Inhibition of Glutathione Synthesis Augments Lysis of Murine Tumor Cells by Sulhydryl-reactive Antineoplastics

A B S T R A C T GSH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of y-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6-to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815 cells either during or immediately after a 1-h pulse with 10 ug/ml of vernolepin increased cytolysis from <3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU] INTRODUCTION Glutathione, the most abundant nonprotein sulfhydryl of mammalian cells, has been shown to play a critical role in cellular defense against a variety of injurious agents (1-3). We have previously studied the role of the GSH redox cycle in tumor cell defense against oxidative injury. In those studies, interference with the GSH redox cycle augmented in vitro cytolysis of tumor cells by macrophages and granulocytes as well as by a model H202-delivery system (glucose oxidase plus glucose) (4, 5). Similar manipulations enhanced the antitumor activity of H202 in vivo (6). GSH also plays a role in protection against toxic electrophiles by thioether formation. In contrast to the cyclic oxidationreduction of GSH during antioxidant defense, restoration of GSH content after detoxification of electrophiles is dependent upon its resynthesis. In this report, we consider the role of GSH and its synthesis in tumor cell defense against sulfhydryl-reactive antineoplastics, in particular, the sesquiterpene lactones (SL) We studied four SL that are active in vitro (vernolepin, helenalin, elephantopin, and eriofertopin, Biochemical assays. Total cellular glutathione (GSH plus GSSG) was measured by a minor modification of the method of Tietze as previously described (5, 28). Total glutathione is expressed as nanomoles of the tripeptide per milligram of cell protein and is referred to as GSH in this report. Extracts of cells treated with compounds that we found to cause GSH depletion were mixed with known amounts of GSH and then assayed to rule out the presence of an inhibitor of the Tietze assay, which might have accounted for the observed loss of GSH. The activity of glucose oxidase was measured with the scopoletin assay for H202 as described (29). Glutathione reductase was assayed by the method of Roos et al. (30). Protein content was determined by the method of Lowry et al. (31) using bovine serum albumin as the standard. Inhibition of glutathione synthesis with BSO. We have previously reported that with these tumors a maximal rate of GSH depletion, and thus maximal inhibition of synthesis, was achieved with 0.2 mM BSO (5). The rate of GSH depletion in the presence of BSO reflects GSH catabolism, efflux, and dilution by cell division in the absence of synthesis. BSO stock solution (20 mM in H20) was stored at 0°C for up to 3 wk before use. Cytolysis assays. Cells were labeled with Na251CrO4 as described (27). Cytolysis was studied in three experimental settings: (a) 4 X 104 labeled cells were incubated with various concentrations of lytic agent or vehicle alone (0.1-1% dimethyl sulfoxide) in the presence or absence of BSO (0.2 mM) in 0.22 ml of MEM, 5% horse serum at 37°C in 5% C02, 95% air for 18 h. (b) Cells (1.5 X 106/ml in MEM, 5% horse serum) were incubated with various concentrations of vernolepin or vehicle alone (0.1% dimethyl sulfoxide) plus Na251CrO4 for 1 h, washed extensively (four centrifugations), GSH At the indicated times (18 h for a and b, 5 h for c), supernatant (0.1 ml for a and c, 0.5 ml for b) was removed after centrifugation for gamma counting and the percent specific release was calculated as described RESULTS Depletion of tumor cell GSH by SL. The possible interaction of cellular GSH with SL was first evaluated. Incubation of P815 mastocytoma cells with vernolepin, helenalin, elephantopin, or eriofertopin for 1 h resulted in 70-97% depletion of GSH Effect of BSO on the lysis of tumor cells by SL. We evaluated the sensitivity of 51Cr-labeled tumor cells to lysis by an 18-h coincubation with SL. The synergistic interaction between BSO and vernolepin was further analyzed by considering the kinetics of onset and reversal of their effects. For this, exposure of cells to vernolepin was limited to a 1-h pulse, followed by an 18-h incubation in its absence, at which time lysis was determined

Induction of Cytotoxic Oxidative Stress by d-Alanine in Brain Tumor Cells Expressing Rhodotorula gracilis d-Amino Acid Oxidase: A Cancer Gene Therapy Strategy

Overview summary Gene-directed enzyme prodrug therapy (GDEPT) is an antineoplastic treatment strategy designed to overcome the systemic toxicity of chemotherapy by specifically expressing a foreign enzyme in malignant cells that converts a nontoxic prodrug into a cytotoxic metabolite. The relative inefficiency of current in situ gene transfer methodology suggests that enzyme/prodrug combinations that produce membrane permeable metabolites will elicit a more favorable therapeutic response. Ideally, the agent produced by the transduced cell “factories” would be cytotoxic toward both proliferating and quiescent cells. We describe a novel GDEPT approach using d-amino acid oxidase from the red yeast Rhodotorula gracilis and d-alanine as a substrate that generates hydrogen peroxide, a reactive metabolite of oxygen that has both these characteristics. We also demonstrate the ability to sensitize tumor cells to this GDEPT protocol by manipulating cellular antioxidant pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63220/1/hum.1998.9.2-185.pd

Towards comprehensive syntactic and semantic annotations of the clinical narrative

Objective: To create annotated clinical narratives with layers of syntactic and semantic labels to facilitate advances in clinical natural language processing (NLP). To develop NLP algorithms and open source components. Methods: Manual annotation of a clinical narrative corpus of 127 606 tokens following the Treebank schema for syntactic information, PropBank schema for predicate-argument structures, and the Unified Medical Language System (UMLS) schema for semantic information. NLP components were developed. Results: The final corpus consists of 13 091 sentences containing 1772 distinct predicate lemmas. Of the 766 newly created PropBank frames, 74 are verbs. There are 28 539 named entity (NE) annotations spread over 15 UMLS semantic groups, one UMLS semantic type, and the Person semantic category. The most frequent annotations belong to the UMLS semantic groups of Procedures (15.71%), Disorders (14.74%), Concepts and Ideas (15.10%), Anatomy (12.80%), Chemicals and Drugs (7.49%), and the UMLS semantic type of Sign or Symptom (12.46%). Inter-annotator agreement results: Treebank (0.926), PropBank (0.891–0.931), NE (0.697–0.750). The part-of-speech tagger, constituency parser, dependency parser, and semantic role labeler are built from the corpus and released open source. A significant limitation uncovered by this project is the need for the NLP community to develop a widely agreed-upon schema for the annotation of clinical concepts and their relations. Conclusions: This project takes a foundational step towards bringing the field of clinical NLP up to par with NLP in the general domain. The corpus creation and NLP components provide a resource for research and application development that would have been previously impossible

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress induces constitutive calcium entry by tacking glutathiones onto the Orai CRAC channel activator STIM1

Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.

GSH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of gamma-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6- to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815cells either during or immediately after a 1-h pulse with 10 micrograms/ml of vernolepin increased cytolysis from less than 3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU]). Of these, only BCNU depleted cellular GSH. Lysis by jatrophone, another GSH-depleting antitumor agent, was increased 21-fold by BSO. Since prolonged incubation with BSO alone results in near-complete GSH depletion without loss of cell viability, SL-mediated cytolysis is probably not a result of GSH depletion. We have demonstrated, however, a critical role for GSH synthetic capacity as a determinant of tumor cell susceptibility to cytolysis by SL. GSH also plays an important role in cellular defense against oxidative injury. Vernolepin, acting as a GSH-depleting agent, markedly sensitized tumor cells to lysis by H2O2 (greater than 6.5-fold increase with 20 micrograms/ml of vernolepin). These findings suggest the possibility that the coordinated deployment of sulfhydryl-reactive antitumor agents, BSO, and oxidative injury might constitute an effective chemotherapeutic strategy

Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.

A B S T R A C T GSH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of y-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6-to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815 cells either during or immediately after a 1-h pulse with 10 ug/ml of vernolepin increased cytolysis from <3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU] INTRODUCTION Glutathione, the most abundant nonprotein sulfhydryl of mammalian cells, has been shown to play a critical role in cellular defense against a variety of injurious agents (1-3). We have previously studied the role of the GSH redox cycle in tumor cell defense against oxidative injury. In those studies, interference with the GSH redox cycle augmented in vitro cytolysis of tumor cells by macrophages and granulocytes as well as by a model H202-delivery system (glucose oxidase plus glucose) (4, 5). Similar manipulations enhanced the antitumor activity of H202 in vivo (6). GSH also plays a role in protection against toxic electrophiles by thioether formation. In contrast to the cyclic oxidationreduction of GSH during antioxidant defense, restoration of GSH content after detoxification of electrophiles is dependent upon its resynthesis. In this report, we consider the role of GSH and its synthesis in tumor cell defense against sulfhydryl-reactive antineoplastics, in particular, the sesquiterpene lactones (SL) We studied four SL that are active in vitro (vernolepin, helenalin, elephantopin, and eriofertopin, Biochemical assays. Total cellular glutathione (GSH plus GSSG) was measured by a minor modification of the method of Tietze as previously described (5, 28). Total glutathione is expressed as nanomoles of the tripeptide per milligram of cell protein and is referred to as GSH in this report. Extracts of cells treated with compounds that we found to cause GSH depletion were mixed with known amounts of GSH and then assayed to rule out the presence of an inhibitor of the Tietze assay, which might have accounted for the observed loss of GSH. The activity of glucose oxidase was measured with the scopoletin assay for H202 as described (29). Glutathione reductase was assayed by the method of Roos et al. (30). Protein content was determined by the method of Lowry et al. (31) using bovine serum albumin as the standard. Inhibition of glutathione synthesis with BSO. We have previously reported that with these tumors a maximal rate of GSH depletion, and thus maximal inhibition of synthesis, was achieved with 0.2 mM BSO (5). The rate of GSH depletion in the presence of BSO reflects GSH catabolism, efflux, and dilution by cell division in the absence of synthesis. BSO stock solution (20 mM in H20) was stored at 0°C for up to 3 wk before use. Cytolysis assays. Cells were labeled with Na251CrO4 as described (27). Cytolysis was studied in three experimental settings: (a) 4 X 104 labeled cells were incubated with various concentrations of lytic agent or vehicle alone (0.1-1% dimethyl sulfoxide) in the presence or absence of BSO (0.2 mM) in 0.22 ml of MEM, 5% horse serum at 37°C in 5% C02, 95% air for 18 h. (b) Cells (1.5 X 106/ml in MEM, 5% horse serum) were incubated with various concentrations of vernolepin or vehicle alone (0.1% dimethyl sulfoxide) plus Na251CrO4 for 1 h, washed extensively (four centrifugations), GSH At the indicated times (18 h for a and b, 5 h for c), supernatant (0.1 ml for a and c, 0.5 ml for b) was removed after centrifugation for gamma counting and the percent specific release was calculated as described RESULTS Depletion of tumor cell GSH by SL. The possible interaction of cellular GSH with SL was first evaluated. Incubation of P815 mastocytoma cells with vernolepin, helenalin, elephantopin, or eriofertopin for 1 h resulted in 70-97% depletion of GSH Effect of BSO on the lysis of tumor cells by SL. We evaluated the sensitivity of 51Cr-labeled tumor cells to lysis by an 18-h coincubation with SL. The synergistic interaction between BSO and vernolepin was further analyzed by considering the kinetics of onset and reversal of their effects. For this, exposure of cells to vernolepin was limited to a 1-h pulse, followed by an 18-h incubation in its absence, at which time lysis was determined