NMR spectroscopy of macrophages loaded with native, oxidized or enzymatically degraded lipoproteins

Oxidized and enzymatically modified low-density lipoproteins (oxLDL and eLDL) play a key role in early stages of atherogenesis. Their uptake by recruited macrophages leads to endolysosomal phospholipidosis or foam cell formation, respectively, each of which is preceded by highly differential lipid restructuring processes. We applied (1)H-NMR spectroscopy (NMRS) to elucidate these structural rearrangements both in consequence of lipoprotein modifications and following phagocytosis. Being specifically sensitive to the mobile lipid subset, NMRS of oxLDL and eLDL revealed a partial and total immobilization of lipids, respectively. NMRS of intact macrophages showed a sixfold increase in mobile lipids in case of loading with eLDL but no significant changes for oxLDL or native LDL. This finding reflected the disparate lipid storage in lipid droplets and in multilamellar endolysosomal clusters when loaded with either eLDL or oxLDL, respectively. Moreover, a significant shift of the degree of saturation towards mainly polyunsaturated fatty acid chains was found for the mobile lipid pool in eLDL-loaded macrophages. Additional analyses of lipid extracts by NMRS and mass spectrometry (MS) reflected these changes in lipid content and in fatty acid composition only partially. In summary, in-cell NMRS represents a unique lipidomics tool to investigate structural changes within the mobile lipid pool following atherogenic triggers that can be not detected by the analysis of lipid extracts by MS or NMRS

An iron-based T1 contrast agent made of iron-phosphate complexes: In vitro and in vivo studies

Abstract A new iron-based T 1 contrast agent consisting of a complex of iron ions coordinated to phosphate and amine ligands (Fe(phos) in short) has been characterized by spectroscopic and magnetic measurements. NMR relaxation studies showed r 1 values to be dependent on the phosphate salt concentration, K2HPO4, present in the medium. r 1 reaches a maximum value of 2.5 mM-1 s-1 for measurements carried out at 7 T and 298 K. 31P MRS, Mssbauer spectroscopy and magnetic measurements of Fe(phos) solutions suggest paramagnetic Fe3+ ions present in the studied iron–phosphate complex. In vitro and in vivo toxicity experiments with C6 cells and CD1 mice, respectively, demonstrated lack of toxicity for Fe(phos) at the highest dose tested in the MRI experiments (12 mM iron for C6 cells and 0.32 mmol iron/kg for mice). Finally, T 1 weighted images of brain tumours in mice have shown positive contrast enhancement of Fe(phos) for tumour afflicted regions in the brain

Cellular and in vivo activity of a novel PI3K inhibitor, PX-866, against human glioblastoma

The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis, and invasion is found in 70%–80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma. We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866 inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression