Teresa Seefeldt

Characterization of a Novel Dithiocarbamate Glutathione Reductase Inhibitor and Its Use as a Tool to Modulate Intracellular Glutathione

Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC50 = 647 μm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm and 0.1 min–1, respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD(+) and NADPH/NADP(+). Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [gamma-glutamylcysteine synthetase and glutathione synthetase].

Increase in thiol oxidative stress via glutathione reductase inhibition as a novel approach to enhance cancer sensitivity to X-ray irradiation

Depletion of the reduced form of glutathione (GSH) has been extensively studied for its effect on sensitizing cancer to radiation. However, little is known about the effects of thiol oxidative stress created through an increase in glutathione disulfide (GSSG) on cancer sensitivity to radiation. In this study, an increase in GSSG was effectively created using 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), an irreversible glutathione reductase (GR) inhibitor. Our results demonstrate that the GSSG increase significantly enhanced cancer sensitivity to X-ray irradiation in four human cancer cell lines (A431, MCF7, NCI-H226, and OVCAR-3). When cells were pretreated with 2-AAPA followed by X-ray irradiation, the IC(50) values for X-ray irradiation of A431, MCF7, NCI-H226, and OVCAR-3 cells were reduced, from 24.2 +/- 2.8, 42.5 +/- 3.0, 43.0 +/- 3.6, and 27.8+/-3.5 Gy to 6.75 +/- 0.9, 8.1 +/- 1.1, 6.75 +/- 1.0, and 12.1 +/- 1.7 Gy, respectively. The synergistic effects observed from the combination of X-rays plus 2-AAPA were comparable to those from the combination of X-rays plus buthionine sulfoximine, a reference compound known to increase cancer sensitivity to radiation. The synergistic effect was correlated with an increase in cell thiol oxidative stress, which was reflected by a five-to sixfold increase in GSSG and 25% increase in total disulfides. No change in GSH or total thiols was observed as a result of GR inhibition.

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1

Context: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. Objective: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. Materials and methods: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. Results: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. Discussion: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. Conclusion: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.

Microtubule S-glutathionylation as a potential approach for antimitotic agents

BackgroundMicrotubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G2/M phase. Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S-glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S-glutathionylation can be a novel approach for antimitotic agents.Methods2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic acid (2-AAPA) was used as a tool to induce microtubule S-glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S-transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S-glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S-glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S-glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines.ResultsOur investigation demonstrated that cell morphology was changed and microtubules were S-glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G2/M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition.ConclusionsThe data from this investigation confirms that microtubule S-glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H2O2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.

Design, synthesis, and biological evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine and its analogs as a novel class of anticancer agents

N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) was identified as a metabolite of sulofenur. Sulofenur was demonstrated to have broad activity against solid tumors in preclinical studies but exhibited disappointing clinical responses due to its high protein binding related adverse effects. NACC exhibited low protein binding and excellent activity against a sulofenur sensitive human colon cancer cell line. In this study, analogs of NACC were synthesized and evaluated with four human cancer cell lines. Two of the NACC analogs showed excellent activity against two human melanoma cell lines, while NACC remains the most potent of the series. All three compounds were more potent than dacarbazine, which is used extensively in treating melanoma. NACC was shown to induce apoptosis without affecting the cell cycle. Further, NACC exhibited low toxicity against monkey kidney cells. The selective anticancer activity, low toxicity, an unknown yet but unique anticancer mechanism and ready obtainability through synthesis make NACC and its analogs promising anticancer agents.

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer

Aspirins potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirins chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxy-benzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirins chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

In-vitro and in-vivo inhibition of melanoma growth and metastasis by the drug combination of celecoxib and dacarbazine.

Celecoxib has been found to be effective in cancer prevention and treatment. Its combination with other chemotherapeutic agents was reported to produce synergistic/additive effects on various cancers. Dacarbazine (DTIC) is one of the most commonly used drugs in the treatment of metastatic melanoma. This investigation aimed to determine the in-vitro and in-vivo effects of the drug combination of celecoxib and DTIC on melanoma growth and metastasis. Melanoma cells B16-F10 and SK-MEL-28, and female C57BL/6 mice were used for the study. Our in-vitro data showed that significant synergistic effects were obtained when celecoxib was used together with various concentrations of DTIC. A study with B16-F10 cells using flow cytometry analysis showed that the drug combination induced significantly more apoptosis than each drug used individually. Our in-vivo results showed that the drug combination was much more effective than each drug used alone for the inhibition of both melanoma growth and metastasis in the B16-F10+C57BL/6 mouse models. For melanoma growth, the median survival rates for phosphate-buffered saline (PBS) (control), celecoxib (30 mg/kg), DTIC-1 (10 mg/kg), DTIC-2 (positive control, 50 mg/kg), and the drug combination (DTIC 10 mg/kg+celecoxib 30 mg/kg) were 6, 6.5, 7.5, 7.5, and 9 days, respectively. For melanoma metastasis, the average number of metastatic tumors in murine lungs was 53.7±10.7, 31.8±18.6, 21.2±21.7, 7.0±9.0, and 0.8±2.0 for PBS, DTIC-1, celecoxib, the drug combination, and DTIC-2. Our results warrant further investigation of the combination as an effective treatment for melanoma patients.

The Role of Antioxidant Vitamins in Cardiovascular Disease

Objective: To review the current data on the role of antioxidant vitamins in cardiovascular disease. Data Sources: Articles were obtained from a MEDLINE search covering all years. Terms used in the search included combinations of antioxidant, vitamin, vitamin E, vitamin C, beta-carotene, and cardiovascular disease. Reference lists from articles were examined for additional references. Study Selection and Data Extraction: Randomized clinical trials were selected and evaluated for study design and data obtained. Observational studies, meta-analyses, and basic science articles were also reviewed for background. Data Synthesis: The use of dietary supplements, including vitamins, minerals, and herbal products, is common among patients in the US. Vitamin E, vitamin C, and beta-carotene are all supplements with reported antioxidant activity. Because oxidative stress has been implicated in the development of cardiovascular disease, there has been significant research at the basic science level as well as observational studies and randomized placebo-controlled trials examining the potential impact of vitamin E, vitamin C, and beta-carotene in the prevention and treatment of cardiovascular disease. While basic science data and data from observational studies have identified benefit from antioxidant vitamin supplementation, randomized clinical trials have failed to support a role for antioxidant vitamin supplementation in the prevention or treatment of cardiovascular disease. Conclusions: Data from randomized clinical trials do not support the use of antioxidant vitamins in cardiovascular disease. Therefore, antioxidant vitamins should not be recommended to patients for prevention or treatment of cardiovascular disease.