EMP2 is a novel therapeutic target for endometrial cancer stem cells.

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against cancer stem cells in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells.

Lipid peroxidation products in biological tissues.

Over the past twenty-years of lipid peroxidation research in this laboratory, considerable effort has gone into development of new methods, with emphasis on measurement of lipid-soluble fluorophores and the volatile hydrocarbons ethane and pentane. Application of these and other methods has been made to biological materials and living animals. Although the various methodologies used in lipid peroxidation research do not necessarily measure the same class of products, and although agreement of results is not always 100%, there is substantial documentation of good correlations between measurements; for example, of trace volatile hydrocarbons with thiobarbituric acid-reacting substances, of pentane production with dietary and/or tissue vitamin E content, and of pentane production with lipid-soluble fluorophores accumulated in spleen as a function of oxidant stress. Individual methodologies do have their inherent limitations. However, measurements of multiple products and their correlations have added significantly to the base of information on biological damage and protection by dietary antioxidants against nutritional and toxicological insults to tissues, cells, and macromolecules as a result of peroxidative and oxidative reactions.

Vitamin E, diethylmaleate and bromotrichloromethane interactions in oxidative damage in vivo.

In vivo interactions of vitamin E with diethylmaleate (DEM) and bromotrichloromethane (CBrCl3) were examined in rats fed a diet either without vitamin E or supplemented with 30 IU dl-alpha-tocopheryl acetate/kg. Groups of rats within each dietary group were given two injections 30 min apart. One group received two injections of the mineral oil carrier. The other groups were injected with either DEM and mineral oil, mineral oil and CBrCl3, or DEM and CBrCl3. The rats were killed 10 min after the second injection. Measurements were made of hepatic GSH, thiobarbituric acid-reactive substances (TBARS) as a lipid peroxidation index, and 11 enzymes as potential markers of oxidant damage. Special focus was placed on reactive cysteine-containing aldehyde dehydrogenase (ALDH). Although dietary vitamin E protected ALDH, the enzyme was highly susceptible to oxidant damage. ALDH activity was correlated with GSH (r = 0.83, p less than 0.001) and there was an inverse relationship between the logarithmic values of ALDH activity and TBARS (r = 0.78, p less than 0.001). Similar results were observed for a number of other enzymes when GSH depletion preceded oxidant treatment. Two-way analysis of variance revealed significant effects of vitamin E and of injection treatments on hepatic GSH. There was a significant interaction between vitamin E and the injection treatments on the activities of five enzymes. The results suggested that vitamin E and GSH functioned together to protect sensitive enzymes against oxidant stress. The sensitive enzymes may be useful markers of hepatic damage in vivo.

Effect of dietary vitamin E and Santoquin on regenerating rat liver.

The influence of dietary vitamin E and Santoquin on lipid peroxidation and liver regeneration in partially-hepatectomized rats was studied. Rats were fed either a basal 10% tocopherol-stripped corn oil diet, the basal diet plus 40 mg dl-alpha-tocopheryl acetate/kg, or the basal diet plus 2 g Santoquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline)/kg. After 6 weeks, rats fed the antioxidant-deficient diet produced more of the lipid peroxidation product, pentane, than did the rats fed antioxidants. Partial hepatectomy was performed after six and one-half weeks or ten weeks of feeding the diets. At 3 and 6 days after surgery, pentane production was significantly elevated over pre-surgery levels in rats fed the antioxidant-deficient or vitamin E-supplemented diets, but not in rats fed the Santoquin-supplemented diet. Six days after surgery, there were fewer thiobarbituric acid reactants in regenerating liver of Santoquin-fed rats than of vitamin-E fed rats or antioxidant-deficient rats. There was no increase in the 6-day level of thiobarbituric acid reactants over the 3-day level in livers of rats fed Santoquin, while there was an increase in livers of the antioxidant-deficient and vitamin E-supplemented rats. Liver sulfhydryl levels were higher at 3 and 6 days post surgery in the Santoquin-fed rats than in the antioxidant-deficient or vitamin E-supplemented rats. Plasma gamma-glutamyl-transpeptidase activity was not different among the groups of rats. Between the third and sixth day following surgery, liver regeneration was significantly stimulated in Santoquin-fed, but not vitamin E-fed rats. After 11 days, a stimulatory, but not statistically significant, effect of vitamin E was found. Although DNA content of liver was higher at 6 days than at 3 days post surgery, it was not different among the dietary groups, indicating that cell proliferation rather than hypertrophy had occurred. Partial hepatectomy could have altered the ability of the liver to metabolize pentane, thus explaining part of the increased production of pentane. However, the results obtained support the interpretation that elevated levels of dietary antioxidants can be beneficial in terms of reduced lipid peroxidation and increased rates of liver regeneration following liver surgery.

Mercury, silver, and gold inhibition of selenium-accelerated cysteine oxidation.

In vivo, cysteine in proteins or glutathione is the major amino acid involved in sulfhydryl oxidation-reduction reactions. An in vitro model of cysteine oxidation accelerated by selenium compounds was used to study the interaction of selenocystine and sodium selenite with metal ions. The interaction of metal ions with selenium compounds inhibited cysteine oxidation. The ionic forms of three toxic soft-acid metals, mercury, silver, and gold, were the most effective inhibitors. The antiarthritic gold drugs, aurothiomalate and aurothioglucose, were of particular interest as they inhibit the activity of selenium-glutathione peroxidase. The effect of gold ligands on gold(I) inhibition of selenocystine-accelerated cysteine oxidation was tested. Sodium cyanide partially reversed inhibition and potassium iodide had no effect. Inhibition of selenium-accelerated oxidation-reduction reactions by soft-acid metal ions may be of biological relevance during toxicities or during antiarthritic gold therapy.

Effect of chronic aurothioglucose treatment of rats on kidney catalase and cytochromes.

Catalase activity and cytochrome content were measured in kidneys of Fisher 344 rats injected with aurothioglucose (ATG) either daily for 3 days or 5 days a week for up to 8 wk. Catalase activity was decreased 39%, 59%, and 48% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. Microsomal cytochrome P-450 levels decreased 71%, 86%, and 80% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. In contrast, cytochrome b5 was significantly increased at 3 days and 2 wk, but not at 8 wk. Microsomal heme contents decreased 44% (p less than 0.001), 34% (p less than 0.001), and 22% (p greater than 0.05) at 3 days, 2 wk, and 8 wk, respectively. The content of mitochondrial cytochromes aa3, b, c1, and c were not affected after 8 wk of ATG treatment. In vitro inhibition of the heme-containing enzyme delta-aminolevulinic acid dehydratase by ATG was reversible in the presence of physiological concentrations of small thiols. Although the activity of this enzyme in kidneys of ATG-treated rats was not measured, its significant inhibition in vivo by ATG appears unlikely. This study demonstrates that there were differential effects of gold on the various cytochromes and that changes in catalase activity paralleled changes in cytochrome P-450 and heme contents in the kidneys of ATG-treated rats. The findings are relevant to nephrotoxicity during chrysotherapy.

A systems model for food purchasing.

The approach taken in this paper of isolating seasonal characteristics of products and then using that information to determine when those products should be ordered to minimize the system's purchasing costs appears to be valid. A drawback to using this approach, however, is acquiring appropriate data. The authors used data which they believed to be appropriate for thier market purchasing environment but which are not necessarily valid for other locations and/or environments. The prospective user of this model must find and obtain data suitable for the environment and isolate those products which appear seasonal. Only a small percentage of the items used possess any seasonal properties. There is also the obvious problem of using past data to determine future outcomes. Past price patterns do not necessarily continue into the future, although they should be at least indications, barring unusual farm and/or market situations. As with all decision modeling approaches, however, this model should be followed with caution. While the approach is valid, the procedure is no substitute for knowledgeable management of food service functions. Results from the model should provide additional input to guide food purchasing decisions but should not be used unequivocably. When property used, however, this procedure will allow food buyers to come closer to the goal of optimally purchasing food products from a systems point of view then is currently possible.

Effect of aurothioglucose on glutathione and glutathione-metabolizing and related enzymes in rat liver and kidney.

The antirheumatic drug aurothioglucose is an inhibitor of the selenoenzyme GSH peroxidase. During chrysotherapy, the decreased levels of erythrocyte GSH and serum sulfhydryls of rheumatoid arthritis patients are normalized concomitant with clinical efficacy. This investigation examined the in vivo and in vitro effect of gold(I) as aurothioglucose on enzymes related to the GSH redox cycle or metabolism. The enzymes measured were GSH peroxidase, GSSG reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase, GSH S-transferase, GSH thiotransferase, glucose-6-phosphate dehydrogenase, superoxide dismutase and catalase. Rats were injected with 30 mumol aurothioglucose/kg body wt. daily for 7 days by intramuscular injection. GSH levels in aurothioglucose-treated rats were 68% higher in erythrocytes (P less than 0.005) and 45% higher in kidney (P less than 0.001) than in control rats. Treatment with aurothioglucose did not elevate plasma or liver GSH. The enzyme activities that were changed by aurothioglucose treatment were GSH peroxidase in kidney (41% decreased, P = 0.005) and liver (13% decreased, P less than 0.05), gamma-glutamyl transpeptidase in kidney (15% decreased, P less than 0.05), and catalase in kidney (58% decreased, P less than 0.001). Kidney glucose-6-phosphate dehydrogenase activity was increased 50% (P less than 0.005) and GSH S-transferase was increased 72% (P less than 0.001). In vitro the only liver enzymes inhibited more than 50% by concentrations of less than 50 microM aurothioglucose were GSH peroxidase (50% inhibited by 25 microM aurothioglucose) and GSH thiotransferase (50% inhibited by 5 microM aurothioglucose). Studies of in vitro enzyme inhibition by aurothioglucose could not be used to predict decreased enzyme activities in vivo. Although decreased activities of two major enzymes that utilize GSH, GSH peroxidase and gamma-glutamyl transpeptidase, coincided with elevated GSH in kidneys of aurothioglucose-treated rats, a direct cause and effect relationship remains speculative.

Respiratory pentane: a measure of in vivo lipid peroxidation applied to rats fed diets varying in polyunsaturated fats, vitamin E, and selenium and exposed to nitrogen dioxide.

Pentane is one decomposition product of omega 6-unsaturated lipid hydroperoxides. The measurement of respiratory pentane is one of the most sensitive in vivo tests of lipid peroxidation. This measurement was applied to test the ability of a low level, short-term exposure of rats of nitrogen dioxide to induce lipid peroxidation. When exposed to 4.48 ppm nitrogen dioxide for 60 min, no increase in pentane production was detected. For 10 weeks prior to exposure, the rats were fed Torula yeast-based diets with 10% stripped lard or 10% stripped corn oil. The ratios of basal pentane production by rats fed the following antioxidants were for corn oil- and lard-fed rats, respectively: 40 I.U. vitamin E/kg and 0 selenium: 0 vitamin E and 0.1 ppm selenium: 0 vitamin E and 0 selenium, 1:2.2:5.8 and 1:1.9:3.5. Pentane production was significantly (P<0.05) greater by corn oil-fed rats than by lard-fed rats only when both vitamin E and selenium were absent from the diet.

Embryonic development of an endoparasitoid, Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media.

Embryos of the parasitoid Microplitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical host Lymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace's, and ExCell 400. The developmental response of M. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace's media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ band stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace's media had a significant effect on eggs attaining germ band stage compared with the Grace's control medium. However, Grace's medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace's control medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Volatile hydrocarbon and carbonyl products of lipid peroxidation: a comparison of pentane, ethane, hexanal, and acetone as in vivo indices.

A study was undertaken to determine whether respiratory hexanal and acetone as well as pentane and ethane could be measured as potential indices of lipid peroxidation in vivo. The tests of induction of lipid peroxidation in rats included injection of iron-dextran and the vitamin E deficiency status. Injection of 460 mg of iron/100 g body wt over a 28-day period increased pentane and ethane production 4- and 6-fold, respectively. Hexanal production was increased 7-fold after injection of 60 mg of iron/100 g body wt, and then it fell back to the preinjection level in spite of continued injection of iron-dextran. Acetone production was lower in iron-injected rats than in controls, and it was ca. 10-fold higher in fasted vitamin E-deficient rats than in vitamin E-supplemented rats, being ca 48 and 5 nmol/100 g/min, respectively. It was observed that halomethane injection did not increase hexanal production, while acetone and pentane production were increased. Pentane and hexanal, but not acetone, were found to arise from decomposition of linoleic acid hydroperoxide in vitro. It was concluded that hydrocarbon gases are better indices of lipid peroxidation than hexanal, which is enzymatically metabolized, and acetone, the production of which is dominated by factors such as altered carbohydrate metabolism.

Retinol inhibition of some proteolytic enzymes.

Automated analyses were used to determine the effect of retinol on the activity of the following proteolytic enzymes: ficin (EC 3.4.4.12), bromelain (EC 3.4.4. 24), trypsin (EC 3.4.4.4.), chymotrypsin A (EC 3.4.4.5), papain (EC 3.4.4.10), clostridiopeptidase A (EC 3.4.4.19), pepsin (EC 3.4.4.1), cathepsin D (EC 3.4.4. 23) from rat-liver and rat-kidney lysosomes and the nonspecific proteolytic enzyme, pronase. Of these proteolytic enzymes only ficin, bromelain, and rat-kidney lysosomal cathepsin D were inhibited significantly by 1x10(-4) M retinol.Some nonproteolytic enzymes not inhibited by retinol were acid phosphatase (EC 3.1.3.2), beta-acetylglucosaminidase (EC 3.2.1.30), arylsulfatase (EC 3.1.6.1), and pyruvate kinase (EC 2.7.1.40). The inhibition of cathepsin D varied with the substrate used, being greater with hemoglobin than with ovalbumin or bovine serum albumin. Carotene and retinol inhibited ficin and cathepsin D to similar extents. Retinol inhibition of ficin was partially reversible. These studies of proteolytic enzyme inhibition by retinol serve as a simple model for studying retinol-protein interactions in vitro.

Effects of dietary vitamin E, selenium, and polyunsaturated fats on in vivo lipid peroxidation in the rat as measured by pentane production.

Starting at 21 days of age, groups of six rats each were fed a basal Torula yeast diet supplemented with 0.4% L-methionine and varying amounts of vitamin E as dl-alpha tocopherol acetate, selenium as sodium selenite, and with either 10% stripped corn oil, stripped lard, or coconut oil. By 7 wk, pentane production by rats fed a corn oil diet deficient in both vitamin E and selenium was twice that by rats fed 0.1 or 1 mg of selenium per kg of the same basal diet. Blood glutathione peroxidase activity after 7 wk was proportional to the logarithm of dietary selenium. Groups of rats fed the vitamin E- and selenium-deficient diets with lard or coconut oil had one-half the pentane production of rats fed the vitamin E- and selenium-deficient corn oil diets. The plasma level of linoleic plus arachidonic acid was 1.8 time greater on a wt % basis in rats fed corn oil than in rats fed lard or coconut oil as the fat source. Pentane production by rats fed 40 i.u. dl-alpha tocopherol acetate per kg of the selenium-deficient corn oil diet was one-sixth of that by rats fed the same diet without vitamin E; the plasma of the rats fed the vitamin E-supplemented corn oil diet had a level of vitamin E that was about six times greater than that of the rats fed the vitamin E-deficient corn oil diet.

Effect of dietary vitamin E on expiration of pentane and ethane by the rat.

An analytical method for the measurement of hydrocarbon gases in the breath of rats is described. The method was used to follow the expiration in rat breath of in vivo formed scission products of hydroperoxides. The major products are pentane from the linoleic acid family and ethane from the linolenic acid family. Rats were fed 0, 11 or 40 i.u. vitamin E acetate/kg diet for 7 wk starting at age 21 days. Data obtained by gas chromatographic analysis of breath samples were analyzed by the Mann-Whitney nonparametric U-test. This statistical analysis showed that pentane evolved by the group of rats not supplemented with vitamin E was significantly higher during the period 1-7 wk than that evolved by either of the two supplemented groups of rats. Ethane from the nonsupplemented group was significantly higher than that from the group supplemented with 40 i.u. vitamin E/kg of diet by 5 wk, and significantly high than both supplemented groups by 6 wk. By 7 wk, pentane production was tenfold greater in the non-supplemented group, and ethane was about twofold greater. There was no significant difference between the groups supplemented with 11 and 40 i.u. vitamin E/kg diet for either ethane or pentane. This new technique, which measures scission products from in vivo lipid peroxidation, promises to be useful for application to many experimental areas where lipid peroxidation is expected or known to occur.

Effect of antioxidants on lipid peroxidation in iron-loaded rats.

Indirect evidence has suggested that lipid peroxidation is associated with iron overload in vivo. As a measure of lipid peroxidation, pentane expired in the breath of rats loaded with an accumulated dose of either 100 mg or 186-200 mg of iron injected intraperitoneally as iron dextran was measured over a 7 to 8 week period, and the effect on pentane production of feeding antioxidant-supplemented diets was determined. By the seventh week of feeding the diets, rats fed 0.3% L-ascorbic acid produced 17% less (P = 0.03) pentane than did rats fed the basal antioxidant-deficient diet, whereas rats fed 0.004% dl-alpha-tocopherol acetate produced 92% less (P less than 0.001). After being fed the basal diet for 7 weeks, iron-loaded rats produced 76 +/- 9 pmol pentane/100 g body wt/min. When synthetic antioxidants were added to the diet at a concentration of 0.25%, the order of effectiveness in decreasing pentane production after 1 week was: N,N'-diphenyl-p-phenylenediamine greater than ethoxyquin greater than butylated hydroxyanisole greater than butylated hydroxytoluene greater than propyl gallate approximately equal to no antioxidant. After removal of either ethoxyquin or N,N'-diphenyl-p-phenylenediamine from the diets for 1 week, pentane production increased to a high level. The total amount of lipid soluble fluorophores in individual spleens of rats fed N,N'-diphenyl-p-phenylenediamine, ethoxyquin, dl-alpha- tocopherol acetate, ascorbic acid and no antioxidant were correlated significantly with the corresponding total integrated amount of pentane produced by the individual rats over the 7 to 8 week period. This study has provided some of the most direct evidence to date that lipid peroxidation is associated with iron overload in vivo.

Are some major in vivo effects of gold related to microenvironments of decreased selenium?

Gold interacts with selenium in vivo, and the normal distribution of selenium among tissues and subcellular compartments changes. Literature evidence shows that many of the effects of gold compounds on the polymorphonuclear neutrophil, macrophage, and lymphocyte cellular components of the immune system are similar to effects observed in these cellular components in selenium-deficient animals. Affected by these two metals are immune functions related to phagocytic cell migration, phagocytosis, microbial killing, lymphocyte mitogenesis/DNA synthesis, arachidonic acid metabolism/prostaglandin synthesis, and immunoglobulin production. The interaction of gold with selenium in vivo may be responsible for some of the multiparameter-based actions of gold compounds used in the treatment of inflammatory diseases such as rheumatoid arthritis. One mechanism by which gold exerts its clinical effects may be related to its interaction with selenium to produce, in specific microenvironments, decreased levels of this essential trace element.

Can serum bilirubin be an index of in vivo oxidative stress?

Bilirubin has been suggested as a physiological antioxidant, and recent studies suggest that its synthesis is induced in response to oxidative stress. Numerous reports in the literature show increases in serum bilirubin when using halogenated hydrocarbons as oxidative stress inducers. Analogously, these increases should also be expected for other inducers. On the other hand, bilirubin is destroyed by the same molecules that induce its production. The measurement of bilirubin may be a useful index of in vivo oxidative stress, although no big differences in bilirubin levels should be expected.

Effect of vitamin E and ozone on pentane and ethane expired by rats.

Pentane and ethane, which arise during lipid peroxidation in vivo, were measured by gas chromatography in breath samples of rats fed for 8 weeks a vitamin E-deficient diet to which had been added 0, 11, or 40 IU vitamin E acetate per kg. Further lipid peroxidation was induced by exposure of individual rats to 1 ppm ozone for 60 min. Nonparametric statistical analysis of the data for pentane expired before exposure of rats to ozone gave alpha values (alpha = 2P) of 0.006 when the O vitamin E group was compared with either of the vitamin E-supplemented groups. For ethane, comparison of the O vitamin E group with the groups supplemented with 11 and 40 IU vitamin E/kg of diet were 0.0294 and 0.0080, respectively. Alpha values less than .05 were considered significant. After a 60-min exposure of rats to 1 ppm ozone, the paired t-test showed pentane to be significantly (P less than .005) increased in only the rats fed the vitamin E-deficient diet.