Denyse Bagrel

Control of Oxidative Posttranslational Cysteine Modifications: From Intricate Chemistry to Widespread Biological and Medical Applications

Cysteine residues in proteins and enzymes often fulfill rather important roles, particularly in the context of cellular signaling, protein-protein interactions, substrate and metal binding, and catalysis. At the same time, some of the most active cysteine residues are also quite sensitive toward (oxidative) modification. S-Thiolation, S-nitrosation, and disulfide bond and sulfenic acid formation are processes which occur frequently inside the cell and regulate the function and activity of many proteins and enzymes. During oxidative stress, such modifications trigger, among others, antioxidant responses and cell death. The unique combination of nonredox function on the one hand and participation in redox signaling and control on the other has placed many cysteine proteins at the center of drug design and pesticide development. Research during the past decade has identified a range of chemically rather interesting, biologically very active substances that are able to modify cysteine residues in such proteins with huge efficiency, yet also considerable selectivity. These agents are often based on natural products and range from simple disulfides to complex polysulfanes, tetrahydrothienopyridines, α,β -unsaturated disulfides, thiuramdisulfides, and 1,2-dithiole-3-thiones. At the same time, inhibition of enzymes responsible for posttranslational cysteine modifications (and their removal) has become an important area of innovative drug research. Such investigations into the control of the cellular thiolstat by thiol-selective agents cross many disciplines and are often far from trivial.

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.

Mangiferin protects against 1-methyl-4-phenylpyridinium toxicity mediated by oxidative stress in N2A cells

1-Methyl-4-phenyl-pyridine ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a Parkinsonian syndrome in humans and animals, a neurotoxic effect postulated to derive from oxidative stress. We report here the first investigation of MPP+-induced oxidative stress in the murine neuroblastoma cell line N2A. Significant cell death was observed following exposure to 0.25 mM MPP+. Markers of oxidative stress included decreased intracellular levels of GSH after 48 h of exposure (85% depletion) as well as an increase in GSSG. Expression of both superoxide dismutase 1 (sod1) and catalase (cat) mRNA was increased, as well the activity of catalase. These cellular effects were, at least partially, reversed by treatment with the natural polyphenol mangiferin. Administration of mangiferin protected N2A cells against MPP+-induced cytotoxicity, restored the GSH content (to 60% of control levels), and down-regulated both sod1 and cat mRNA expression. Together, these results suggest that the protective effect of mangiferin in N2A cells is mediated by the quenching of reactive oxygen intermediates. Therefore, mangiferin could be a useful compound in therapies for degenerative diseases, including Parkinsons disease, in which oxidative stress plays a crucial role.

The glutathione-related detoxification system is increased in human breast cancer in correlation with clinical and histopathological features

Purpose: The glutathione detoxification pathway includes glutathione S-transferase (GST) and peroxidase (GPX) isoenzymes as well as glutathione reductase (GSSR). Though well established from cultured cancer cell lines, its involvement in resistance is still unclear in the tumours. This study aimed to describe the parameters that influence the glutathione contents and associated activities in breast cancer. Methods: The components of the glutathione pathway were measured in the tumours from 41 women with primary breast cancer in comparison with those in the matched tumour-free samples. Appropriate statistical studies (regression analysis, Wilcoxon signed rank test) explored the influence of clinical and prognostic factors. Results: Reduced and total glutathione contents were largely increased (P < 0.0001) and all related activities were significantly enhanced in the tumours. Interindividual variations were described, probably due to various parameters (age, menopause, axillary lymph node status, S and G2 + M cell fractions, ER, cathepsin-D and c-ErbB-2 expressions) that influence particular components of the glutathione pathway, especially the glutathione levels. Conclusions: The breast tumours improved their redox status and detoxification capacities depending on various parameters of significance for cell proliferation and aggressiveness, which supports the involvement of the glutathione pathway in malignant cell resistance to oxidative stress and apoptosis.

Apoptosis/necrosis switch in two different cancer cell lines: Influence of benzoquinone- and hydrogen peroxide-induced oxidative stress intensity, and glutathione

Depending on the strength of oxidative stress, cells exhibit proliferative, apoptotic or necrotic responses. We have investigated whether the severity of glutathione (GSH) depletion could determine the type of cell death using 1,4-benzoquinone (BQ) and H(2)O(2) in two different tumor cell lines (human mammary gland carcinoma MCF-7 and rat hepatoma H5-6). BQ-treated surviving cells showed an increase in GSH, but no detectable oxidized glutathione (GSSG) nor reactive oxygen species (ROS) augmentation. Alternatively, H(2)O(2) depressed GSH. BQ induced mostly apoptosis, up to 90% cell elimination, while necrosis was prominent in H(2)O(2)-treated cultures. The resistance of BQ-treated cells to necrosis could be due to increased cellular GSH and formation of BQ-GSH conjugates which are less toxic than free BQ, minimal toxicity being provided by GS4-BHQ. This ability of certain cancer cells to tightly keep the apoptotic pathway may have therapeutic applications for oxidation-based drugs.

The glutathione-related detoxification pathway in the human breast: a highly coordinated system disrupted in the tumour tissues

Glutathione and the associated enzymes, glutathione S-transferases, peroxidases, and reductase, have been implicated in cancer chemoresistance. This pathway was investigated in paired cancerous and peritumoral breast samples from 41 women. The tumours exhibited a higher redox status as deduced from increased transferase, peroxidase, and reductase activities and from higher total and reduced glutathione contents. Several components were strongly correlated in peritumoral tissues, suggesting a highly co-ordinated glutathione pathway that appeared disrupted in breast tumours with only a few correlations left. Therefore, resistance could spontaneously result from deregulated variations in the glutathione pathway, which might be relevant to the malignant disease progression.

Antiproliferative effect of natural tetrasulfides in human breast cancer cells is mediated through the inhibition of the cell division cycle 25 phosphatases

For many years, in vitro and in vivo studies have reported that organosulfur compounds (OSCs), naturally found in Allium vegetables, are able to suppress the proliferation of various tumor cells. In spite of recent advances, the specific molecular mechanisms involved in OSC activity are still unclear. Considering the antiproliferative effects observed in cancer cells, we postulated that OSCs might target the cell division cycle (Cdc) 25 phosphatases which are crucial enzymes of the cell cycle. Our findings suggest phosphatases Cdc25 as possible targets of naturally occuring polysulfides contributing to their anticancer properties. We report on the inhibitory activity of tetrasulfides occurring naturally in garlic and onion towards the human Cdc25 phosphatases. Diallyl- and dipropyltetrasulfides have emerged as interesting irreversible inhibitors of the Cdc25 isoforms A and C in vitro. Furthermore, growth of both sensitive (MCF-7) and resistant (Vcr-R) human breast carcinoma cells was significantly decreased by these tetrasulfides. The observed antiproliferative effect appeared to be associated with a G2-M cell cycle arrest.

Impact of chronic treatment with red wine polyphenols (RWP) on cerebral arterioles in the spontaneous hypertensive rat.

Several reports suggest that consumption of red wine is associated with a lower risk of stroke. We investigated the chronic effect of red wine polyphenols (RWP) on the functional and structural characteristics of cerebral arterioles in chronic hypertension, which is an important risk factor of stroke. Spontaneous hypertensive rats (SHR) were treated with RWP extract (100 mg/kg/day in drinking water) for 10 weeks. We measured the effect of agonist- and hypotension-induced changes in internal diameter of cerebral arterioles using an open cranial window technique. Wall mechanical parameters were determined in deactivated cerebral arterioles. The activity of antioxidant enzymes in plasma was determined. Adenosine diphosphate-induced vasodilatation was decreased by 48% in SHR and normalized in SHR treated with RWP. RWP had no effect on hypotension-induced dilatation. RWP decreased the wall thickness/external diameter ratio by 13% and significantly shifted the stress-strain relationship of the arteriole wall to the left. There was a decrease in glutathione-S-transferase and glutathione peroxidase after treatment of RWP in SHR. In summary, chronic oral administration of RWP to SHR improved endothelium-dependent dilatation, normalized wall stress and diameter, and altered the systemic antioxidant state. These effects of RWP could be useful in the prevention of stroke in hypertensive patients.

Synthesis and biological evaluation of novel coumarin-based inhibitors of Cdc25 phosphatases.

The cell division cycle 25 (Cdc25) family of proteins are dual specificity phosphatases that activate cyclin-dependent kinase (CDK) complexes, which in turn regulate progression through the cell division cycle. Overexpression of Cdc25 proteins has been reported in a wide variety of cancers; their inhibition may thus represent a novel approach for the development of anticancer therapeutics. Herein we report new coumarin-based scaffolds endowed with a selective inhibition against Cdc25A and Cdc25C, being 6a and 6d the most efficient inhibitors and worthy of further investigation as anticancer agents.

Coumarin polysulfides inhibit cell growth and induce apoptosis in HCT116 colon cancer cells

Coumarins and coumarin derivatives as well as diallyl polysulfides are well known as anticancer drugs. In order to find new drugs with anticancer activities, we combined coumarins with polysulfides in the form of di-coumarin polysulfides. These novel compounds were tested in the HCT116 colorectal cancer cell line. It turned out that they reduced cell viability of cancer cells in a time and concentration dependent manner. Cells tested with these coumarin polysulfides accumulate in the G(2)/M phase of the cell cycle and finally they go into apoptosis. A decrease in bcl-2 level, and increase in the level of bax, cytochrome c release into the cytosol, cleavage of caspase 3/7and PARP suggested that coumarin polysulfides induced the intrinsic pathway of apoptosis. Comparison of these new coumarin compounds with the well known diallyl polysulfides revealed that the coumarin disulfides were more active than the corresponding diallyl disulfides. The activities of the coumarin tetrasulfides and the corresponding diallyl tetrasulfides are similar. The novel coumarin compounds regulated the phosphatase activity of the cell cycle regulating cdc25 family members, indicating that these phosphatases are implicated in the induction of cell cycle arrest and possibly in apoptosis induction as well. In addition, coumarin polysulfides also down-regulated the level of cdc25C, which also contributed to the arrest in the G(2)-phase of the cell cycle.