An in-vivo pilot study into the effects of FDG-mNP in cancer in mice.

PURPOSE: Previously, fluorodeoxy glucose conjugated magnetite nanoparticles (FDG-mNPs) injected into cancer cells in conjunction with the application of magnetic hyperthermia have shown promise in new FDG-mNPs applications. The aim of this study was to determine potential toxic or unwanted effects involving both tumour cells and normal tissue in other organs when FDG-mNPs are administered intravenously or intratumourally in mice. MATERIALS AND METHODS: FDG-mNPs were synthesized. A group of six prostate-tumour bearing mice were injected with 23.42 mg/ml FDG-mNPs (intravenous injection, n = 3; intratumoural injection into the prostate tumour, n = 3). Mice were euthanized and histological sampling of tissue was conducted for the prostate tumour, as well as for lungs, lymph nodes, liver, kidneys, spleen, and brain, at 1 hour (n = 2) and 7 days (n = 4) post-injection. A second group of two normal (non-cancerous) mice received the same injection intravenously into the tail vein and were euthanised at 3 and 6 months post-injection, respectively, to investigate if FDG-mNPs remained in organs at those time points. RESULTS: In prostate-tumour bearing mice, FDG-mNPs concentrated in the prostate tumour, while relatively small amounts were found in the organs of other tissues, particularly the spleen and the liver; FDG-mNP concentrations decreased over time in all tissues. In normal mice, no detrimental effects were found in either mouse at 3 or 6 months. CONCLUSION: Intravenous or intratumoural FDG-mNPs can be safely administered for effective cancer cell destruction. Further research on the clinical utility of FDG-mNPs will be conducted by applying hyperthermia in conjunction with FDG-mNPs in mice.

Roles of electro-acupuncture in glucose metabolism as assessed by 18F-FDG/PET imaging and AMPKα phosphorylation in rats with ischemic stroke.

Targeted energy metabolism balance contributes to neural survival during ischemic stroke. Herein, we tested the hypothesis that electro­acupuncture (EA) can enhance cerebral glucose metabolism assessed by 18F­fluorodeoxyglucose/positron emission tomography (18F­FDG/PET) imaging to prevent propagation of tissue damage and improve neurological outcome in rats subjected to ischemia and reperfusion injury. Rats underwent middle cerebral artery occlusion (MCAO) and received EA treatment at the LI11 and ST36 acupoints or non­acupoint treatment once a day for 7 days. After EA treatment, a significant reduction in the infarct volume was determined by T2­weighted imaging, accompanied by the functional recovery in CatWalk and Rota-rod performance. Moreover, EA promoted higher glucose metabolism in the caudate putamen (CPu), motor cortex (MCTX), somatosensory cortex (SCTX) regions as assessed by animal 18F­FDG/PET imaging, suggesting that three­brain regional neural activity was enhanced by EA. In addition, the AMP­activated protein kinase α (AMPKα) in the CPu, MCTX and SCTX regions was phosphorylated at threonine 172 (Thr172) after ischemic injury; however, phosphorylation of AMPK was further increased by EA. These results indicate that EA could promote AMPKα phosphorylation of the CPu, MCTX and SCTX regions to enhance neural activity and motor functional recovery after ischemic stroke.

Predictive value of F-18 FDG PET/CT quantization parameters for progression-free survival in patients with diffuse large B-cell lymphoma.

OBJECTIVES: To evaluate the predictive significance of F-18 FDG PET/CT quantization parameters for progression-free survival (PFS) in patients with diffuse large B cell lymphoma (DLBCL) before chemotherapy. METHODS: We conducted a retrospective study involving 60 patients with DLBCL between January 2010 and August 2014 who had undergone F-18 FDG PET/CT scan prior to treatment. Maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), total lesion glycolysis (TLG), and number of enlarged lymph nodes (>2 cm) were measured. The primary outcome measure was PFS. Spearman rank correlation analysis, univariate and multivariate Cox regression models, receiver operating characteristic (ROC) analysis, and Kaplan-Meir survival curves were used. RESULTS: Spearman analysis determined that the MTV and TLG values were positively related to Ann Arbor stage, National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) score, and lactate dehydrogenase (LDH) level. The number of enlarged lymph nodes was positively related only to LDH level. The SUVmax value and clinical characteristics were not related. Univariate Cox regression determined that the MTV and TLG values, number of enlarged lymph nodes, and NCCN-IPI score were predictive factors. Multivariate Cox regression determined that the MTV and TLG values and number of enlarged lymph nodes predicted PFS independently of the NCCN-IPI score. The SUVmax value was not predictive of PFS. According to the cut-off determined from ROC analysis, lower MTV and TLG values were highly predictive of favorable PFS. CONCLUSIONS: In contrast to SUVmax, the MTV and TLG may be significant prognostic markers for PFS in DLBCL.

Episodic autobiographical memory in amnestic mild cognitive impairment: what are the neural correlates?

Autobiographical memory in amnestic Mild Cognitive Impairment (aMCI) is characterized by impaired retrieval of episodic memories, but relatively preserved personal semantic knowledge. This study aimed to identify (via FDG-PET) the neural substrates of impaired episodic specificity of autobiographical memories in 35 aMCI patients compared with 24 healthy elderly controls. Significant correlations between regional cerebral activity and the proportion of episodic details in autobiographical memories from two life periods were found in specific regions of an autobiographical brain network. In aMCI patients, more than in controls, specifically episodic memories from early adulthood were associated with metabolic activity in the cuneus and in parietal regions. We hypothesized that variable retrieval of episodic autobiographical memories in our aMCI patients would be related to their variable capacity to reactivate specific sensory-perceptual and contextual details of early adulthood events linked to reduced (occipito-parietal) visual imagery and less efficient (parietal) attentional processes. For recent memories (last year), a correlation emerged between the proportion of episodic details and activity in lateral temporal regions and the temporo-parietal junction. Accordingly, variable episodic memory for recent events may be related to the efficiency of controlled search through general events likely to provide cues for the retrieval of episodic details and to the ability to establish a self perspective favouring recollection.

Effects of vitamin E supplementation on plasma membrane permeabilization and fluidization induced by chlorpromazine in the rat brain.

Neurotransmitter receptors play a key role in most research on antipsychotic drugs, but little is known about the effects of these drugs on the plasma membrane in the central nervous system. Therefore, we investigated whether chlorpromazine (CPZ), a typical phenothiazine antipsychotic drug, affects the plasma membrane integrity in the rat brain, and if so, whether these membrane alterations can be prevented by dietary supplementation with vitamin E, which has been shown to be an antioxidant and also a membrane-stabilizer. Leakage of [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG)-6-phosphate from rat striatal slices and decrease in 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy were used as indexes for plasma membrane permeabilization and fluidization, respectively. CPZ induced leakage of [(18)F]FDG-6-phosphate from striatal slices, and the leakage was delayed in the vitamin E-supplemented group compared to that in the normal diet group. The decrease in plasma membrane anisotropy induced by CPZ was significantly attenuated by vitamin E supplementation. Chronic treatment with alpha-phenyl-N-tert-butyl nitrone, a free radical scavenger, had no effect on CPZ-induced plasma membrane permeabilization, and the treatment with CPZ did not induce lipid peroxidation. CPZ can reduce plasma membrane integrity in the brain, and this reduction can be prevented by vitamin E via its membrane-stabilizing properties, not via its antioxidant activity.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

Chemotherapy-induced normalization of FDG uptake by colorectal liver metastases does not usually indicate complete pathologic response.

Dramatic responses are being observed in colorectal cancer liver metastases treated with newer chemotherapeutic regimens. These have been associated with normalization of [(18)F]fluoro-2-deoxy-D-glucose (FDG) uptake (complete metabolic response) on follow-up Positron Emission Tomography with [(18)F]fluoro-2-deoxy-D-glucose (FDG-PET) scans in some patients. It is unclear how often complete metabolic response is indicative of complete tumor destruction. We analyzed a subset of patients who had neoadjuvant chemotherapy for hepatic metastases from colorectal adenocarcinoma. Inclusion criteria were: (1) FDG-avid hepatic lesions before initiation of chemotherapy; (2) complete metabolic response of the same lesions after chemotherapy; and (3) histopathologic examination of hepatic lesions. Complete pathologic response was defined as no histologically identifiable viable tumor. Fourteen patients fit the inclusion criteria. All had synchronous, hepatic-only colorectal metastases. On microscopic examination, complete pathologic response to the neoadjuvant regimen was found in only 5 of 34 lesions (15%) and in only 3 of the 14 patients (21%). Seven lesions had complete metabolic response and disappeared on computed tomography (CT); of these, six still contained viable tumor. We conclude that complete metabolic response on FDG-PET after neoadjuvant chemotherapy is an unreliable indicator of complete pathologic response. Therefore, currently, curative resection of liver metastases in these patients should not be deferred on the basis of FDG-PET findings.

Clotrimazole decreases glycolysis and the viability of lung carcinoma and colon adenocarcinoma cells.

Glycolysis is known to be the primary energy source in most cancer cells. We investigated here the effect of clotrimazole (1-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. We found that clotrimazole induced a significant, dose- and time-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP, and cell viability. These findings suggest that clotrimazole causes a reduction in glycolysis and ATP levels, which eventually leads to cell destruction after 3 h of treatment. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, this substance is most promising agent in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cancer cell growth.

Excretion of a phosphorus-containing carbohydrate by Streptomyces sp. A50.

A new phosphorus-containing compound (1) was detected by (31)P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(alpha)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(alpha)-2-(N-acetyl)glucosamine, exhibited a pK(a) value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2D-homonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy.

Pentose phosphate pathway alterations in multi-drug resistant leukemic T-cells: 31P NMR and enzymatic studies.

31P NMR studies were carried out on the parental drug-sensitive human T-lymphoblastoid cell line CCRI-CEM (CEM) and its multi-drug-resistant (MDR) CEM-VBL100 variants, to assess the role of the pentose phosphate (PP) in MDR expression. CEM and CEM-VBL100 were incubated in the presence of 2-deoxyglucose, as recently proposed by our group (Clin. Chim. Acta 208: 39, 1992). Accumulation of 2-deoxyglucose 6-phosphate was much lower in the drug-resistant than in sensitive cells, indicating PP shunt activation in the MDR variants. This result was confirmed by enzymatic analyses, which demonstrated that, with respect to the parental line, the MDR variant was characterized by a) unaltered hexokinase activity; b) higher glucose 6-phosphate dehydrogenase activity; c) increased levels of reduced glutathione and marked increase of glutathione peroxidase activity after cell exposure to an oxidizing agent (tert-butylhydroperoxide). These results support the view that cell detoxification mechanisms mediated by the pentose phosphate pathway may contribute to the expression of MDR in tumours.

Reaction of the isosteric methylenephosphonate analog of alpha-D-glucose 1-phosphate with phosphoglucomutase. Induced-fit specificity revisited.

The phospho form of phosphoglucomutase reacts with the isosteric methylenephosphonate analog of alpha-D-glucose 1-phosphate to produce the corresponding analog of alpha-D-glucose 1,6-bisphosphate plus the dephosphoenzyme. In a coupled reaction, kcat/Km = 1.7 x 10(3) M-1 s-1, which is about 2 x 10(-5) times that for the corresponding reaction with alpha-D-glucose 1-phosphate. The decrease in kcat/Km is divided more or less evenly between less efficient PO3- transfer and decreased binding, although smaller phosphates and phosphonates bind approximately equally. There is a much smaller difference in the binding of glucose 1-methylenephosphonate 6-phosphate and glucose 1,6-bisphosphate to the dephosphoenzyme: the binding ratio is < 1:35 when the glucose ring is oriented similarly. Preferred binding patterns for a number of substrates/inhibitors, studied by 31P NMR and UV-difference spectroscopy, suggest that in the ground state the phosphonate group is tolerated to a much greater extent at the catalytic subsite than at the phosphate-binding subsite, where binding specificity appears to be directed toward a tetrahedral-PO3(2-) group attached to a bridging atom that can act as a hydrogen-bond acceptor. Binding specificity at the catalytic subsite apparently is directed toward a different array, possibly (-O...PO3...O-)2-. Some of these results are considered in terms of a modified version of the "induced fit" concept of enzymic specificity, which is reexamined in view of implied thermodynamic restrictions. The internal rearrangement whereby the positions of the anionic groups of the phosphate/phosphonate are exchanged is compared with the analogous rearrangements involving glucose 1,6-bisphosphate and 1,4-butanediol bisphosphate. The supplementary material describes a three-step synthesis of 1-deoxy-alpha-D-glucose 1-methylenephosphonate together with a procedure for phosphorylating the phosphonate to produce an analog of alpha-D-glucose 1,6-bisphosphate and also describes a facile procedure for the qualitative conversion of organic phosphonates to inorganic phosphate.

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of bovine heart hexokinase by 2-deoxy-D-glucose-6-phosphate: characterization by 31P NMR and metabolic implications.

The glucose analog, 2-deoxy-D-glucose (2DG), has been used widely for studying the initial steps in the metabolism of glucose by radio-isotope tracer methods and by 31P NMR. In the rat heart perfused with acetate/2DG (both 5 mM) plus insulin, trapping of phosphorus by 2-deoxy-D-glucose-6-phosphate (2DG6P) results in a steady state exhibiting high 2DG6P (55 mM) and low ATP concentrations but near-normal function, as observed in an earlier 31P NMR study. In order to understand how the 2DG6P concentration is stabilized, we studied the inhibition of a mammalian hexokinase by 2DG6P in vitro by a 31P NMR technique. Inhibition, previously unobserved, was found. It is similar to inhibition by G6P in that it is competitive with ATP and not competitive with 2DG, but the inhibition constant (1.4 mM) is much larger. The experimental protocol includes provisions for enzymatic destruction of stray inhibitors such as G6P. The results show that the high 2DG6P and low ATP concentrations found in the steady state of the perfused heart should strongly reduce the rate of phosphorylation of sugars by hexokinase.

Brain slice glucose utilization.

The metabolism of 2-deoxyglucose has been studied in 540 micron and 1,000 micron hypothalamic brain slices. Slice 2-deoxyglucose (2DG) and 2-deoxyglucose-6-phosphate (2DG6P) levels were measured after tissue homogenization and perchloric acid extraction. By analyzing the uptake and washout kinetics with nonlinear least-squares methods, we have determined the rate constants for three-, four-, or five-parameter kinetic models and obtained a value for the in vitro lumped constant (LC). The kinetic analysis reveals a small, slowly decaying, 2DG component that is not predicted by any of the models. If this component is treated as a separate, parallel compartment, then the four- and five-parameter models are essentially equivalent. To compare our data to prior in vivo data, we combined 2DG and 2DG6P to produce Ci*, the total slice radioactivity, and analyzed the first 45 min of uptake. These data were fit best by a three-parameter model and the slowly decaying pool was not identified. Calculation of glucose utilization from total tissue radioactivity, measured by whole slice homogenization and by image analysis of autoradiograms, showed excellent correlation between the two methods. Image analysis of radioactivity in the suprachiasmatic nucleus, which is present in these slices, revealed a spontaneous diurnal variation in in vitro glucose utilization in close quantitative agreement with prior in vivo measurements. The kinetic analysis of the 1,000 micron slice was qualitatively similar to that of the 540 micron slice but revealed an increase in the LC and a large decrease in k1 as well as the expected large increase in the hexokinase rate constant, k3. Overall, in vitro glucose utilization increased by about 60%. These results are consistent with our prior studies of the 1,000 micron slice and support our interpretation that the 1,000 micron slice is an excellent in vitro model for brain ischemia without infarction.

Phosphate/hexose 6-phosphate antiport in Streptococcus lactis.

After growth in appropriate media, resting cells of Streptococcus lactis 7962 showed a rapid exchange between external and internal pools of inorganic phosphate. This exchange was not found in other strains of S. lactis (ML3, 133, or K1) or in Streptococcus faecalis. Phosphate exchange in S. lactis 7962 did not require other anions or cations in the assay medium, nor was phosphate influx affected by the membrane potential and pH gradient formed during glycolysis. Thus, the exchange reaction was independent of known ionic drivers (H+, Na+, OH-, etc.). Experiments testing inhibitions of phosphate entry suggested that alternative substrates for exchange included arsenate, as well as the 6-phosphates of glucose, 2-deoxyglucose, fructose, mannose, or glucosamine, and direct studies with 2-deoxyglucose 6-phosphate verified that resting cells could accumulate this sugar phosphate to levels expected for exchange with internal phosphate. Two other observations supported the idea of an exchange between phosphate and sugar phosphate. First, early addition of the heterologous substrate blocked entry of the test compound, whereas later addition caused efflux of preaccumulated material. Second, expression of phosphate exchange and 2-deoxyglucose 6-phosphate transport varied in parallel. Both activities were found at high levels after growth in medium supplemented with rhamnose or arabinose, at intermediate levels with addition of galactose, and at low levels after growth with glucose, fructose, or mannose. We conclude that these findings describe a novel anion antiporter that mediates the exchange of phosphate (arsenate) and sugar 6-phosphates.