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Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid- β(1-40) accumulates around blood vessels. In neurons, amyloid- β impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid- β(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR ( NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid- β(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid- β(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid- β(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid- β(1-40) and impaired in 5x-FAD mice.

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.

We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.

Increased activity of the main carbohydrate pathways (glycolysis, pentose phosphate, and hexosamine biosynthetic pathways) is one of the hallmarks of metabolic diseases such as cancer. Sulfoquinovosyl diacylglycerol is a sulfoglycolipid found in the human diet that possesses anticancer activity that is absent when its carbohydrate moiety (glucose 6-sulfonate or sulfoquinovose) is removed. This work used bacterial systems to further understand the metabolism of this sugar through three main carbohydrate processing pathways and how this could influence its biological activity. Using 13C NMR spectroscopy and enzyme assays, we showed that glucose 6-sulfonate cannot enter the pentose phosphate pathway, hence decreasing pentose and nucleotide biosyntheses. In glycolysis, glucose 6-sulfonate only provides one pyruvate per monosaccharide molecule, decreasing the flux of this pathway by half when compared to glucose 6-phosphate. Glucose 6-sulfonate can enter the hexosamine biosynthetic pathway by producing glucosamine 6-sulfonate, which is a reported antibacterial agent that competitively inhibits hexosamine production. All these interactions with carbohydrate routes might help explain the observed anticancer activity that glucose 6-sulfonate has in vitro. This adds to our knowledge of how vegetables rich in glucose 6-sulfonate can also act as metabolic inhibitors of pathways that are increased in metabolic diseases.

The structural rationale, synthesis and evaluation of an inhibitor designed to block glucosamine synthesis by competitively inhibiting the action of glutamine: fructose-6-phosphate amidotransferase and subsequently reducing the transformation of any glucosamine-6-phosphate formed to UDP-N-acetylglucosamine are described. The inhibitor 2-acetamido-2,6-dideoxy-6-sulfo-D-glucose (D-glucosamine-6-sulfonate) is an analog of glucosamine-6-phosphate in which the phosphate group in the latter is replaced with a sulfonic acid group. The inhibitor is designed to function by three different modes which together reduce UDP-N-acetylglucosamine synthesis. This reduction was confirmed by evaluating the effect of the inhibitor on bacterial cell-wall synthesis and by demonstrating that it inhibits acetylation of glucosamine-6-phosphate competitively and by acting as a surrogate substrate. Inhibition of glucosamine production or suitably activated glucosamine in bacteria leads to disruption of the peptidoglycan structure, which results in softening, bulging, deformation, fragility and lysis of the cells. These modifications were documented by scanning electron microscopy for bacteria treated with the inhibitor. They were observed for inhibitor concentrations in the 20 mg/mL range for Escherichia coli and Bacillus subtilis and the 5 mg/mL range for Rhizobium trifolii.

The cytotoxicity of the dichloromethane crude extract (DCE), obtained from the aerial parts of Pothomorphe umbellata (L.) Miq (Piperaceae), was evaluated against nine human cancer cell lines (MCF-7, NCI-ADR/RES, OVCAR-3, PC-3, HT-29, NCI-H460, 786-O, UACC-62, K-562). The DCE presented antiproliferative activity with good potency against all cell lines at low concentrations (between 4.0 and 9.5 microg/mL) and with selectivity (1.55 microg/mL) for the leukemia cell line (K-652). DCE (100, 200, 300 and 400 mg/kg, ip) was also evaluated in the Ehrlich ascites tumor model. Both the survival number and the life span of the animals that died increased by at least 45 and 50%, respectively (8 animals per group), demonstrating P. umbellata extract potential anticancer activity. The results of the in vivo antitumor activity prompted the fractionation of the crude extract. The crude extract was submitted to dry column chromatography with dichloromethane-methanol (99:1). The column effluent fractions were extracted with methanol, dried under vacuum yielding fractions FR1 (less polar), FR2 (medium polarity), and FR3 (polar), which were analyzed for their growth inhibition or cytotoxic properties by a 48-h sulforhodamine B cell viability assay by measuring the total protein content. FR1 demonstrated high potency and cytotoxicity, a result compatible with the high toxicity of oxalic acid; FR2, containing 4-nerolidylcathecol, presented the lowest cytotoxic activity compared to the other two fractions but with selectivity for prostate cancer cell line; FR3, containing a mixture of steroids described in the literature as possessing various biological activities, also presented potent anticancer in vitro activity. These results suggest that P. umbellata DCE in vivo antitumor activity may be a consequence of the activity of different active principles.

A series of R-(+)-limonene derivatives bearing a substituted thiourea moiety (3-13) and five S-methyl analogs (14-18) were synthesized and evaluated for their in vitro antiproliferative activity against human cancer cell lines. Compounds bearing aromatic substituents (3-6) exhibit cytotastic activity in the full panel of cell lines tested, with GI50 values in the range of 2.5 to 24 µmol L-1. Compounds 3, 10, 12 and 16 were the most active with GI50 values in the range of 0.41 to 3.0 µmol L-1, against different cell lines. No presente trabalho descrevemos a síntese e a avaliação da atividade antiproliferativa, frente a linhagens de células tumorais humanas, de derivados do R-(+)-limoneno (3-18) contendo uma unidade tiouréia substituída. Os derivados com substituintes arílicos (3-6) exibiram atividade citostática frente a todas linhagens testadas, com inibição de 50% do crescimento celular (GI50) em concentrações na faixa de 2,5 a 24 µmol L-1. Os compostos 3, 10, 12 e 16 foram os mais ativos, com GI50 na faixa de 0,41 a 3,0 mmol L-1, frente a diferentes linhagens celulares.