODS AND MATERIALS: Patients with nondisseminated NPC at initial presentation (n = 630) were enrolled in this study. All patients had magnetic resonance imaging of the head and neck and were treated with conformal radiotherapy. Among them, 93% had concurrent chemotherapy, and 76% had postradiation chemotherapy. The extent of the primary tumor, age at diagnosis, primary tumor size, tumor and nodal classification, histology, and serum lactate dehydrogenase (LDH) level before treatment were included in the analysis for building a prognostic scoring system. The end point for this study was LR control. RESULTS: The prognostic score was defined as the number of adverse prognostic factors present at diagnosis. Four factors had similarly independent prognostic effects (hazard ratio, 2.0-2.6): age >40 years, histologic WHO type I-II, serum LDH level > or =410 U/L, and involvement of two or more sites of the following anatomic structures, i.e., sphenoid floor, clivus marrow, clivus cortex, prevertebral muscles, and petrous bone. The score predicted the 5-year probability of LR control as follows: 0 (15% of the patients), 100%; 1 (42% of the patients), 93%; 2 (29% of the patients), 83%; 3 or higher (13% of the patients), 71%. CONCLUSION: This scoring system is useful in the decision-making for individual patients and the design of clinical trials to improve LR control for advanced-stage NPC.

Evaluation of 31P high-resolution magic angle spinning of intact tissue samples.

The first detailed evaluation is presented of high-resolution (31)P MRS using magic angle spinning (MAS) of intact tissue samples and comparison with the conventional method of studying tissue extracts. The main motivation is that MAS leaves the sample intact at the end of the study for histopathological evaluation. While MAS of tissue samples has previously been demonstrated for (1)H MRS, (31)P MRS is better suited to study of the phospholipid metabolites of importance in cancer. Samples of rhabdomyosarcoma and RIF-1 experimental tumours were maintained at 4 degrees C, spun at 3 kHz and measured in 28-min acquisitions at 11.7 and 14 T. Metabolite stability was evaluated using four sequential 28-min acquisitions. High-resolution MRS was performed on extracts of the same tissue samples. (31)P HR-MAS yielded well-resolved high-resolution spectra, showing peaks from phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate, glycerophosphoethanolamine and glycerophosphocholine, with linewidths in the range 3-20 Hz. In tumour samples there was no significant change in peak areas over a 2-h period, while peaks sensitive to pH (inorganic phosphate, PE and PC) showed a small change in chemical shift, corresponding to a change of 0.13 +/- 0.06 pH units. Tissue metabolite concentrations showed good agreement with concentrations measured from extracts of the same pieces of tissue. For calculation of metabolite concentrations, the measurement of a reference compound in a separate measurement is more robust than using the signal from a reference compound in the rotor with the sample. Compared with performing tissue extracts, use of MAS of intact tissue samples requires less preparation, is quicker and permits the same sample to be used for subsequent histopathology. The methodology has particular application in studying phospholipid metabolism in cancer and in monitoring tumour response to treatment, where concentrations of phospholipid-related metabolites are found to alter following response to a wide range of anti-cancer therapies.

Muscle metabolites, detected in urine by proton spectroscopy, correlate with disease damage in juvenile idiopathic inflammatory myopathies.

OBJECTIVE: To assess for novel markers of muscle damage using urinary muscle metabolites by 1H magnetic resonance spectroscopy in patients with juvenile idiopathic inflammatory myopathy (IIM). METHODS: Creatine (Cr), choline (Cho), betaine (Bet), glycine (Gly), trimethylamine oxide (TMAO), and several other metabolites were measured in first morning void urine samples from 45 patients with juvenile IIM and from 35 healthy age-matched controls, and correlated with measures of myositis disease activity and damage. Urinary metabolite to age-adjusted creatinine (Cn) ratios were examined. RESULTS: Age-adjusted initial Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios were higher in patients with juvenile IIM than controls (P < 0.01). Cr:Cn ratios showed significant correlations with physician-assessed global disease damage (Spearman rs = 0.37; P = 0.01), Steinbrocker functional class (rs = 0.35; P = 0.02), serum Cr (rs = 0.72; P = 0.001), and lactate dehydrogenase (rs = 0.34; P = 0.03) levels. Cho:Cn (rs = 0.3; P = 0.05), Gly:Cn (rs = 0.33; P = 0.03), and TMAO:Cn (rs = 0.36; P = 0.02) ratios showed a significant correlation with serum aldolase levels. Cho:Cn ratios also showed a significant correlation with aspartate aminotransferase levels (rs = 0.35; P = 0.02). A linear regression model was used to evaluate the factors influencing urinary Cr:Cn ratios in the 43 patients with data sets available at the initial visit. The regression model explained 73% of the variation in Cr:Cn ratios. The most significant factor was the physician-assessed global disease damage (R2 = 0.50, P = 0.015). CONCLUSION: Urinary Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios are elevated in juvenile IIM and Cr:Cn correlates strongly with global disease damage. The Cr:Cn ratio may have potential utility as a marker of myositis disease damage.

Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

N-acetyl aspartate estimation: a potential method for determining neuronal loss in the transmissible spongiform encephalopathies.

Neurodegenerative pathology is typical of the transmissible spongiform encephalopathies (TSEs), and is thought to underlie clinical disease. Some morphometric studies have shown early focal neurone loss, but the full extent of TSE induced neuronal loss in the central nervous system is not known, and can only be accurately estimated using intensive morphometric techniques. We have used a murine scrapie model in which we determined the levels of N-acetyl aspartate (NAA), a putative neuronal marker, by both high-performance liquid chromatography and high resolution, proton magnetic resonance spectroscopy in samples taken sequentially from the hippocampus. This scrapie model develops severe neuronal loss in the hippocampus, and the NAA levels showed a significant positive correlation with our previous morphometric estimates of neurone number. NAA measurement may therefore provide a practical alternative to intensive morphometric techniques in the investigation of neurodegeneration in the TSEs.

Urinary levels of creatine and other metabolites in the assessment of polymyositis and dermatomyositis.

BACKGROUND: A simple and reliable method is needed to assess disease activity and monitor the efficacy of therapy in polymyositis (PM) and dermatomyositis (DM). This study used in vitro proton ((1)H) magnetic resonance spectroscopy (MRS) to explore whether excretion of urinary metabolites can be used as a reliable marker of disease in PM and DM patients. METHODS: Urine samples were obtained from PM/DM patients (n=34), healthy controls (50) and subjects with known muscle-wasting conditions including adult-onset muscular dystrophy (8), stroke patients (10), rheumatoid arthritis (RA) patients on steroids (13) and not on steroids (16) and patients with alcoholic myopathy (12). Levels of urinary metabolites were then correlated with creatine kinase (CK) activities and quadriceps muscle strength. RESULTS: Creatine was detected in the urine in 26 of 35 patients with PM/DM, four of 60 cases with other medical disorders (including one with adult-onset dystrophy, one with a stroke and two with RA who were not on steroids) and 10 of 50 healthy controls. The urinary creatine/creatinine ratio exceeded 0.4 in 20 patients with PM/DM but no patients with other medical disorders and no healthy controls. These differences were highly significant (P<0.001) by Kruskal-Wallis test (comparing all groups) and by Mann-Whitney U-tests (comparing individual groups with PM/DM cases). Citrate, glycine, choline-containing compounds and taurine levels were significantly increased in PM/DM when compared with controls. There were positive correlations between CK activities and choline-containing compounds (r=0.78, P=0.0006) and also between CK activities and betaine (r=0.57, P=0.026). CONCLUSIONS: This study shows significant differences in the urinary levels of creatine, choline-containing metabolites, betaine and citrate in PM/DM subjects compared with controls, although further work is required to elucidate the underlying metabolic processes.

Proposed preliminary core set measures for disease outcome assessment in adult and juvenile idiopathic inflammatory myopathies.

In order to develop a preliminary core set of disease outcome measures for use in clinical trials of idiopathic inflammatory myopathies (IIM), we evaluated those measures used in previous trials, assessed the validation of published instruments and discussed these at an international consensus conference. The initial proposals were further refined by a multidisciplinary group of adult and paediatric specialists experienced in IIM using the Delphi method. The proposed preliminary core set of disease activity measures consists of five domains: physician and patient/parent global assessments of disease activity; muscle strength; physical function; serum activity of muscle enzymes; and an assessment tool to capture extra-skeletal muscle disease activity. The group recommended further development of a core set of disease damage measures for assessment of persistent changes in anatomy, pathology and function of at least 6 months' duration. The group recommended that patient-reported outcomes should include generic health-related quality of life assessments using the Medical Outcomes Study 36-item Short Form (SF-36) health survey in adult IIM patients and a validated quality of life instrument for paediatric patients. We propose the core set of outcome measures as a minimum group of assessments to include in all IIM therapeutic studies. The use of this core set should assist in standardizing outcome measurement and in optimizing therapeutic trials in myositis.

On-line identification of 3,4-methylenedioxymethamphetamine in human urine by non-aqueous capillary electrophoresis-fluorescence spectroscopy at 77 K.

The analytical profiles for 3,4-methylenedioxymethamphetamine (3,4-MDMA) and related amphetamines in urine samples are described for non-aqueous capillary electrophoresis-fluorescence spectroscopy. 3,4-MDMA was detected and identified on-line, using a cryogenic molecular fluorescence technique at 77 K. Under optimized conditions, baseline separation of the selected compounds was achieved in less than 12 min. Precision was evaluated by measuring the repeatability and intermediate precision of the migration times and corrected peak areas. The non-aqueous CE separation conditions and the spectral characteristics of 3,4-MDMA with respect to solvent and temperature effects are also discussed.

A comparative study of outcome in myositis and other musculoskeletal disorders assessed using the Nottingham health profile.

OBJECTIVE: This study evaluated the comparative impact of myositis and other musculoskeletal disorders on general health using the Nottingham health profile (NHP) as a generic measure of health status. METHODS: A prospective observational study of 113 females with myositis, 142 females with rheumatoid arthritis, 45 females with spinal osteoporosis and 96 females with knee osteoarthritis. RESULTS: All mean NHP section scores were higher in myositis and other musculoskeletal disorders compared to population mean values. Section scores for energy and social isolation were high in myositis compared to all other disorders. Scores for physical disability in myositis were similar to RA. Pain scores were higher in RA and OA compared to myositis. Backwards linear regression models explained 26-42% of the variation in energy and social isolation scores. Emotion and physical section scores were the major determinants and the pattern was similar in all disorders. Disease duration and age had little effect. CONCLUSIONS: Myositis is not simply a disease with physical problems but has wide ranging effects on social and emotional well being. Until disease-specific instruments are available, a generic measure like the NHP can be used to assess problems other than muscle pain and loss of strength.

Development of a capillary electrophoresis-77 K luminescence detection system for online spectral identification.

We have demonstrated that capillary electrophoresis (CE) can be easily interfaced with 77 K luminescence spectroscopy (LS) for separation and online spectral identification of structurally similar analytes. This novel CE-LS apparatus consists of a regular CE system, instrumentation for LS and a specially designed capillary Dewar. When the separating molecules traverse into the cryostat detection window, liquid nitrogen is added, freezing the separating analyte zones within the capillary. At low temperature, detection limits are improved via signal averaging and the inherent increase in quantum yield at 77 K. We present the first application of the CE-LS system to structural isomers (2,3- and 3,4-methylenedioxymethamphetamine) and stereoisomers (trans- and cis-resveratrol). With this approach, the CE-LS interfacing provides a sensitive, accurate, rapid, simple and economic methodology for analytical chemistry.