Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-¹⁴C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the ¹H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 ± 0.9 and 5.2 ± 2.4 nmol/10⁶ cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 ± 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to ≤0.2. Two-dimensional {phosphocholine/total choline, [total choline]} and {glycerophosphocholine/total choline, [total choline]} maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 ± 0.1 nmol/10⁶ cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-¹⁴C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher (∼5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)–mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)–mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKα (but not ChoKβ) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A₂ activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKα mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC byosinthesis and degradation, respectively. Cancer Res; 70(5); 2126–35

Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.

Summary: Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS‐A and CS‐B present an altered redox balance with increased steady‐state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K‐ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild‐type genes. High‐resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS‐A and CS‐B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with β1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA–induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA–induced agglutination and cell death. All of the CGs tested inhibited L-PHA–induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC₅₀ of 60 ± 20 nmol/L. Digoxin also increased the fraction of some concanavalin A–binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc₁Man₃GlcNAc₂Fuc₁ and GlcNAc₂Man₃GlcNAc₂Fuc₁ oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation–mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients. [Cancer Res 2008;68(16):6688–96]

Cancer treatment options are increasing. However, even among the same tumor histotype, interpatient tumor heterogeneity should be considered for best therapeutic result. Metabolomics represents the last addition to promising “omic” sciences such as genomics, transcriptomics, and proteomics. Biochemical transformation processes underlying energy production and biosynthetic processes have been recognized as a hallmark of the cancer cell and hold a promise to build a bridge between genotype and phenotype. Since breast tumors represent a collection of different diseases, understanding metabolic differences between molecular subtypes offers a way to identify new subtype-specific treatment strategies, especially if metabolite changes are evaluated in the broader context of the network of enzymatic reactions and pathways. Here, after a brief overview of the literature, original metabolomics data in a series of 92 primary breast cancer patients undergoing surgery at the Istituto Nazionale dei Tumori of Milano are reported highlighting a series of metabolic differences across various molecular subtypes. In particular, the difficult-to-treat luminal B subgroup represents a tumor type which preferentially relies on fatty acids for energy, whereas HER2 and basal-like ones show prevalently alterations in glucose/glutamine metabolism.