Metabolic characteristics of imatinib resistance in chronic myeloid leukaemia cells.

BACKGROUND AND PURPOSE: Early detection of resistance development is crucial for imatinib-based treatment in chronic myeloid leukaemia (CML) patients. We aimed to distinguish metabolic markers of cell resistance to imatinib. EXPERIMENTAL APPROACH: Two human imatinib-sensitive CML cell lines: LAMA84-s and K562-s, and their resistant counterparts: LAMA84-r and K562-r (both resistant to 1 microM imatinib), and K562-R (5 microM) were analysed by nuclear magnetic resonance spectroscopy to assess global metabolic profiling, including energy state, glucose and phospholipid metabolism. KEY RESULTS: We found, by Western blotting and flow cytometry, that the levels of Bcr-Abl tyrosine kinase and multi-drug resistance p-glycoprotein were inconsistent among resistant clones. On the other hand, phospholipid metabolism and lactate production were highly predictive for cell response to imatinib. As previously reported, sensitive cells showed significantly decreased glycolytic activity (lactate) and phospholipid synthesis (phosphocholine) as well as increased phospholipid catabolism (glycerophosphocholine) after 24 h of 1 microM imatinib treatment, which correlated with inhibition of cell proliferation and induction of apoptosis. In contrast to their sensitive counterparts, the K562-r, K562-R and LAMA84-r maintained increased phospholipid synthesis and glycolytic lactate production in the presence of 1 microM (K562-r and LAMA84-r) and 5 microM (K562-R) imatinib. CONCLUSIONS AND IMPLICATIONS: Specific metabolic markers for early detection of imatinib resistance, including increased glycolytic activity and phospholipid turnover, can be identified in resistant clones. Once validated in human isolated leukocytes, they may be used to monitor the responsiveness of CML patients to treatment.

Abnormalities in glucose uptake and metabolism in imatinib-resistant human BCR-ABL-positive cells.

The development of imatinib resistance has become a significant therapeutic problem in which the etiology seems to be multifactorial and poorly understood. As of today, clinical criteria to predict the development of imatinib resistance in chronic myelogenous leukemia (CML), other than rebound of the myeloproliferation, are under development. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib because imatinib-resistant gastrointestinal stromal KIT-positive tumors reveal highly elevated glucose uptake in radiologic images. We used nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry to assess (13)C glucose uptake and metabolism (glycolysis, TCA cycle, and nucleic acid ribose synthesis) during imatinib treatment in CML cell lines with different sensitivities to imatinib. Our results show that sensitive K562-s and LAMA84-s BCR-ABL-positive cells have decreased glucose uptake, decreased lactate production, and an improved oxidative TCA cycle following imatinib treatment. The resistant K562-r and LAMA84-r cells maintained a highly glycolytic metabolic phenotype with elevated glucose uptake and lactate production. In addition, oxidative synthesis of RNA ribose from (13)C-glucose via glucose-6-phosphate dehydrogenase was decreased, and RNA synthesis via the nonoxidative transketolase pathway was increased in imatinib-resistant cells. CML cells which exhibited a (oxidative/nonoxidative) flux ratio for nucleic acid ribose synthesis of >1 were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited a (oxidative/nonoxidative) flux ratio of <0.7. The changes in glucose uptake and metabolism were accompanied by intracellular translocation of GLUT-1 from the plasma membrane into the intracellular fraction in sensitive cells treated with imatinib, whereas GLUT-1 remained located at the plasma membrane in LAMA84-r and K562-r cells. The total protein load of GLUT-1 was unchanged among treated sensitive and resistant cell lines. In summary, elevated glucose uptake and nonoxidative glycolytic metabolic phenotype can be used as sensitive markers for early detection of imatinib resistance in BCR-ABL-positive cells.

Use of the 1-mm micro-probe for metabolic analysis on small volume biological samples.

Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and (1)H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 microl: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 x 10(6): chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

The metabolites citrate, myo-inositol, and spermine are potential age-independent markers of prostate cancer in human expressed prostatic secretions.

OBJECTIVES: Due to specific physiological functions, prostatic tissues and fluids have unique metabolic profiles. In this study, proton nuclear magnetic resonance spectroscopy ((1)H-NMRS) is used to assess potential metabolic markers of prostate cancer (PCa) in human expressed prostatic secretions (EPS). METHODS: Metabolic profiles of EPS from 52 men with PCa and from 26 healthy controls were analyzed using quantitative (1)H-NMRS. The metabolites quantified included citrate, spermine, myo-inositol, lactate, alanine, phosphocholine, glutamine, acetate, and hydroxybutyrate. Logistic regression (LR) was used to model the risk of PCa based on metabolite concentrations while adjusting for age. RESULTS: The average age of the EPS donors with PCa was 58.0+/-7.0 years and 52.2+/-12.1 for the healthy donors. The median Gleason score for the men with PCa was 7 (range 5-9). The LR models indicated that the absolute concentrations of citrate, myo-inositol, and spermine were highly predictive of PCa and inversely related to the risk of PCa. The areas under the receiver operating characteristic curves (AUROC) for citrate, myo-inositol and spermine were 0.89, 0.87, and 0.79, respectively. At 90% sensitivity, these metabolites had specificities of 74%, 51%, and 34%, respectively. The LR analysis indicated that absolute levels of these three metabolites were independent of age. CONCLUSIONS: The results indicate that citrate, myo-inositol and spermine are potentially important markers of PCa in human EPS. Further, the absolute concentrations of these metabolites in EPS appear to be independent of age, increasing the potential utility of these markers due to elimination of age as a confounding variable.

Glutamine-mediated attenuation of cellular metabolic dysfunction and cell death after injury is dependent on heat shock factor-1 expression.

BACKGROUND: Cellular metabolic dysfunction is associated with occurrence of multiple-organ failure after critical illness. Glutamine (GLN) attenuates cellular metabolic dysfunction in critical illness models. The mechanism of this protection is unclear. We previously demonstrated that GLN's benefit in critical illness might be due to enhanced heat shock protein (HSP) expression. We hypothesize that GLN's attenuation of cellular metabolic dysfunction is dependent on presence of heat shock factor-1 (HSF-1). METHODS: HSF-1 wild-type and knockout mouse embryonic fibroblasts (HSF-1+/+ and HSF-1-/-) were used in all experiments. Cells were not treated, or were treated with 8 mmol/L GLN and immediately exposed to heat stress injury (45 degrees C for 45 minutes). Cells were harvested for metabolic analysis by nuclear magnetic resonance (NMR) at 24 hours postinjury. Cell survival was assessed using the MTS assay. RESULTS: GLN treatment in HSF-1+/+ cells led to significant attenuation of decreases in adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio, phosphomonoester/phosphodiester (PME/PDE) ratio, and cell survival observed in non-GLN-treated HSF-1+/+ cells. In HSF-1-/- cells, the beneficial effect of GLN on preservation of ATP/ADP ratio, PME/PDE proliferation, and cell survival was lost. GLN-treated HSF-1-/- cells had a significant increase in extracellular lactate concentrations vs GLN-treated HSF+/+ cells. CONCLUSIONS: GLN treatment attenuated cellular metabolic dysfunction and improved cell membrane recovery only in HSF-1+/+ cells. Cellular injury, as measured by lactate release and cell survival assay, was improved by GLN treatment in HSF-1+/+ cells alone. Thus, GLN's beneficial effect on cellular metabolic dysfunction and cell survival appears to be dependent on HSF-1 expression.

Metabolic profiling of livers and blood from obese Zucker rats.

BACKGROUND/AIMS: Obesity frequently leads to changes in fatty acid metabolism with subsequent fatty infiltration in the liver. METHODS: In this study, metabolic profile of the livers and blood from lean and obese Zucker rats was established based on quantitative nuclear magnetic resonance spectroscopy (NMR) analysis. RESULTS: (1)H NMR on liver lipid extracts indicated significantly increased concentrations of total fatty acids and triglycerides. (31)P NMR on liver extracts revealed that obese livers have a compromised energy balance (low [ATP/ADP]) with decreased mitochondrial activity. Simultaneously, increased glycolytic activity was detected. The most pronounced differences were highly increased methionine and decreased betaine concentrations in obese animals. This suggests a significant alteration in methionine metabolism, which may be in part responsible for the development of steatosis, induction of mitochondrial dysfunction, and increased vulnerability of fatty livers to ischemia/reperfusion injury. A trend towards decreased hepatic glutathione concentrations as well as a reduced [PUFA/MUFA] ratio were present in the obese group, indicating increased oxidative stress and lipid peroxidation. CONCLUSIONS: In conclusion, NMR analysis on blood and liver tissue from obese Zucker rats reveals specific metabolic abnormalities in mitochondrial function and methionine metabolism, which result in a decreased hepatic energy state.

Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers.

Livers from obese donors often have fatty infiltrates and are more susceptible to ischemia-reperfusion injury and subsequent graft dysfunction. This often leads to the exclusion of organs from obese donors. We investigated whether ischemic preconditioning (IP, 10 min ischemia, 10 min reperfusion) preserves cellular metabolism in livers from obese Zucker rats during cold ischemia. Liver samples (-IP and +IP) were collected from obese and control lean rats at different time points of cold ischemia (CI) and analyzed by magnetic resonance spectroscopy (1H- and 31P-MRS) to assess whether IP improves hepatic cellular metabolism. IP significantly improved high energy metabolism in IP livers from obese rats when compared with obese controls during the first hours of CI. At 4 h of cold storage, obese IP livers were not different from control lean non-IP livers. The beneficial metabolic effect of IP on livers form obese rats, however, was absent at 8 h of reperfusion. In contrast, in livers from lean rats, IP resulted in improved high-energy metabolism during the entire observation period of 8 h. In a later part of the study, IP of liver grafts from obese rats before 4 h of cold storage improved recipient survival after graft transplantation. IP of liver grafts from obese rats before 4 h of CI increases 24-h survival of recipient animals from 25% to 88%.