Integrating systemic and molecular levels to infer key drivers sustaining metabolic adaptations.

Metabolic adaptations to complex perturbations, like the response to pharmacological treatments in multifactorial diseases such as cancer, can be described through measurements of part of the fluxes and concentrations at the systemic level and individual transporter and enzyme activities at the molecular level. In the framework of Metabolic Control Analysis (MCA), ensembles of linear constraints can be built integrating these measurements at both systemic and molecular levels, which are expressed as relative differences or changes produced in the metabolic adaptation. Here, combining MCA with Linear Programming, an efficient computational strategy is developed to infer additional non-measured changes at the molecular level that are required to satisfy these constraints. An application of this strategy is illustrated by using a set of fluxes, concentrations, and differentially expressed genes that characterize the response to cyclin-dependent kinases 4 and 6 inhibition in colon cancer cells. Decreases and increases in transporter and enzyme individual activities required to reprogram the measured changes in fluxes and concentrations are compared with down-regulated and up-regulated metabolic genes to unveil those that are key molecular drivers of the metabolic response.

Antitumoral effect of phenazine N5,N10-dioxide derivatives on Caco-2 cells.

We studied the in vitro antitumoral effect of a series of phenazine di- N-oxide derivatives, named 2-chloroacetylamino-7(8)-nitrophenazine N(5), N(10)-dioxide (1), 2-amino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (2), 2-chloroacetylamino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (3), and 2-amino-7(8)-methoxyphenazine N(5), N(10)-dioxide (4), on Caco-2 cells. These phenazine N(5), N(10)-dioxide derivatives belong to our in-house chemical library. The products were selected according to their stereoelectronic characteristics and taking into account their differential cytotoxicity against V79 cells. Human colorectal adenocarcinoma cell line Caco-2 was used to study the cell growth inhibition capacity of these compounds, their capacity of altering the cell cycle and possible induction of apoptosis, DNA fragmentation, and genotoxic damage. The IC 50 after 24 h of incubation was lower for 1, 2, and 3 (4.8, 46.8, and 8.2 microM, respectively) than for 4 (474.7 microM). Compound 1 induced arrest in the G2/M phase at 24 and 48 h of treatment and apoptosis at the highest doses at 24 h of treatment. These facts were corroborated with caspase 3, caspase 9, and cytochrome c activation and DNA fragmentation at 24 h of treatment. The derivatives studied induced neither significant single strand breaks nor oxidative damage at the different studied times. We concluded that among the series of N(5), N(10)-dioxide phenazine derivatives analyzed, 1, which contains a nitro moiety and a chloroacetamide group, is the most promising as an antitumoral compound.

Effect of new antioxidant cysteinyl-flavanol conjugates on skin cancer cells.

Novel catechin derivatives obtained from grape procyanidins and l-cysteine scavenge free radicals by hydrogen atom donation, rather than electron transfer, and reduce cell viability in A375 and M21 melanoma cells. In particular, 4beta-(S-cysteinyl)epicatechin 3-O-gallate has a free radical scavenging capacity as strong as that of tea (-)-epigallocatechin gallate and causes a significant S-phase cell-cycle arrest in both cell lines at doses higher than 100 microM. The other cysteinyl compounds do not affect normal cell cycle distribution. The gallate derivative also induces apoptosis in melanoma cells more strongly than the other derivatives and the parent (-)-epicatechin do. The gallate compound seems to trigger nuclear condensation and fragmentation, which is confirmed by DNA laddering. Interestingly, they do not induce apoptosis in keratinocytes (HaCaT).

Metabolic strategy of boar spermatozoa revealed by a metabolomic characterization.

Metabolomic characteristics in boar spermatozoa were studied using [1,2-(13)C(2)]glucose and mass isotopomer analysis. In boar spermatozoa, glycolysis was the main pathway of glucose utilization producing lactate/pyruvate, whereas no gluconeogenesis was seen. Slight glycogen synthesis through the direct pathway and some incorporation of pyruvate into the Krebs cycle also took place. Neither RNA ribose-5-phosphate nor fatty acid synthesis from glucose occurred despite the detection of pyruvate dehydrogenase activity. In contrast to the known metabolic activities in dog sperm, boar spermatozoa have low levels of energy production and biosynthetic activities suggesting two different metabolic profiles for the two different phenotypes.

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.

A novel cyclometallated Pt(II)-ferrocene complex induces nuclear FOXO3a localization and apoptosis and synergizes with cisplatin to inhibit lung cancer cell proliferation.

Cisplatin is a platinum-based compound that acts as an alkylating agent and is used to treat a variety of malignant tumors including lung cancer. As cisplatin has significant limitations in the clinic, alternative platinum compounds such as cycloplatinated complexes have been considered as attractive anti-tumor agents. Here, we report the antiproliferative activity of a novel diastereomerically pure cycloplatinated complex (Sp,1S,2R)-[Pt{(κ(2)-C,N)[(η(5)-C5H3)-CH[double bond, length as m-dash]N-CH(Me)-CH(OH)-C6H5]Fe(η(5)-C5H5)}Cl(DMSO)] 6a, against A549 non-small cell lung cancer. Mechanistic studies revealed that compound 6a induces nuclear translocation of a FOXO3a reporter protein as well as endogenous FOXO3a in U2OS and A549 cells, respectively. Accordingly, treatment of A549 cells with compound 6a activates the intrinsic caspase pathway and dramatically increases the percentage of apoptotic cells. Furthermore, 6a displays a synergistic antiproliferative effect when applied together with cisplatin. Compound 6a is also active in other cancer cell lines including NCI-H460 large cell lung cancer cells. Importantly, antiproliferative activity of the platinacycle 6a on the non-tumor and non-proliferating 3T3-L1 cell line is weaker than in all cancer cell lines tested.

The stable isotope-based dynamic metabolic profile of butyrate-induced HT29 cell differentiation.

Stable isotope-based dynamic metabolic profiling is applied in this paper to elucidate the mechanism by which butyrate induces cell differentiation in HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with [1,2-13C2]glucose or [1,2-13C2]butyrate as single tracers to observe the changes in metabolic fluxes in these cells. In HT29 cells, increasing concentrations of butyrate inhibited glucose uptake, glucose oxidation, and nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon utilization for de novo fatty acid synthesis and tricarboxylic acid cycle flux was replaced by butyrate. We also demonstrated that these changes are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The results suggest that the mechanism by which colon carcinoma cells acquire a differentiated phenotype is through a replacement of glucose for butyrate as the main carbon source for macromolecule biosynthesis and energy production. This provides a better understanding of cell differentiation through metabolic adaptive changes in response to butyrate in HT29 cells, demonstrating that variations in metabolic pathway substrate flow are powerful regulators of tumor cell proliferation and differentiation.