The synthetic molecule stauprimide impairs cell growth and migration in triple-negative breast cancer.

Stauprimide, a semi-synthetic derivative of staurosporine, is known mainly for its potent differentiation-enhancing properties in embryonic stem cells. Here, we studied the effects of stauprimide in cell growth and migration of triple-negative breast cancer cells in vitro, evaluating its potential antitumoral activity in an orthotopic mouse model of breast cancer in vivo. Our results from survival curves, EdU incorporation, cell cycle analysis and annexin-V detection in MDA-MB-231 cells indicated that stauprimide inhibited cell proliferation, arresting cell cycle in G2/M without induction of apoptosis. A decrease in the migratory capability of MDA-MB-231 was also assessed in response to stauprimide. In this work we pointed to a mechanism of action of stauprimide involving the modulation of ERK1/2, Akt and p38 MAPK signalling pathways, and the downregulation of MYC in MDA-MB-231 cells. In addition, orthotopic MDA-MB-231 xenograft and 4T1 syngeneic models suggested an effect of stauprimide in vivo, increasing the necrotic core of tumors and reducing metastasis in lung and liver of mice. Together, our results point to the promising role of stauprimide as a putative therapeutic agent in triple-negative breast cancer.

Quantitative (1) H MR spectroscopic imaging of the prostate gland using LCModel and a dedicated basis-set: correlation with histologic findings.

Proton magnetic resonance spectroscopic imaging ((1) H-MRSI) has been advocated as a valuable tool for prostate cancer diagnosis. However, a barrier to widespread clinical use of this technique is the lack of robust quantification methods that yield reproducible results in an institution-independent manner. The main goal of this study was to develop a standardized and fully automated approach (LCModel-based) for quantitative prostate (1) H-MRSI. To this end, a dedicated basis set was constructed by the combination of simulated (citrate, Cit; choline, Cho, and creatine, CR) and experimentally acquired (spermine, Spm) spectra. The overlapping Spm, Cho, and Cr could be resolved and quantified individually, thus allowing for the independent assessment of glandular (Cit and Spm) and proliferative (Cho) components. Several metabolite ratios were calculated and compared to the histologic findings of prostatectomy specimens from 10 prostate cancer patients with Gleason scores (3 + 3) and (3 + 4). The Cho mole fraction and the Cho/(Cit + Spm) ratio were found to best discriminate between prostate cancer and healthy tissue. The comparison between the quantitative MRSI results and the histologic findings suggests that no correlation exists between the detected metabolic alterations and the Gleason score of low-grade tumors.

Perturbation of mouse glioma MRS pattern by induced acute hyperglycemia.

(1)H MRS is evolving into an invaluable tool for brain tumor classification in humans based on pattern recognition analysis, but there is still room for improvement. Here we propose a new approach: to challenge tumor metabolism in vivo by a defined perturbation, and study the induced changes in MRS pattern. For this we recorded single voxel (1)H MR spectra from mice bearing a stereotactically induced GL261 grade IV brain glioma during a period of induced acute hyperglycemia. A total of 29 C57BL/6 mice were used. Single voxel spectra were acquired at 7 T with point resolved spectroscopy and TE of 12, 30 and 136 ms. Tumors were induced by stereotactic injection of 10(5) GL261cells in 17 mice. Hyperglycemia (up to 338 +/- 36 mg/dL glucose in the blood) was induced by intraperitoneal bolus injection. Maximal increases in glucose resonances of up to 2.4-fold were recorded from tumors in vivo. Our observations are in agreement with extracellular accumulation of glucose, which may suggest that glucose transport and/or metabolism are working close to their maximum capacity in GL261 tumors. The significant and specific MRS pattern changes observed when comparing euglycemia and hyperglycemia may be of use for future pattern-recognition studies of animal and human brain tumors by enhancing MRS-based discrimination between tumor types and grades.

Targeting of lanthanide(III) chelates of DOTA-type glycoconjugates to the hepatic asyaloglycoprotein receptor: cell internalization and animal imaging studies.

The characterization of a new class of hydrophilic liver-targeted agents for gamma-scintigraphy and MRI, consisting, respectively, of [(153)Sm](3+) or Gd(3+) complexes of DOTA monoamide or bisamide linked glycoconjugates (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), is reported. In vitro studies show high uptake of radiolabeled [(153)Sm]-DOTAGal(2) by the human hepatocyte carcinoma cell line Hep G2 containing the asialoglycoprotein receptor (ASGP-R), which is decreased to less than 50% by the presence of its high-affinity ligand asialofetuin (ASF). In vivo biodistribution, gamma-imaging and pharmacokinetic studies on Wistar rats using the [(153)Sm](3+)-labeled glycoconjugates show a high uptake in the receptor-rich organ liver of the radiolabeled compounds containing terminal galactosyl groups, but very little uptake for those compounds with terminal glycosyl groups. Blocking the receptor in vivo reduced liver uptake by 90%, strongly suggesting that the liver uptake of these compounds is mediated by their binding to the asyaloglycoprotein receptor (ASGP-R). This study also demonstrated that the valency increase improves the targeting capability of the glycoconjugates, which is also affected by their topology. However despite the specific liver uptake of the radiolabeled galactose-bearing multivalent compounds, the animal MRI assessment of the corresponding Gd(3+) chelates shows liver-to-kidney contrast effects which are not significantly better than those shown by GdDTPA. This probably results from the quick wash-out from the liver of these highly hydrophilic complexes, before they can be sufficiently concentrated within the hepatocytes via receptor-mediated endocytosis.

Intracellular water motion decreases in apoptotic macrophages after caspase activation.

Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.

Mapping extracellular pH in rat brain gliomas in vivo by 1H magnetic resonance spectroscopic imaging: comparison with maps of metabolites.

The value of extracellular pH (pH(e)) in tumors is an important factor in prognosisand choice of therapy. We demonstrate here that pH(e) can be mappedin vivo in a rat brain glioma by (1)H magnetic resonance spectroscopic imaging (SI) of the pH buffer (+/-)2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA). (1)H SI also allowed us to map metabolites, and, to better understand the determinants of pH(e), we compared maps of pH(e), metabolites, and the distribution of the contrast agent gadolinium1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraaceticacid (Gd-DOTA). C6 cells injected in caudate nuclei of four Wistar rats gave rise to gliomas of approximately 10 mm in diameter. Three mmols of IEPA were injected in the right jugular vein from t = 0 to t = 60 min. From t = 50 min to t = 90 min, spin-echo (1)H SI was performed with an echo time of 40 ms in a 2.5-mm slice including the glioma (nominal voxel size, 2.2 microl). IEPA resonances were detected only within the glioma and were intense enough for pH(e) to be calculated from the chemical shift of the H2 resonance in almost all voxels of the glioma. (1)H spectroscopic images with an echo time of 136 ms were then acquired to map metabolites: lactate, choline-containing compounds (tCho), phosphocreatine/creatine, and N-acetylaspartate. Finally, T(1)-weighted imaging after injection of a bolus of Gd-DOTA gave a map indicative of extravasation. On average, the gradient of pH(e) (measured where sufficient IEPA was present) from the center to the periphery was not statistically significant. Mean pH(e) was calculated for each of the four gliomas, and the average was 7.084 +/- 0.017 (+/- SE; n = 4 rats), which is acid with respect to pH(e) of normal tissue. After normalization of spectra to their water peak, voxel-by-voxel comparisons of peak areas showed that N-acetylaspartate, a marker of neurons, correlated negatively with IEPA (P < 0.0001) and lactate (P < 0.05), as expected of a glioma surrounded by normal tissue. tCho (which may indicate proliferation) correlated positively with pH(e) (P < 0.0001). Lactate correlated positively with tCho (P < 0.0001), phosphocreatine/creatine (P < 0.001), and Gd-DOTA (P < 0.0001). Although lactate is exported from cells in association with protons, within the gliomas, no evidence was observed that pH(e) was significantly lower where lactate concentration was higher. These results suggest that lactate is produced mainly in viable, well-perfused, tumoral tissue from which proton equivalents are rapidly cleared.

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy.

Ex vivo ¿(13)C, (2)H¿ NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ¿(13)C, (2)H¿ NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.

Structural studies of 5-ethyl-2'-deoxyuridine by selective pulse 1H DPFGSE NOE spectroscopy and PM3 calculations.

The solution conformation of 5-ethyl-2'-deoxyuridine (EDU) has been calculated from the vicinyl proton-proton NMR coupling constants and nuclear Overhauser (NOE) distances using excitation sculpting of selective pulses (Double Pulsed Field Gradient Spin Echo NOE) at 500 MHz and molecular modelling (PM3) studies.