Gilbert Arthur

1-O-octadecyl-2-O-methyl-glycerophosphocholine inhibits the transduction of growth signals via the MAPK cascade in cultured MCF-7 cells.

1-O-Octadecyl-2-O-methyl-glycerophosphocholine (ET18-OCH3) is an ether lipid with selective antiproliferative properties whose mechanism of action is still unresolved. We hypothesized that since ET18-OCH3 affects a wide variety of cells, its mechanism of action was likely to involve the inhibition of a common widely used pathway for transducing growth signals such as the mitogen-activated protein kinase (MAPK) cascade. To test this, we established conditions whereby quiescent MCF-7 cells took up ET18-OCH3 in sufficient quantities that inhibited cell proliferation subsequent to the addition of growth medium and examined the activation of components of the MAPK cascade under these conditions. ET18-OCH3 inhibited the sustained phosphorylation of MAPK resulting in a decrease in the magnitude and duration of activation of MAPK in cells stimulated with serum or EGF. ET18-OCH3 had no effect on the binding of EGF to its receptors, their activation, or p21ras activation. However, an interference in the association of Raf-1 with membranes and a resultant decrease in Raf-1 kinase activity in membranes of ET18-OCH3-treated cells was observed. ET18-OCH3 had no direct effect on MAPK or Raf-1 kinase activity. A direct correlation between ET18-OCH3 accumulation, inhibition of cell proliferation, Raf association with the membrane, and MAPK activation was also established. These results suggest that inhibition of the MAPK cascade by ET18-OCH3 as a result of its effect on Raf-1 activation may be an important mechanism by which ET18-OCH3 inhibits cell proliferation.

Acyl specificity of hamster heart CDP-choline 1,2-diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis

The acyl specificity of 1,2-diacylglycerol: CDP-choline phosphocholine transferase (EC 2.7.8.2) for the formation of phosphatidylcholine with the appropriate acyl groups in hamster heart was investigated. Enzyme activity was determined in the microsomal fraction with 1,2-diacylglycerols of known acyl content. Maximum enzyme activity was obtained with diacylglycerol containing a monoenoic acyl group at the C-2 position of the glycerol moiety, regardless of the acyl group at the C-1 position. The specificity of the enzymes was also investigated by perfusing the isolated hamster heart with labelled glycerol. Comparison of the molecular species of the labelled diacylglycerols and phosphatidylcholine subsequent to perfusion revealed that the specificity of phosphocholine transferase was not limited to the monoenoic species of diacylglycerol. The difference in specificity observed between the in vitro assay and the perfusion study may partly be attributed to the presence of detergent in the enzyme assay mixture (to facilitate solubility of diacylglycerol). It is concluded that in the hamster heart, phosphocholine transferase has only limited ability to select the appropriate acyl groups for phosphatidylcholine biosynthesis. It appears that the majority of the newly formed phosphatidylcholine in the heart via the CDP-choline pathway is subsequently resynthesized by deacylation-reacylation process.

Lovastatin protects human neurons against Aβ-induced toxicity and causes activation of β-catenin–TCF/LEF signaling

Abstract Alzheimers disease (AD) is characterized by cognitive decline due to excess amyloid beta peptide (Aβ), neurofibrillary tangles, and neuronal loss. Aβ promotes neuronal apoptosis in AD by activating glycogen synthase kinase-3β (GSK-3β), leading to degradation of β-catenin and inactivation of Wnt signaling. β-Catenin interacts with the T-cell factor (TCF)/Lymphoid enhancer factor (LEF)-nuclear complex to mediate Wnt signaling and cell survival. Statins are associated with decreased prevalence of AD. Lovastatin has been shown to decrease the production of Aβ and to promote neuronal survival. The mechanisms of how statins promote neuronal survival are unclear. We propose that the neuroprotective effect of lovastatin may be due to inactivation of GSK-3β activity, resulting in induction of Wnt signaling. Here, we report that lovastatin prevented Aβ-induced apoptosis in human SK-NSH cells. This was accompanied by reduction in active GSK-3β, and increased nuclear translocation of β-catenin, TCF-3, and LEF-1. Lovastatin treatment induced an increase in TCF/LEF–chloramphenicol acetyl transferase (CAT) gene reporter activity. More importantly, β-catenin and TCF were required for the neuroprotective function of lovastatin. Our results suggest that lovastatin protects neuronal cells from Aβ-induced apoptosis and causes reduction in GSK-3β activity, resulting in activation of Wnt signaling.

The distribution and acyl composition of plasmalogens in Guinea pig heart

In guinea pig heart homogenate, 34% of both choline and ethanolamine phosphoglycerides were in the form of plasmalogens (1-alkenyl, 2-acyl glycerophospholipid). Plasmalogens accounted for 39% of the choline phosphoglycerides and 36% of the ethanolamine phosphoglycerides in the mitochondrial fraction. Ethanolamine plasmalogen was the major ethanolamine phosphoglyceride (63%) in the guinea pig heart microsomal fraction. A high arachidonyl content was found in both diacyl and 1-alkenyl, 2-acyl glycerophosphoethanolamine. The C-2 fatty acyl profiles of the diacyl and 1-alkenyl, 2-acyl choline phosphoglycerides differed considerably from each other in the homogenate as well as in the subcellular fractions. Significant differences in the C-2 fatty acyl profiles also were observed in diacyl and 1-alkenyl, 2-acyl ethanolamine phosphoglycerides. Such differences suggest there is no direct metabolic relationship between the diacyl glycerophosphocholine (-ethanolamine) and its plasmalogen analog.

Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity

Functional and physical heterogeneity of polyclonal IgE has been reported. Extremely low serum concentrations of IgE have limited the study of these important differences. We have purified polyclonal dog IgE and developed polyclonal and monoclonal (mAb C2) anti-dog IgE antibodies. In this study chromatofocusing of dog IgE revealed two biologically active IgE fractions: IgE1 eluted at pH 5.0, and IgE2 eluted at pH 4.7. The two IgE subforms (IgEs) exhibited typical IgE characteristics: positive in the 48-hour passive cutaneous anaphylaxis response, heat-labile, identical molecular weight, and reactive to polyclonal anti-dog IgE. However, the two IgEs were found to be significantly heterogeneous. IgE1 bound to protein A and did not react with mAb C2 in ELISA and isoelectric focusing-immunoblotting, whereas IgE2 did not bind to protein A and reacted with mAb C2. Further, in sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting, IgE2, but not IgE1, reacted with seven well-defined mAb anti-human IgE antibodies and an mAb anti-mouse IgE antibody, even though both IgE1 and IgE2 reacted with polyclonal anti-human and anti-mouse IgE. Neuraminidase or endoglycosidase treatment did not abolish the differential antigenicity and charge of IgE1 and IgE2, although the antigenicity of IgE2 was significantly reduced after incubation with endoglycosidase. These data suggest that carbohydrate moieties are not involved in the observed differences in antigenicity and charge and that the two IgE molecules represent distinct isotypes. In studies with seven purified IgE fractions obtained from different ragweed-allergic dogs, the distribution of ragweed IgE2 varied 200-fold, whereas ragweed total IgE levels varied only fourfold. This raises the possibility of a relationship between different IgEs and the allergic response.

Plasmalogenase in hamster heart

In this study, the presence of plasmalogenase for the hydrolysis of the alk-1-enyl bond at the C-1 position of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) in the hamster heart was examined. A new spectrophotometric assay was developed for this study, in which the aldehyde released by the hydrolysis of the plamalogenase was oxidized to carboxylic acid by the action of aldehyde dehydrogenase, with the production of the molar equivalent of NADH. The results obtained from the spectrophotometric assay were comparable to those obtained by determining the rate of ethanolamine plasmalogens utilized during the reaction. However, the sensitivity of the spectrophotometric assay for plasmalogenase was shown to be 25-fold higher than with the methods described previously and enzyme activity could be detected with 1 micrograms of microsomal protein. Hamster heart plasmalogenase activity was located exclusively in the microsomal fraction, and the enzyme displayed a pH optimum at 8.5. The enzyme showed no absolute requirement for divalent metallic cations.

The effect of fenofibrate treatment on endothelium-dependent relaxation induced by oxidative modified low density lipoprotein from hyperlipidemic patients

The objective of the research project was to investigate whether fenofibrate treatment may alter the biochemical content of the oxidized LDL and consequently its ability to impair the endothelium-dependent relaxation in hyperlipidemic patients. We hypothesized that fenofibrate treatment of hyperlipidemic patients may attenuate the ability of their oxidized LDL to impair the endothelium-dependent relaxation of the blood vessels as a consequence of fenofibrate-induced changes to the content and composition of lysoPC in the LDL molecule.Hyperlipidemic patients (Type IIb and Type IV) were recruited from the Lipid Clinic, HSC, Winnipeg, Canada, for this study. A blood sample was taken immediately after the recruitment, a second sample was taken after 6 weeks of dietary treatment, and a third sample was taken after 8 weeks of fenofibrate treatment. LDL was isolated from the plasma and oxidized by copper sulfate. Fenofibrate was shown to be highly effect in the reduction of total cholesterol, LDL cholesterol and triglycerides in these patients. Fenofibrate treatment also caused the attenuation of impairment of endothelium-dependent relaxation by the oxidized LDL from these patients. A slight reduction of lysophosphatidylcholine level was also found in the oxidized LDL of the fenofibrate treated patients, relative to LDL isolated after dietary treatment. In addition there were no changes in the fatty acid levels of the lysophosphatidylcholine isolated from LDL. Taken together, our results suggest that while the reduced lysophosphatidylcholine levels may contribute to the attenuated impairment of the endothelium-dependent relaxation of the aortic ring, other unidentified factors impacted by fenofibrate are likely to contribute to the attenuated effects.

Purification and Identification of Polyclonal IgE Antibodies from Ragweed-Sensitized Dog Sera

We have purified and characterized polyclonal dog IgE. Serum IgE was precipitated by (NH4)2SO4 and then purified by two different procedures. Ion exchange on DEAE-Sephacel, followed by HPLC using Tonen hydroxylapatite and then Protein G-Sepharose, produced a highly purified IgE fraction (No. 1) free of IgG, IgA and IgM as measured by ELISA, but recovery of IgE as measured by passive cutaneous anaphylaxis was low. Gel filtration on Sephacryl S-300, Con A-Sepharose and Protein G-Sepharose recovered 18% of initial IgE, 0.02% IgG, 0.4% IgM and 0.3% IgA. This IgE fraction (No. 2) was used to induce antibody production in rabbits. Western blot analysis was then performed for dog IgE fractions No. 1 and 2. Using the rabbit anti-dog IgE, a prominent IgE band with an apparent molecular mass of 226 kD was identified in fractions No. 1 and 2 subjected to nonreducing SDS-PAGE. This band also reacted with anti-human IgE, but not with anti-dog IgG or anti-dog IgA. Under reducing conditions the approximate molecular mass for the IgE & chain, estimated by Western blot using rabbit anti-dog IgE, was 73 kD, providing a molecular mass of 196 kD for dog IgE.

Ultrashort cationic lipopeptides and lipopeptoids: Evaluation and mechanistic insights against epithelial cancer cells

Peptides present an attractive scaffold for the development of new anticancer lead agents due to their accessibility and ease of modification. Synthetic ultrashort cationic lipopeptides, with four amino acids or less conjugated to a fatty acid, were developed to retain the biological activity of longer peptides in a smaller molecular size. Herein, we report the activity of amphiphilic lipotripeptides, lipotripeptoids and lipotetrapeptides against breast (MDA-MB-231, JIMT-1), prostate (DU145) and pancreas (MiaPaCa2) epithelial cancer cell lines. The lipotripeptide C16-KKK-NH2 and lipotetrapeptide C16-PCatPHexPHexPCat-NH2 were identified to possess anticancer activity. The latter lipotetrapeptide possess a short polyproline scaffold consisting of only two L-4R-aminoproline (PCat) and two L-4R-hexyloxyproline (PHex). However, all the prepared lipotripeptoids lack anticancer activity. The amphiphilic C16-PCatPHexPHexPCat-NH2 exhibited similar anticancer potency to the surfactant benzethonium chloride while superior activity was observed in comparison to myristylamine. Mechanistic studies revealed that the peptides do not lyse ovine erythrocytes nor epithelial cancer cells, thus ruling out necrosis as the mechanism of cell death. Surprisingly, the two lipopeptides exhibit different mechanisms of action that result in cancer cell death. The lipotripeptide C16-KKK-NH2 was found to induce caspase-mediated apoptosis while C16-PCatPHexPHexPCat-NH2 kills tumor cells independent of caspases.

Cytotoxic properties of d-gluco-, d-galacto- and d-manno-configured 2-amino-2-deoxy-glycerolipids against epithelial cancer cell lines and BT-474 breast cancer stem cells

Glycosylated antitumor ether lipids (GAELs) 6 and 7 containing a α- or β-D-gluco-configured 2-amino-2-deoxy (2-NH2-Glc) sugar moiety linked to a glycerolipid aglycone kill cancer cell lines via a non-apoptotic mechanism that could be exploited to kill cancer stem cells. To test this hypothesis and develop novel potent GAEL analogs, we synthesized GAELS which contain D-galacto- and D-manno-configured 2-amino-2-deoxy sugar moieties (2-NH2-Gal or 2-NH2-Man) and investigated their cytotoxicity against human epithelial cancer cell lines and cancer stem cells derived from BT-474 breast cancer cells. Within the class of D-galacto-configured GAELs, we prepared both O- and S-glycosidic linkages as well as their corresponding α- and β-anomers and screened against breast (BT-474, JIMT-1 and BT-549), pancreas (MiaPaCa2) and prostate cancer (DU145, PC3) cancer cell lines. The α-anomeric 2-NH2-Gal-based lipid 1 was the most active of all the compounds tested with CC50 values of 4.4-8 μM and is the most active GAEL synthesized to date. The β-anomer 2 was 4->5-fold less active than 1. Replacement of the α-O-glycosidic by an α-S-glycosidic linkage resulted in a 2-4-fold reduction in activity, while the β-S-glycolipid 4 was inactive. In comparison, α-configured 2-NH2-Man-based glycerolipid 5 displayed very little activity with CC50 > 30 μM. The effect of the most active GAELs, 1, 6, or 7, on cancer stem cell viability revealed that all three inhibited the formation of tumorspheres from BT-474 cancer stem cell lines, caused the disintegration of preformed tumorspheres and resulted in total loss of cell viability of the cancer stem cells at concentrations of 20 μM. In contrast, the related antitumor ether lipid gold standard, edelfosine that is in clinical development was much less effective in preventing tumorsphere formation and affecting the viability of the cancer stem cells. Taken together our study demonstrates that α-GAEL anomers are more potent than their corresponding β-anomers and that the nature of the CHO moiety as well as the glycosidic bond significantly affects activity. The study also showed that GAELs are effective in killing CSCs while the apoptosis-inducing edelfosine is not.