Molecular correlates of MRS-based 31 phosphocreatine muscle resynthesis rate in healthy adults.

Dynamic phosphorus MRS (31 P-MRS) is a method used for in vivo studies of skeletal muscle energetics including measurements of phosphocreatine (PCr) resynthesis rate during recovery of submaximal exercise. However, the molecular events associated with the PCr resynthesis rate are still under debate. We assessed vastus lateralis PCr resynthesis rate from 31 P-MRS spectra collected from healthy adults as part of the CALERIE II study (caloric restriction), and assessed associations between PCr resynthesis and muscle mitochondrial signature transcripts and proteins (NAMPT, NQO1, PGC-1α, and SIRT1). Regression analysis indicated that higher concentration of nicotinamide phosphoribosyltransferase (NAMPT) protein, a mitochondrial capacity marker, was associated with faster PCr resynthesis. However, PCr resynthesis was not associated with greater physical fitness (VO2 peak) or messenger ribonucleic acid levels of mitochondrial function markers such as NQO1, PGC-1α, and SIRT1, suggesting that the impact of these molecular signatures on PCr resynthesis may be minimal in the context of an acute exercise bout. Together, these findings suggest that 31 P-MRS based PCr resynthesis may represent a valid non-invasive surrogate marker of mitochondrial NAMPT in human skeletal muscle.

The aspartate metabolism pathway is differentiable in human hepatocellular carcinoma: transcriptomics and (13) C-isotope based metabolomics.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer related mutations in HCC, integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. messenger RNA, mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry based metabolomics analysis of stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compared with non-tumor liver (p value < 0.0001). In addition, branched-chain amino acid metabolism (p value < 0.0001) and tricarboxylic acid metabolism (p value < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p value = 0.0013), succinate (p value = 0.0001), lactate (p value = 0.0114), glycerophosphoethanolamine (p value = 0.015), and inorganic phosphate (p value = 0.0001). However, (13) C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-(13) C6 ]glucose metabolism or [U-(13) C5 ,(15) N2 ]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a (13) C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via (13) C labeling, to investigate targeted therapeutics against these enzymes, and to optimize the in vivo performance of (13) C MRI techniques.

(31) P and (1) H MRS of DB-1 melanoma xenografts: lonidamine selectively decreases tumor intracellular pH and energy status and sensitizes tumors to melphalan.

In vivo (31) P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg intraperitoneally) exhibit a decrease in intracellular pH (pH(i) ) from 6.90 ± 0.05 to 6.33 ± 0.10 (p < 0.001), a slight decrease in extracellular pH (pH(e) ) from 7.00 ± 0.04 to 6.80 ± 0.07 (p > 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p < 0.001) relative to the baseline level. Both bioenergetics and pH(i) decreases were sustained for at least 3 h following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p > 0.05) at 20 min post-LND, with no significant change in pH(e) and a small transient decrease in bioenergetics (32.9 ± 10.6%, p > 0.05) at 40 min post-LND. No changes in pH(i) or adenosine triphosphate/inorganic phosphate were detected in the brain (pH(i) , bioenergetics; p > 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p > 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by (1) H MRS with a selective multiquantum pulse sequence with Hadamard localization increased approximately three-fold (p = 0.009). Treatment with LND increased the systemic melanoma response to melphalan (LPAM; 7.5 mg/kg intravenously), producing a growth delay of 19.9 ± 2.0 days (tumor doubling time, 6.15 ± 0.31 days; log(10) cell kill, 0.975 ± 0.110; cell kill, 89.4 ± 2.2%) compared with LND alone of 1.1 ± 0.1 days and LPAM alone of 4.0 ± 0.0 days. The study demonstrates that the effects of LND on tumor pH(i) and bioenergetics may sensitize melanoma to pH-dependent therapeutics, such as chemotherapy with alkylating agents or hyperthermia.

Human Hepatocellular Carcinoma Metabolism: Imaging by Hyperpolarized 13C Magnetic Resonance Spectroscopy.

PURPOSE: Most cancers exhibit high levels of aerobic glycolytic metabolism with diminished levels of mitochondrial oxidative phosphorylation even in the presence of normal or near-normal levels of oxygen ("Warburg effect"). However, technical challenges have limited the development of non-invasive in vivo imaging techniques for monitoring glycolytic metabolism of hepatocellular carcinoma (HCC) and quantitatively evaluating the impact of this effect on the growth and therapy of this disease. Thus, there is a critical need to develop non-invasive techniques for longitudinal assessment of the metabolism and treatment response of patients with unresectable HCCs. PROCEDURES: This article discusses a novel method, "Hyperpolarized 13C MRS imaging", for achieving this objective and thus improving the prognosis of HCC patients. The primary objective has been to characterize in vivo metabolic biomarkers as determinants of HCC metabolism and treatment response of unresectable HCC tumors or viable HCC cells. RESULTS: This innovative technique capitalizes on a new technology that increases the sensitivity of MRS detection of crucial metabolites in cancer cells. CONCLUSION: It is anticipated that this innovative approach will lead to improved methods, both for the diagnosis and staging of HCCs and for the facilitation of the development of enzyme targeted therapies and other therapeutic interventions.

Preclinical study of treatment response in HCT-116 cells and xenografts with (1) H-decoupled (31) P MRS.

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G(2) /M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G(2) /M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton ((1) H)-decoupled phosphorus ((31) P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83 ± 5% G(2) /M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo (1) H-decoupled (31) P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies.

In vivo MRSI of hyperpolarized [1-(13)C]pyruvate metabolism in rat hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1-(13)C]alanine in an orthotopic HCC using single-voxel hyperpolarized [1-(13)C]pyruvate MRS. In the present study, we implemented a three-dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real-time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1-(13)C]alanine and [1-(13)C]lactate after a bolus intravenous injection of [1-(13)C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1-(13)C]pyruvate conversion to [1-(13)C]alanine significantly superseded that of [1-(13)C]pyruvate conversion to [1-(13)C]lactate, potentially serving as a marker of HCC tumors.

Multimodality imaging of abnormal vascular perfusion and morphology in preclinical 9L gliosarcoma model.

BACKGROUND: This study demonstrates that a dynamic susceptibility contrast-magnetic resonance imaging (DSC-MRI) perfusion parameter may indicate vascular abnormality in a brain tumor model and reflects an effect of dexamethasone treatment. In addition, X-ray computed tomography (CT) measurements of vascular tortuosity and tissue markers of vascular morphology were performed to investigate the underpinnings of tumor response to dexamethasone. METHODOLOGY/PRINCIPAL FINDINGS: One cohort of Fisher 344 rats (N = 13), inoculated intracerebrally with 9L gliosarcoma cells, was treated with dexamethasone (i.p. 3 mg/kg/day) for five consecutive days, and another cohort (N = 11) was treated with equal volume of saline. Longitudinal DSC-MRI studies were performed at the first (baseline), third and fifth day of treatments. Relative cerebral blood volume (rCBV) was significantly reduced on the third day of dexamethasone treatment (0.65 ± .13) as compared to the fifth day during treatment (1.26 ±.19, p < 0.05). In saline treated rats, relative CBV gradually increased during treatment (0.89 ±.13, 1.00 ± .21, 1.13 ± .23) with no significant difference on the third day of treatment (p>0.05). In separate serial studies, microfocal X-ray CT of ex vivo brain specimens (N = 9) and immunohistochemistry for endothelial cell marker anti-CD31 (N = 8) were performed. Vascular morphology of ex vivo rat brains from micro-CT analysis showed hypervascular characteristics in tumors, and both vessel density (41.32 ± 2.34 branches/mm(3), p<0.001) and vessel tortuosity (p<0.05) were significantly reduced in tumors of rats treated with dexamethasone compared to saline (74.29 ± 3.51 branches/mm(3)). The vascular architecture of rat brain tissue was examined with anti-CD31 antibody, and dexamethasone treated tumor regions showed reduced vessel area (16.45 ± 1.36 µm(2)) as compared to saline treated tumor regions (30.83 ± 4.31 µm(2), p<0.001) and non-tumor regions (22.80 ± 1.11 µm(2), p<0.01). CONCLUSIONS/SIGNIFICANCE: Increased vascular density and tortuosity are culprit to abnormal perfusion, which is transiently reduced during dexamethasone treatment.