Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

Salivary levels of tumor necrosis factor-alpha in oral lichen planus

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.     

Metabolic profile of NO-flurbiprofen (HCT1026) in rat brain and plasma: a LC-MS study

Male rats were given the nitrooxybutyl ester of flurbiprofen HCT1026 (15 mg/Kg) by oral administration and plasma and brain levels of the parent drug and its potential metabolites (HCT1027 and flurbiprofen) were determined at different times post-administration by a validated HPLC method (UV-DAD detection; LOQ: 0.13 nmoles/ml and 0.3 nmoles/g respectively in plasma and brain tissue). Structural confirmation of the analytes was achieved by MS monitoring of their de-protonated (negative ion mode) or cationized/protonated (positive ion mode) molecular ions and of the relative fragment ions obtained by collision-induced dissociation (CID) experiments. The results indicate that flurbiprofen is the only metabolite found at measurable levels in both plasma and brain, while HCT1026 or its de-nitrated metabolite HCT1027 were always below the limit of detection at all the observation times. The same was observed after administration of the higher dose of HCT1026 (100 mg/Kg, i.p.). In orally-treated animals the time-course of flurbiprofen formation strictly parallels that of NOx (nitrite/nitrate) in plasma but not in brain, where the levels were always in the range of the controls. These data indicate that the NO molecule is not released from the parent drug within the brain.     

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.     

The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalators

Fifteen platinum(II)-based metallointercalators have been synthesised that utilise substituted 1,10-phenanthroline (phen) ligands, including 5-chloro-1,10-phenanthroline (5-Cl-phen), 5-methyl-1,10-phenanthroline (5-CH3-phen), 5-amino-1,10-phenanthroline (5-NH2-phen), 5-nitro-1,10-phenanthroline (5-NO2-phen) and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), and achiral ethylenediamine (en) and the chiral ancillary ligands 1S,2S-diaminocyclohexane (S,S-dach) and 1R,2R-diaminocyclohexane (R,R-dach). Their cytotoxicity in the L1210 murine leukaemia cell line was determined using growth inhibition assays. The most cytotoxic metal complexes are those that contain S,S-dach ancillary ligands and 5-CH3-phen intercalating ligands. One metallointercalator [Pt(5-CH3-phen)(S,S-dach)]Cl2 (5MESS), displays a 5-10-fold increase in cytotoxicity compared to the clinical agent cisplatin. From DNA binding experiments there appears to be no significant difference between any of the metal complexes, indicating that neither DNA binding affinity nor the mode of binding/DNA adduct formed is the sole determinant of the cytotoxicity of this family of platinum(II)-based metallointercalators.     

A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila

Drosophila MSL-2 is the limiting component of the dosage compensation complex. Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (SXL) binding to sites in both the 5' and the 3' untranslated regions (UTRs). Here, we uncover the mechanism by which SXL achieves tight control of translation initiation. SXL binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by SXL bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA.