Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Regulation of phospholipid biosynthesis in {\it Saccharomyces cerevisiae\/} by CDP-diacylglycerol synthase

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS) while phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol-1-phosphate). In this study a gene (CDS1) encoding CDP-DAG synthase in S. cerevisiae was isolated and identified. The CDS1 gene encodes the majority, if not all, of the synthase activity, and is essential for cell growth. Overexpression of the CDS1 gene resulted in an elevation in the apparent initial rate of synthesis and also steady-state level of PI relative to PS in both wild type yeast and the cds1 mutant. Down-regulation of CDS1 expression resulted in an inositol excretion phenotype and an opposite effect on the above phospholipid synthesis in the cds1 mutant. This regulation of phospholipid biosynthesis is mediated by changes of the phospholipid biosynthetic enzymes via a mechanism independent of the expression of the INO2-OPI1 regulatory genes. Reduction in the level of CDP-DAG synthase activity resulted in an increase in PS synthase activity which followed a similar change in the CHO1/PSS (encodes PS synthase) mRNA level. INO1 (encodes inositol-1-phosphate synthase) mRNA also increased but only after CDP-DAG synthase activity fell below the wild type level. PI synthase activity followed the decrease of the CDP-DAG synthase activity, but there was no parallel change in the level of PIS1 mRNA. A G$\sp{305}$/A$\sp{305}$ point mutation within the CDS1 gene which causes the cdg1 phenotype was identified. A human cDNA clone encoding CDP-DAG synthase activity was characterized by complementation of the yeast cds1 null mutant. ^

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^