Study of Glutathione S-transferase-P1 in cancer blood plasma after extraction by affinity magnetic nanoparticles and monitoring by MALDI-TOF, IM-Q-TOF and LC-ESI-Q-TOF MS.

Glutathione S-transferase P1 (GST-P1) is considered as a detoxification enzyme and can be upregulated in several cancers. Therefore, qualification and/or quantification of GST-P1 in biological fluids can be noteworthy in cancer diagnostic and/or prognostic methods. Whereas costly immunoassays methods are routinely used for clinical analysis, long analysis time per sample is still considered as their disadvantages. To create a fast, efficient, and economical GST-P1 qualification and/or quantification technique, we developed an affinity magnetic nanoparticle-MS method. In proposed method there is no need for any pretreatment for reducing the complexity of sample and depletion of high abundant proteins that are used in routinely immunoassays methods. After enrichment of GST-P1 from blood plasma samples by affinity magnetic nanoparticle (without any pretreatment), the final eluent was analyzed using MALDI-TOF, IM-Q-TOF and LC-ESI-Q-TOF MS. For the first time this study demonstrates the suitability of affinity magnetic nanoparticle-MS method for qualification/quantification of GST-P1 from acute lymphoblastic leukemia blood plasma samples with the limit-of-detection 0.0094 ppm in less than 5 h. Our finding showed that in these blood plasma samples the level of GST-P1 can be up to six times more than healthy children.

Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine.

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC50 value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells.

The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin.

Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71% and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver-Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 µM and K i (CDNB) as 0.26 ± 0.03 µM.

Evaluation of the in vitro inhibitory impact of hypericin on placental glutathione S-transferase pi.

St John's Wort (SJW) extracts are herbal products which are available without prescription in most countries and widely used in the treatment of mild to moderate depression. Since it is a herbal product and available without prescription, use of SJW is common among pregnant and/or lactating woman. The principal of the study was to clarify the effects of hypericin, one of the components of SJW, on glutathione S-transferase-pi (GST-pi) purified from human placenta. The K (m) values of GST-pi were 0.21 ± 0.03 mM for glutathione (GSH) and 2.29 ± 0.54 mM for 1-chloro-2,4-dinitrobenzene (CDNB). At fixed [GSH], the V (m) value calculated was about 3 times higher than the conditions in which [CDNB] was fixed; 201 ± 30 U/mg protein versus 74 ± 3 U/mg protein. At constant substrate concentrations (1 mM), an average IC (50) value of 0.70 ± 0.02 µM was obtained. Hypericin inhibited GST-pi competitively with respect to both substrates. When GSH was the varied substrate a K (i) value of 0.31 ± 0.05 µM was found; when CDNB was the varied substrate, a K (i) value of 0.85 ± 0.02 µM was obtained. On the basis of these data considering transplacental transfer of hypericin and immature hepatic clearance of the baby, using this herbal product may cause abnormalites due to the inhibition of one of the most important placental detoxification enzymes, GST-pi.

The inhibition characteristics of human placental glutathione S-transferase-π by tricyclic antidepressants: amitriptyline and clomipramine.

Tricyclic antidepressants (TCAs) are the non-selective amine re-uptake inhibitors, well absorbed from small intestine, cross the blood-brain barrier, distributed in the brain, and are bound to glutathione S-transferase-π (GST-π). TCAs can pass through placenta, accumulate in utero baby, and cause congenital malformations. Thus, the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of GST-π with amitriptyline and clomipramine was investigated. The K (m) values for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) were found to be 0.16 ± 0.04 and 3.60 ± 1.67 mM, respectively. The V (m) values were varying according to the fixed substrate; [CDNB] fixed, 53 ± 3 and [GSH] fixed 182 ± 63 U/mg protein. At variable [GSH] and variable [CDNB], the k (cat) values of 7.0 × 10(6) and 1.42 × 10(7) s(-1) and the k (cat)/K (m) values of 4.38 × 10(10) and 3.94 × 10(9 )M(-1 )s(-1) were obtained, respectively. At fixed [CDNB] and variable [GSH], amitriptyline (K (s) = 0.16 ± 0.03 mM; α = 2.08; and K (i) = 1.75 ± 0.37 mM) and clomipramine (K (s) = 0.24 ± 0.05 mM; α = 1.57; and K (i) = 3.90 ± 2.26 mM) showed linear mixed-type inhibition whereas when the varied substrate is CDNB, amitriptyline (K (i) = 4.90 ± 0.68 mM) and clomipramine (K (i) = 3.37 ± 0.39 mM) inhibition were noncompetitive. The inhibition of GST-π by TCAs means the destruction of its protective role against toxic electrophiles. The effect of antidepressants on fetus will be much severe, thus, the antidepressant therapy of pregnant women should be done with caution.

Purification and biochemical characterization of glutathione S-transferase from Down syndrome and normal children erythrocytes: a comparative study.

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was determined in ten DS and ten healthy children matched for age (3-10 years). DS group exhibited significantly lower GST value (2.7 units/gHb) as compared to controls (6.6 units/gHb) (40.9%). GST activity was significantly decreased to 40.9% in the DS group as compared to controls. Also GSH concentration was significantly decreased to 60.6% in the DS group compared to the controls. Glutathione transferase was purified from erythrocytes of normal and DS pooled blood samples by affinity chromatography with specific activity of 23.7% and 7.9%, respectively. The effect of freezing and thawing, storage time of freezing and GSH concentration on the stability of the enzyme were examined. Normal GST exhibited a pH optimum at pH 7 followed by sharp decrease, however DS GST exhibited pH optimum between pH 7.5 and 8. The Km values for 1-chloro-2,4-dinitrobenzene (CDNB) and GSH were 0.205 mM and 0.786 mM, respectively, for normal GST, and 0.318 mM and 1.307 mM, respectively for DS GST. The activation energy (Ea) was calculated to be 2.25 and 4.25 cal/mol for normal GST and 3.8 cal/mol for DS GST. Normal and DS GST were inhibited by the same inhibitors (hematin, bromosulfophthalein and cibacron blue), but with different degree. On kinetic basis, the individuals with lower overall GST activity and slight differences in some kinetic characters are at greater risk from xenobiotic contamination as compared to those with higher overall GST activity observed in normal individuals.

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1.

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 µM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 µM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 µM. The IC(50) value of 1 was 6.8 µM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K(i) values of 8.8 and 0.21 µM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.

Human ocular carotenoid-binding proteins.

Two dietary carotenoids, lutein and zeaxanthin, are specifically delivered to the human macula at the highest concentration anywhere in the body. Whenever a tissue exhibits highly selective uptake of a compound, it is likely that one or more specific binding proteins are involved in the process. Over the past decade, our laboratory has identified and characterized several carotenoid-binding proteins from human retina including a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein, a member of the steroidogenic acute regulatory domain (StARD) family as a lutein-binding protein, and tubulin as a less specific, but higher capacity site for carotenoid deposition. In this article, we review the purification and characterization of these carotenoid-binding proteins, and we relate these ocular carotenoid-binding proteins to the transport and uptake role of serum lipoproteins and scavenger receptor proteins in a proposed pathway for macular pigment carotenoid delivery to the human retina.

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system.

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain.

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin--derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine--derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.