Addition Of Buthionine Sulfoximine Research Articles

Abstract A103: Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile

Abstract The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy. Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.

Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile.

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

Lowering glutathione level by buthionine sulfoximine enhances in vivo photodynamic therapy using chlorin e6-loaded nanoparticles

Synergetic effects in mechanisms are important for successful combination therapy. Herein, we used buthionine sulfoximine (BSO) to reduce the cytosolic concentration of glutathione (GSH), a natural scavenger of reactive oxygen species (ROS). We then performed photodynamic therapy (PDT) using chlorin e6-loaded poly(ethylene glycol)-block-poly(D,L lactide) nanoparticles (Ce6-PEG-PLA-NPs). Upon laser irradiation, cytotoxic ROS generated from Ce6-PEG-PLA-NPs killed tumor cells effectively due to the reduced concentration of GSH with addition of BSO. When intravenously injected into tumor-bearing mice, Ce6-PEG-PLA-NPs resulted in efficient delivery of Ce6 to tumor tissues, as shown in near-infrared fluorescence (NIRF) imaging. The accumulation of Ce6 in tumor tissue was more than 2 folds increased by Ce6-PEG-PLA-NPs at every time points. The synergetic effect of reduced GSH synthesis by BSO and efficient delivery of Ce6-PEG-PLA-NPs to tumor tissue was observed in a mouse model after laser irradiation. The combination of BSO and Ce6-PEG-PLA-NPs resulted in complete suppression of tumor, while BSO or Ce6-PEG-PLA-NPs-treated tumors grew to about 1800 and 400 mm3, respectively. The overall results suggest that the combination of BSO and photosensitizer-loaded NPs is effective and demonstrates promise for tumor therapy.

Exogenous Glutathione Enhances Mercury Tolerance by Inhibiting Mercury Entry into Plant Cells.

Despite the increasing understanding of the crucial roles of glutathione (GSH) in cellular defense against heavy metal stress as well as oxidative stress, little is known about the functional role of exogenous GSH in mercury (Hg) tolerance in plants. Here, we provide compelling evidence that GSH contributes to Hg tolerance in diverse plants. Exogenous GSH did not mitigate the toxicity of cadmium (Cd), copper (Cu), or zinc (Zn), whereas application of exogenous GSH significantly promoted Hg tolerance during seed germination and seedling growth of Arabidopsis thaliana, tobacco, and pepper. By contrast, addition of buthionine sulfoximine, an inhibitor of GSH biosynthesis, severely retarded seed germination and seedling growth of the plants in the presence of Hg. The effect of exogenous GSH on Hg specific tolerance was also evident in the presence of other heavy metals, such as Cd, Cu, and Zn, together with Hg. GSH treatment significantly decreased H2O2 and O2- levels and lipid peroxidation, but increased chlorophyll content in the presence of Hg. Importantly, GSH treatment resulted in significantly less accumulation of Hg in Arabidopsis plants, and thin layer chromatography and nuclear magnetic resonance analysis revealed that GSH had much stronger binding affinity to Hg than to Cd, Cu, or Zn, suggesting that tight binding of GSH to Hg impedes Hg uptake, leading to low Hg accumulation in plant cells. Collectively, the present findings reveal that GSH is a potent molecule capable of conferring Hg tolerance by inhibiting Hg accumulation in plants.

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

Redox-Mediated Suberoylanilide Hydroxamic Acid Sensitivity in Breast Cancer.

Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

BIMELis a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

sBackgroundArsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.MethodsHL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.ResultsHL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.ConclusionsThe enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

Low glutathione regulates gene expression and the redox potentials of the nucleus and cytosol in Arabidopsis thaliana

Reduced glutathione (GSH) is considered to exert a strong influence on cellular redox homeostasis and to regulate gene expression, but these processes remain poorly characterized. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities. The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 and -320 mV. Growth in the presence of the GSH-synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately -260 mV. GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECATE), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH-synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO. Inhibition of auxin transport had no effect on organ GSH levels, but exogenous auxin decreased the root GSH pool. We conclude that GSH depletion significantly increases the redox potentials of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses, but not oxidative stress.

The regulation of cytotoxicity and cyclooxygenase‐2 expression by 2‐hydroxy‐ethyl methacrylate in human osteoblasts are related to intracellular glutathione levels

To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P<0.05). The 50% inhibition concentration of HEMA was approximately 3mmol L(-1) . HEMA was found to induce COX-2 protein expression in U2OS cells (P<0.05). The addition of OTZ acted as a protective effect on HEMA-induced cytotoxicity and COX-2 expression (P<0.05). In contrast, the addition of BSO enhanced HEMA-induced cytotoxicity and COX-2 expression (P<0.05). Taken together, the levels of HEMA that were tested inhibited cell growth on U2OS cells. HEMA has a significant potential for periapical toxicity. The activation of COX-2 protein expression may be one of the mechanisms of HEMA-induced periapical inflammation. These inhibitory effects were associated with intracellular GSH levels.

Thioredoxin expression is reduced at the plasma membrane of CD4+ T-cells in mid-life adults compared to young adults

Thioredoxin is a 2-cys oxidoreductase enzyme involved in maintaining cellular redox state. A number of human thioredoxin isoforms have been described; Trx1 is located in the cytoplasm, on the exofacial surface and as a secreted protein whereas Trx2 is located in the mitochondria. Secreted Trx1 has previously been implicated in T-cell signaling. As T-cell regulation declines with age we have investigated the age effects on T-cell Trx1. In the present study, we demonstrate an increase in Trx1 expression (p=0.0317; n=9) on total lymphocytes from healthy mid-life adults (age, 50-78) when compared to young adults (age, 25-30), however, a decrease in the level of Trx1 (p=0.0024) on the cell surface of CD4+ T-cells was observed. To further investigate the mechanisms underpinning the age-related decrease in cell surface Trx1, Jurkat T-cell line was treated with or without the addition of buthionine sulfoximine (BSO) to modulate intracellular GSH levels. BSO reduced cellular GSH content in a dose dependent manner with a 50% decrease after 25μ M BSO for 24 hours. BSO treatment is also associated with an increase in Trx1 expression (1.5-fold increase) as measured by western blot analysis. However, with a selective pull-down of cell surface proteins we observed a decrease in thioredoxin expression (3-fold decrease) at the plasma membrane. Furthermore, BSO treatment decreased mitogenic activation of Jurkats as measured by PHA-induced interleukin-2 secretion. These data suggest that in response to GSH depletion Jurkat T-cells increase cellular levels of Trx1 and alter subcellular localization resulting in decreased surface expression of Trx1. This may reflect adaptation by retention of thioredoxin in the cytoplasm to maintain intracellular protein integrity. Redox homeostasis within the immune synapse is vital for efficient antigen mediated T-cell activation; the impact of decreased expression of thioredoxin with age is currently unclear but it could impact on T-cell function.

Cytotoxicity of dentine bonding agents on human pulp cells is related to intracellular glutathione levels

To evaluate ex vivo the mechanisms of cytotoxicity of dentine bonding agents in human pulp cells in vitro. Human pulp cells were obtained from impacted third molars with informed consent and then cultured using an explant technique. Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity was judged using an assay of tetrazolium bromide reduction. To determine whether glutathione (GSH) levels were important in the cytotoxicity of dentine bonding agents, cells were pretreated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Three replicates of each dentine bonding agents were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (anova). Tests of differences of the treatments were analysed by Duncan's test. Clearfil SE Bond, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (P<0.05). The cytotoxicity was upregulated by dentine bonding agents in the following order: PB>SB>CB. Addition of OTZ extracellularly protected the pulp cells from dentine bonding agents-induced cytotoxicity (P<0.05). Addition of BSO enhanced pulp cell death on dentine bonding agents-induced cytotoxicity (P<0.05). Dentine bonding agents have significant potential for pulpal toxicity. GSH depletion could be the mechanism for dentine bonding agents-induced cytotoxicity.

Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels

To evaluate the mechanisms of cytotoxicity of chlorhexidine (CHX) in human osteoblastic cells in vitro. Cytotoxicity, cell proliferation and collagen synthesis assays were performed to elucidate the toxic effects of CHX on the human osteoblastic cell line U2OS. To determine whether glutathione (GSH) levels were important in the cytotoxicity of CHX, cells were pre-treated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. CHX demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P < 0.05). The 50% inhibition concentration of CHX was approximately 0.005%. CHX also inhibited cell proliferation and collagen synthesis (P < 0.05). The addition of OTZ acted as a protective effect on the CHX-induced cytotoxicity (P < 0.05). In contrast, the addition of BSO enhanced the CHX-induced cytotoxicity (P < 0.05). The levels of CHX tested inhibited cell growth, proliferation and collagen synthesis on U2OS cells. CHX has significant potential for periapical toxicity. GSH depletion might be one of the mechanisms underlying CHX cytotoxicity.

Potentiation of arsenic trioxide cytotoxicity by Parthenolide and buthionine sulfoximine in murine and human leukemic cells

To possibly increase the in vitro cytotoxic activity of arsenic trioxide (ATO) by combining it with Parthenolide (PRT), a known NF-kappaB inhibitor and buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. Several cell lines representing various hematological malignancies were treated in vitro with the study drugs alone or in combinations. Flow cytometry was used to assess cell death rates and reative oxygen species production. Glutathione and ATP levels were determinded using a photometric and a luminometric assay, respectively. Cell death was characterised by fluorescence microscopy and DNA fragmentation analysis. PRT increased cytotoxicity of ATO in seven out of eight cell lines. Addition of buthionine sulfoximine (BSO) further potentiated cytotoxicity of the combined treatment. When combined with PRT and BSO, clinically achievable concentrations of ATO (2.5 microM) induced cytotoxicity rates of 80-98% after 24 h. Importantly, lymphocytes from healthy donors were largely unaffected by these treatment modalities, also after growth stimulation in cell culture. N-acetylcysteine inhibited the cytotoxic effects of the triple combination. Treatment of leukemic cells with ATO, PRT and BSO rapidly depleted cells from glutathione, induced oxidative stress and decreased intracellular ATP levels. Cell death showed characteristics of necrosis presumably as a result of ATP loss. Based on the observed selectivity towards malignant cells this combination may offer a therapeutic option applicable to different kinds of leukemia.

Cytotoxicity of formaldehyde on human osteoblastic cells is related to intracellular glutathione levels

Formaldehyde that leaches out of formaldehyde-releasing root canal sealers, specifically from setting material extruded into the periapical region may participate in the development of periapical inflammation or the continuation of a pre-existing periapical lesion. However, the effects of formaldehyde on human osteoblasts have not been investigated. The aim of this study was to evaluate the mechanisms of cytotoxicity of formaldehyde on human osteoblastic cell line U2OS in vitro. Cytotoxicity and cell proliferation assays were performed to elucidate the adverse effects of formaldehyde on U2OS cells. Formaldehyde demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (p<0.05). The 50% inhibition concentration of formaldehyde was about 3 mM. Formaldehyde also inhibited cell proliferation during a 3-day culture period (p<0.05). To determine whether glutathione (GSH) levels were important in the cytotoxicity of formaldehyde, we pretreated cells with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. The addition of OTZ acted as a protective effect on the formaldehyde-induced cytotoxicity (p<0.05). In contrast, the addition of BSO enhanced the formaldehyde-induced cytotoxicity (p<0.05). Taken together, the levels of formaldehyde tested inhibited cell growth and proliferation on U2OS cells. Formaldehyde has significant potential for periapical toxicity. These inhibitory effects were associated with intracellular GSH levels.

Effects of a Bacterial Lipid Byproduct on Human Pulp Fibroblasts In Vitro

Butyrate, a short chain fatty acid, is a metabolic lipid byproduct of various root canal pathogens, such as Porphyromonas endodontalis. However, little is known about the effects of butyrate on cultured human pulp fibroblasts. H33258 fluorescence, flow cytometry, and protein synthesis assays were used to investigate the pathobiologic effects of butyrate on cultured human pulp fibroblasts. Butyrate exhibited cytotoxic effects on human pulp fibroblasts in a concentration-dependent manner (p < 0.05). The addition of butyrate resulted in G2/M phase arrest (p < 0.05). Butyrate also inhibited protein synthesis in a dose-dependent manner (p < 0.05). To determine whether glutathione (GSH) levels were important in the cytotoxicity of butyrate, we pretreated cells with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. The addition of OTZ acted as a protective effect on the butyrate-induced cytotoxicity (p < 0.05). In contrast, the addition of BSO enhanced the butyrate-induced cytotoxicity (p < 0.05). These results indicate that butyrate is cytotoxic to human pulp fibroblasts by inhibiting cell growth, cell-cycle kinetics, and protein synthesis. These inhibitory effects were associated with intracellular GSH levels.

Opposing Effects of Glutathione Depletion and Follicle-Stimulating Hormone on Reactive Oxygen Species and Apoptosis in Cultured Preovulatory Rat Follicles

Oxidative stress and depletion of the antioxidant glutathione (GSH) trigger apoptosis in many systems. Previous work showed that antioxidants prevented apoptosis as effectively as FSH in preovulatory follicles. We aimed to test the hypotheses that follicular reactive oxygen species (ROS) initiate apoptosis and that follicular GSH protects against apoptosis. Preovulatory follicles were isolated from ovaries of immature rats primed with pregnant mare serum gonadotropin. Negative control (0-h) follicles were processed immediately. Others were cultured for 2 to 48 h with 1) medium alone, 2) 75 ng/ml ovine FSH, or 3) FSH plus 100 mum buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Total GSH concentrations declined in follicles cultured without FSH for 48 h, whereas FSH increased GSH levels above those observed at 0 h. BSO suppressed GSH to undetectable levels. Treatment with FSH prevented apoptosis in granulosa cells, measured by terminal dUTP transferase-mediated nick-end-labeling and activated caspase 3 immunohistochemistry. Addition of BSO partially and significantly reversed the antiapoptotic effect of FSH on granulosa cells; supplementation of GSH completely prevented BSO-induced granulosa cell apoptosis. Whole-follicle ROS production, measured as dichlorofluorescein and rhodamine fluorescence using confocal microscopy, was significantly increased by 4 h of culture and increased further thereafter. FSH significantly suppressed ROS production, and the addition of BSO partially overcame this effect of FSH. These findings provide evidence that oxidative stress induces apoptosis in preovulatory follicles and that the antiapoptotic effect of FSH is mediated in part by stimulation of follicular GSH synthesis and suppression of ROS production.

Nitric oxide decreases IGF-1 receptor function in vitro; glutathione depletion enhances this effect in vivo

Insulin-like growth factor-1 (IGF) helps maintain healthy articular cartilage; however, arthritic cartilage becomes less responsive to the anabolic actions of IGF. We previously showed that high concentrations of nitric oxide (NO) decrease IGF receptor tyrosine phosphorylation and response to IGF in intact chondrocytes. The current studies evaluate direct effects of NO on IGF receptor kinase (IGF-RK) in vitro. NO from S-nitroso-N-acetyl-d,l-penicillamine (SNAP) or 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) inhibits IGF-RK auto- and substrate phosphorylation in a dose and time dependent manner. There is a linear correlation between inhibition of auto- and substrate phosphorylation (r(2)=0.98). Increasing either dithiothreitol or reduced glutathione (GSH) content of the phosphorylation buffer to protect thiol groups blocks NO inhibition of IGF-RK substrate phosphorylation. Increased S-nitrosylation of cysteines in IGF-RK after exposure to SNAP suggests that NO may react with sulfhydryl groups, form S-nitrosothiols, which may result in functional modifications. NO blockade of IGF-1 stimulated proteoglycan synthesis in intact cells is enhanced when chondrocyte glutathione is depleted. The in vitro system shows that there can be direct effects of NO on IGF-RK that modify receptor function; the intact cell studies suggest that the mechanisms identified in vitro may be important in intact chondrocyte insensitivity to IGF-1 in cells exposed to NO.

Reduced Glutathione is a Novel Regulator of Vernalization-Induced Bolting in the Rosette Plant Eustoma grandiflorum

The transition from the vegetative rosette stage to the reproductive growth stage (bolting) in the rosette plant Eustoma grandiflorum has a strict requirement for vernalization, a treatment that causes oxidative stress. Since we have shown that reduced glutathione (GSH) and its biosynthesis are associated with bolting in another rosette plant Arabidopsis thaliana, we here investigated whether a similar mechanism governs the vernalization-induced bolting of E. grandiflorum. Addition of GSH or its precursor cysteine, instead of vernalization, induced bolting but other thiols, dithiothreitol and 2-mercaptoethanol, did not. The inductive effect of vernalization on bolting was nullified by addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, without decreasing the plant growth rate. BSO-mediated inhibition of bolting was reversed by addition of GSH but not by cysteine. These indicate that vernalization-induced bolting involves GSH biosynthesis and is specifically regulated by GSH. Plant GSH increased during the early vernalization period along with the activity of gamma-glutamylcysteine synthetase that catalyzes the first step of GSH biosynthesis, although there was little change in amounts of GSH precursor thiols, cysteine and gamma-glutamylcysteine. These findings strongly suggest that vernalization stimulates GSH synthesis and synthesized GSH specifically determines the bolting time of E. grandiflorum.

Mechanisms of cytotoxicity of nicotine in human periodontal ligament fibroblast cultures in vitro.

The use of tobacco products significantly contributes to the progression of periodontal disease and poor response to healing following periodontal therapy. The purpose of this study was to determine the effects of nicotine, a major component of cigarette smoking, on human periodontal ligament fibroblast (PDLF) growth, proliferation, and protein synthesis to elucidate its role in periodontal destruction associated with its use. Human PDLFs were derived from three healthy individuals undergoing extraction for orthodontic reasons. At a concentration higher than 2.5 mM, nicotine was found to be cytotoxic to human PDLFs (P < 0.05). Nicotine also significantly inhibited cell proliferation and decreased protein synthesis in a dose-dependent manner. At concentrations of 50 and 200 microM, nicotine suppressed the growth of PDLFs by 48% and 86% (P < 0.05), respectively. A 10-mM concentration level of nicotine significantly inhibited the protein synthesis to only 44% of these in the untreated control (P < 0.05). Furthermore, the effects of antioxidants (superoxide dismutase (SOD); catalase and 2-oxothiazolidine-4-carboxylic acid (OTZ) and buthionine sulfoximine (BSO) were added to search for the possible mechanism of action, as well as a method for the prevention, of cigarette smoking-associated periodontal diseases. The addition of OTZ, a precursor of cysteine that metabolically promotes GSH synthesis, acted as a protective effect on the nicotine-induced cytotoxicity. However, SOD and catalase did not decrease the nicotine-induced cytotoxicity. In contrast, the addition of BSO, a cellular GSH synthesis inhibitor, enhanced the nicotine-induced cytotoxicity. These results indicate that thiol depletion could be the mechanism for nicotine cytotoxicity. The levels of nicotine tested inhibited cell growth, proliferation, and protein synthesis on human PDLFs. This suggests that nicotine itself might augment the destruction of periodontium associated with cigarette smoking. In addition, these inhibitory effects were associated with intracellular thiol levels. Factors that induce glutathione synthesis of human PDLF may be used for further chemoprevention of cigarette smoking-related periodontal diseases.