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The phytochemical investigation of Ochrosia eliptica leaves resulted in isolation and identification of eight compounds; lupeol, lupeol acetate, uvaol, ursolic acid, β-sitosterol glucoside, rutin, 8-methoxy and 9-methoxy ellipticine . The ethanolic extract and fractions were studied for their cytotoxic, antioxidant and anti-inflammatory activities. The cytotoxic activity was performed on human mammary adenocarcinoma (MCF7), its multidrug-resistant ‎ counterpart (VCREMS), estrogen receptor negative human metastatic breast ‎ adenocarcinoma cells (MDA-MB-231) and the non-cancerous, immortalized by ‎ telomerase, human breast epithelial cell line (hTERT-HME1). Additionally, the inhibitory potential on Cdc25s proteins was determined. The results showed that the dichloromethane (DCM) fraction and the major alkaloid; 9-methoxyellipticine exhibited high inhibitory activity against all tested cell lines particularly MCF7 and VCREMS cell lines, whereas t he DCM fraction showed a significant inhibitory action on Cdc25 A isoform. In contrast, the n-butanol fraction and 9-methoxyellipticine displayed the highest antioxidant potential. The DCM fraction showed significant anti-inflammatory activity compared to indomethacin. This work comprises the first comprehensive work to be conducted on O. elliptica leaves showing its potential in multiple biological activities .

Twenty one sesquiterpenes were isolated from the organic extract of the Caribbean gorgonian Pseudopterogogia rigida. Among them, seven bisabolanes (1, 2, 9–11, 13 and 14), six cadinanes (15–20) and one sesquiterpene featuring a new tricyclic carbon skeleton (21) are new natural products, while two (6 and 7) are reported for the first time from a natural source. The structures of the new compounds were established by detailed analyses of their NMR and MS data. The isolated metabolites were evaluated for their inhibitory activity against CDC25 phosphatases, which represent possible targets related to cancer therapy. Compounds 4 and 18 were the most active with IC50 values of 1.88 and 3.44 μM, respectively, against the CDC25A isoform.

The 5th Biologie Prospective Santorini Conference explored the themes of systems biology, nutrigenomics and pharmacogenomics, all of which are related to personalized health, personalized therapy and personalized medicine. The conference started with a satellite meeting on genome-wide scan studies where the need for simplified models, the quality of the phenotypes and the input of epigenetics were dominant remarks. All of the omics approaches were then applied during the 3 days’ sessions to multifactorial diseases (e.g., diabetes, atherosclerosis, cancer and inflammation) and often focused on gene–gene and gene–environment interactions. Afterwards, a fundamental session on drug metabolism, theranostics and pharmacogenetics and their practical aspects showed that the translation to clinical practice is finally happening although much slower than expected.

The cell division cycle 25 phosphatases (CDC25A, B, and C; E.C. 3.1.3.48) are key regulator of the cell cycle in human cells. Their aberrant expression has been associated with the insurgence and development of various types of cancer, and with a poor clinical prognosis. Therefore, CDC25 phosphatases are a valuable target for the development of small molecule inhibitors of therapeutic relevance. Here, we used an integrated strategy mixing organic chemistry with biological investigation and molecular modeling to study novel quinonoid derivatives as CDC25 inhibitors. The most promising molecules proved to inhibit CDC25 isoforms at single digit micromolar concentration, becoming valuable tools in chemical biology investigations and profitable leads for further optimization. [Formula: see text].

International audience; Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1–9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin–quinone structure. © 2013 Wiley Periodicals, Inc.

The reactivity of 4-(1-butoxyvinyl)-2H-chromen-2-one (1) and (E)-4-(2-butoxyvinyl)-2H-chromen-2-one (2) as diene in thermal Diels–Alder cycloaddition reactions with several electron-poor dienophiles is reported. Among several dienophiles used in this study 1,4-benzoquinone afforded cycloadducts 11-butoxy-1H-naphtho[1,2-c]chromene-1,4,5-trione (3e) and 1H-naphtho[1,2-c]chromene-1,4,5-trione (4g) that showed Cdc25 phosphatase-inhibition activity at low micromolar values, with both compounds more effective against Cdc25 A and Cdc25 C isoforms.

Twenty one sesquiterpenes were isolated from the organic extract of the Caribbean gorgonian Pseudopterogogia rigida. Among them, seven bisabolanes (1, 2, 9–11, 13 and 14), six cadinanes (15–20) and one sesquiterpene featuring a new tricyclic carbon skeleton (21) are new natural products, while two (6 and 7) are reported for the first time from a natural source. The structures of the new compounds were established by detailed analyses of their NMR and MS data. The isolated metabolites were evaluated for their inhibitory activity against CDC25 phosphatases, which represent possible targets related to cancer therapy. Compounds 4 and 18 were the most active with IC50 values of 1.88 and 3.44 μM, respectively, against the CDC25A isoform.

Previously, we presented the chemical design of a promising series of antimalarial agents, 3-[substituted-benzyl]-menadiones, with potent in vitro and in vivo activities. Ongoing studies on the mode of action of antimalarial 3-[substituted-benzyl]-menadiones revealed that these agents disturb the redox balance of the parasitized erythrocyte by acting as redox cyclers—a strategy that is broadly recognized for the development of new antimalarial agents. Here we report a detailed parasitological characterization of the in vitro activity profile of the lead compound 3-[4-(trifluoromethyl)benzyl]-menadione 1c (henceforth called plasmodione) against intraerythrocytic stages of the human malaria parasite Plasmodium falciparum . We show that plasmodione acts rapidly against asexual blood stages, thereby disrupting the clinically relevant intraerythrocytic life cycle of the parasite, and furthermore has potent activity against early gametocytes. The lead's antiplasmodial activity was unaffected by the most common mechanisms of resistance to clinically used antimalarials. Moreover, plasmodione has a low potential to induce drug resistance and a high killing speed, as observed by culturing parasites under continuous drug pressure. Drug interactions with licensed antimalarial drugs were also established using the fixed-ratio isobologram method. Initial toxicological profiling suggests that plasmodione is a safe agent for possible human use. Our studies identify plasmodione as a promising antimalarial lead compound and strongly support the future development of redox-active benzylmenadiones as antimalarial agents.

International audience; Cell division cycle dual phosphatases (CDC25) are essential enzymes that regulate cell progression in cell cycle. Three isoforms exist as CDC25A, B and C. Over-expression of each CDC25 enzyme is found in cancers of diverse origins. Thiazolidinone derivatives have been reported to display anti-proliferative activities, bactericidal activities and to reduce inflammation process. New 2-(thienothiazolylimino)-1,3-thiazolidin-4-ones were synthesized and evaluated as inhibitors of CDC25 phosphatase. Among the molecules tested, compound 6 inhibited CDC25A with an IC50 estimated at 6.2 +/- 1.0 mu M. The binding of thiazolidinone derivative 6 onto CDC25A protein was reversible. In cellulo, compound 6 treatment led to MCF7 and MDA-MB-231 cell growth arrest. To our knowledge, it is the first time that such 4-thiazolidinone derivatives are characterized as CDC25 potential inhibitor.

Quinones and quinones-like compounds are potential candidates for the inhibition of CDC25 phosphatases. The combination of MALDI-MS analyses and biological studies was used to develop a rapid screening of a targeted library of indeno[1,2-b]indoloquinone derivatives. The screening protocol using MALDI-TOFMS and MALDI-FTICRMS highlighted four new promising candidates. Biological investigations showed that only compounds 5c–f inhibited CDC25A and -C phosphatases, with IC50 values around the micromolar range. The direct use of a screening method based on MALDI-MS technology allowed achieving fast scaffold identification of a new class of potent inhibitors of CDC25 phosphatases. These four molecules appeared as novel molecules of a new class of CDC25 inhibitors. Assessment of 5c–e in an MRC5 proliferation assay provided an early indicator of toxicity to mammalian cells. Compound 5d seems the most promising hit for developing new CDC25 inhibitors.

International audience; A one-pot synthesis of novel benzopyran-4-ones is described. In a tandem reaction, organobase-catalysed Michael addition of (RCOCH2COR2)-C-1 on chromone-3-carboxylic acid led to decarboxylation and pyran-4-one ring opening of the latter. This was followed by chromone- and/or chromanone ring closure of the resulting Michael adducts when R-1 is an ortho-hydroxyaryl group. Antioxidant testing of 14 derivatives identified strong antiradical properties of chromanones 3o-r (2.1-3.1 mu mol Trolox equiv./mu mol compound in the DPPH assay). Chromanones 3p and 3r and 2-styrylchromone 3k were also most potent in inducing the cytoprotective Keap1-Nrf2 signalling pathway in a reporter gene assay (fivefold induction at concentrations

CDC25 (cell division cycle 25) phosphatases are essential for cell cycle control under normal conditions and in response to DNA damage. They are represented by three isoforms, CDC25A, B and C, each of them being submitted to an alternative splicing mechanism. Alternative splicing of many genes is affected in response to genotoxic stress, but the impact of such a stress on CDC25 splicing has never been investigated. In this study, we demonstrate that genotoxic agents (doxorubicin, camptothecin, etoposide and cisplatin), alter the balance between CDC25C splice variants in human breast cancer cell lines both at the mRNA and protein levels. This modulation occurs during the response to moderate, sub‐lethal DNA damage. Our results also suggest that the CDC25C splice variants expression shift induced by a genotoxic stress is dependent on the ATM/ATR signaling but not on p53. This study highlights the modulation of CDC25C alternative splicing as an additional regulatory event involved in cellular response to DNA damage in breast 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.

Considering that oxidative stress is strongly implicated in the toxicity of chemotherapy, much effort is focused on the research of diverse antioxidants as protective agents. An efficient synthesis of three novel benzophenones containing 1,3-thiazol moiety (6a-c) is described. Their antioxidant power was evaluated in vitro and in three cell lines (the cancerous MCF7 and the non-cancerous hTERT-HME1 mammary cells, and the H9c2 cardiomyoblastic cells). One analogue 5-(2,5-dihydroxybenzoyl)-2(3H)-benzothiazolone (6c), displayed an important antioxidant activity, a low cytotoxicity, and could decrease reactive oxygen species production generated by tert-butyl hydroperoxide (tBHP) in all three cell lines. Interestingly, 6c was able to protect the non-cancerous cells against tBHP-induced death. Further studies are underway to determine its relevance as an adjuvant in oxidative stress inducing chemotherapy.

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.

Dual-specific phosphatases Cdc25 play a critical role in the cell cycle regulation by activating kinases of Cdk/cyclin complexes. Three Cdc25 isoforms (A, B and C) have been identified in mammalians. Cdc25A and B display oncogenic properties and are over-expressed in different tumors. Cdc25 phosphatases are therefore attractive targets for therapeutic strategies. Novel maleic anhydride derivatives bearing a fatty acid chain of variable size have been synthesized and tested for their Cdc25 inhibitory potential using an in vitro assay. We report biological activity of ineffective, moderate, and efficient inhibitors on breast cancer cells (MCF7) and its counterpart resistant to vincristine (Vcr-R). The most potent compounds induced Cdk2 inhibition and accumulation in G0/G1 phase of the cell cycle. Moreover, apoptosis was triggered within 48-h treatment, without oxidative burst and modulation of the Bax to Bcl-2 ratio. When used as pre-treatments, these derivatives were also able to potentiate adriamycin and cisplatin toxicity in both cell lines. Thus, maleic anhydride derivatives may mediate apoptosis through a cell cycle blockage via inhibition of Cdc25. This class of inhibitors may present potential interest in therapeutic strategies against cancer.

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1-9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin-quinone structure.

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.

Aurones [2-benzylidenebenzofuran-3(2H)-ones] are either natural or synthetic compounds, belonging to the flavonoid family. They are isomeric to flavones and provide a bright yellow color to the pla...

Reactive oxygen species regulate protein functionality. Cell cycle CDC25 phosphatases are targets of such oxidative regulation in vitro. We sought to evaluate if a thioredoxin (trx)-dependent redox regulation of CDC25 exists in cancer cells. For that purpose, we used MCF7 and MDA-MB 231 breast cancer cells, which express trx1 differentially, together with two trx/thioredoxin reductase (trxR) inhibitors, Auranofin and Acrolein. Auranofin could induce a full trxR inhibition associated with ROS production in both cell lines. Acrolein could provoke similar effects only in MDA-MB 231 cells with a low trx1 expression. Simultaneous trx1 oxidation and trxR inactivation occurred only in the presence of Acrolein and resulted in a G2-M cell cycle arrest, without full CDC25 inhibition in MDA-MB 231 cells. Our data suggest that the maintenance of CDC25 activity does not fully rely on the trx system in breast cancer cells, even in the presence of a major oxidative stress.

La production des especes reactives de l’oxygene (ERO) est fortement impliquee dans les mecanismes de toxicite des medicaments utilises en chimiotherapie et donc dans la generation de leurs effets secondaires. L’emploi d’agents protecteurs couples a la chimiotherapie fait donc l’objet de recherches intensives en vue d’une utilisation optimale des molecules anticancereuses. L’objectif de ce travail est de selectionner et d’isoler parmi les substances naturelles, des molecules nouvelles, presentant un pouvoir antioxydant, protecteur des cellules non cancereuses, mais n’affectant pas l’efficacite des substances anticancereuses vis-a-vis des cellules tumorales.

The increased use of animals in fundamental and applied research due to the remarkable drug development in the 20th century has been an important matter of concern for people at large, but also for the scientific community. This led Russel and Burch to examine the decisions which could meliorate this situation, and they proposed, in 1959, the principle of the 3Rs (Reduce, Refine, and Replace) nowadays largely admitted as an ethical and incontrovertible principle (Russell & Bursch 1959). Alternatives to animal experiments (Scheme 1) then knew a fantastic boom with the permanent objective of a high scientific quality in order to prevent, treat and cure human illness. Reaching the equilibrium between in vitro and in vivo models, observing the 3Rs rules, is very difficult. Effectively, in vitro systems allow an excellent control of all parameters of the experiments, and then, good quantifications. More the models are simple, more they are easy to handle, but more they also are dedifferentiated and keep away from the in vivo situation.

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.

Background CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results CDC25 spliced transcripts are differentially expressed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 transcript is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

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 G 2 -M cell cycle arrest.

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.

Viral immortalization (SV40) of adult rat hepatocytes was performed in controlled culture conditions (isolation method, collagen matrix, defined culture medium, short-time culture before virus infecti

Considering that oxidative stress is strongly implicated in the toxicity of chemotherapy, much effort is focused on the research of diverse antioxidants as protective agents. An efficient synthesis of three novel benzophenones containing 1,3-thiazol moiety (6a-c) is described. Their antioxidant power was evaluated in vitro and in three cell lines (the cancerous MCF7 and the non-cancerous hTERT-HME1 mammary cells, and the H9c2 cardiomyoblastic cells). One analogue 5-(2,5-dihydroxybenzoyl)-2(3H)-benzothiazolone (6c), displayed an important antioxidant activity, a low cytotoxicity, and could decrease reactive oxygen species production generated by tert-butyl hydroperoxide (tBHP) in all three cell lines. Interestingly, 6c was able to protect the non-cancerous cells against tBHP-induced death. Further studies are underway to determine its relevance as an adjuvant in oxidative stress inducing chemotherapy.

A hepatic cell line has been immortalized after simian vacuolating virus 40 infection of adult rat hepatocytes maintained in defined culture conditions. This cell line, designated SVHep B4, expressed nuclear large T antigen, exhibited an extended lifespan (50 subcultures) and had a hepatocyte-like morphology. Expression and regulation of drug metabolizing enzymes were studied in long-term cultures of SVHep B4 cells. Significant activities of phase I and phase II enzymes were detected. gamma-Glutamyltransferase, a marker often increased in neoplastic and dedifferentiated hepatocytes, showed a low activity whereas the hepatospecific enzyme tyrosine aminotransferase was expressed at levels similar to those in liver. Responsiveness of drug metabolizing enzymes to inducers was investigated with phenobarbital, dexamethasone and methylcholanthrene. IIB and IA subfamilies of cytochrome P450 were increased, respectively, by phenobarbital (170%) and methylcholanthrene (500%). Glucuronidation of 1-naphthol was increased by phenobarbital (140%) and 3-methylcholanthrene (160%). Phenobarbital, methylcholanthrene and dexamethasone were found to increase significantly gamma-glutamyltransferase while tyrosine aminotransferase activity was enhanced by dexamethasone. Stable expression and inducibility of drug metabolizing enzymes in long-term cultures of the SVHep B4 cell line demonstrate that immortalization of adult hepatocytes represents a promising tool for drug biotransformation studies in vitro.

Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.

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.

Carboxylic acid-containing drugs are metabolized mainly through the formation of glucuronide and coenzyme A esters. These conjugates have been suspected to be responsible for the toxicity of several nonsteroidal anti-inflammatory drugs because of the reactivity of the electrophilic ester bond. In the present study we investigated the reactivity of ketoprofenyl-acylglucuronide (KPF-OG) and ketoprofenyl-acyl-coenzyme A (KPF-SCoA) toward cytosolic rat liver glutathione S-transferases (GST). We observed that KPF-SCoA, but not KPF-OG inhibited the conjugation of 1-chloro-2,4-dinitrobenzene and 4-nitroquinoline N-oxide catalyzed by both purified cytosolic rat liver GST and GST from FAO and H5-6 rat hepatoma cell lines. Photoaffinity labeling with KPF-SCoA suggested that the binding of this metabolite may overlap the binding site of 4-methylumbelliferone sulfate. Furthermore, high-performance liquid chromatography and mass spectrometry analysis showed that both hydrolysis and transacylation reactions were observed in the presence of GST and glutathione. The formation of ketoprofenyl-S-acyl-glutathione could be kinetically characterized (apparent K(m) = 196.0 +/- 70.6 microM). It is concluded that KPF-SCoA is both a GST inhibitor and a substrate of a GST-dependent transacylation reaction. The reactivity and inhibitory potency of thioester CoA derivatives toward GST may have potential implications on the reported in vivo toxicity of some carboxylic acid-containing drugs.

The use of doxorubicin (DOX) in the treatment of solid tumors is limited by cardiotoxicity essentially due to oxidative stress generation. The aim of this study was to identify coumarin derivatives displaying a protective antioxidant activity without affecting DOX antitumoral efficiency. A set of eighteen coumarinic derivatives was synthesized. Their antioxidant power was evaluated in vitro with the FRAP (ferric reducing ability of plasma) method and in human breast adenocarcinoma MCF7 cells using H 2 DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) in a cytometric analysis. 4-Methyl-7,8-dihydroxycoumarin was found to exhibit an important antioxidant strength, a low cytotoxicity, and could decrease ROS (reactive oxygen species) production generated by DOX treatment without affecting DOX cytotoxicity in MCF7 cells.

An efficient synthesis of dialkylsubstituted maleic anhydrides 1a–j is described. The inhibitory potential of these original anhydride derivatives was tested toward the three human isoforms A, B and C of dual specific phosphatases Cdc25. A micromolar range inhibition of Cdc25s was observed with the maleic anhydrides bearing simple alkyl side chains longer than C9, to reach the optimal activity with a C17 chain length.

Incubation of aortic rings in a culture medium produces phenomena similar to those observed with aging, i.e. oxidative stress and inflammation leading to increased nitric oxide (NO)-mediated dilation and decreased arterial sensitivity to vasoconstrictor agents. We evaluated whether melatonin protects aortic rings from such a decrease in vasoreactivity. Two concentrations of melatonin were used: 10(-8) M, EC50 for vascular MT1-MT2 receptors, and 10(-5) M, reported as anti-oxidant. Anti-oxidant capacity, inducible nitric oxide synthase (iNOS) expression and isometric contraction of thoracic aorta rings (Wistar rats) evoked by norepinephrine (NE) were assessed. Three days of incubation of aortic rings induced iNOS expression and a fall in NE-evoked contraction. When melatonin was added to the organ bath, it (10(-5) M) increased (+96%, P0.05), but did not restore (compared with freshly isolated rings) NE-evoked contraction. Three days of treatment with melatonin increased (10(-8) M, +99%) or restored (10(-5) M, +216%) NE-evoked contraction (compared with freshly isolated rings). The beneficial effects of 10(-8) and 10(-5) M melatonin on NE-evoked contraction were abolished in the presence of luzindole (2 x 10(-6) M, a melatonin receptor antagonist). The incubation-induced increase in iNOS expression was reduced following 3 days of melatonin administration (10(-8) and 10(-5) M). Melatonin (10(-5) M) increased catalase activity (6550 +/- 256, P0.05 vs. nontreated fresh aortic rings 5554 +/- 444 nmol min(-1) mg protein(-1)). In conclusion, melatonin counteracts the incubation-induced loss of agonist-evoked contraction of aortic rings by a specific receptor-mediated phenomenon involving iNOS expression; at higher melatonin concentrations, an anti-oxidant effect is probably also involved.

The impact of the anti-cancer drugs cisplatin (CDDP) and adriamycin (ADR) was investigated on sensitive and resistant MCF-7-derived human breast cancer cells. Cytotoxicity was evaluated by MTT assay, reactive oxygen species (ROS), apoptosis and necrosis by flow cytometry, glutathione (GSH) by HPLC, and Bcl-2, Bax and PARP expression by Western blot. A perturbation of ROS and intracellular GSH levels, and the enhancement of both apoptosis and necrosis were observed in sensitive cells. Transfected MCF-7 cells overexpressing the anti-apoptotic Bcl-2 protein, as well as MCF-7-derived vincristine-resistant cell line (Vcr-R) were resistant to both drugs. This resistance was clearly associated with an unaltered GSH level and with the inhibition of an early GSH efflux. Vcr-R cell resistance seemed to rely on a different mechanism, since it was found to be independent of Bcl-2 expression. Since Bcl-2 overexpression confers the strongest degree of resistance of MCF-7-derived cells, our observations further highlight Bcl-2 as a prime pharmacological target to sensitize cancer cells to chemotherapeutic agents.

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 Parkinson's disease, in which oxidative stress plays a crucial role.

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

The histone deacetylase inhibitor sodium butyrate induces several gene products that modify cellular metabolism. Here, we investigated its ability to modulate glutathione-related detoxification enzymes in the breast cancer cell line MCF-7 and a derivative resistant to vincristine (VCREMS). We found that sodium butyrate induced glutathione S-transferase and glutathione-dependent peroxidase activities and triggered glutathione depletion. Expression of MRP1, an ATP-dependent GS-X pump, was unmodified. Moreover, isobologram analysis showed that sodium butyrate sensitized VCREMS to doxorubicin-mediated toxicity. Verapamil, an inhibitor of MRP1, did not significantly affect this chemosensitizing effect, suggesting that the observed toxicity stems from multifactorial mechanisms. Interestingly, synergism between sodium butyrate and doxorubicin was more pronounced in resistant VCREMS cells than in parental sensitive MCF-7 cells.

Thieno analogues of kenpaullone have been synthesized using an established method. We investigated the effect of five structural analogues of kenpaullone on vincristine sensitive and resistant MCF7 (human mammary adenocarcinoma) cells. One analogue, 8-Bromo-6,11-dihydro-thieno-[3',2':2,3]azepino[4,5-b]indol-5(4H)-one (3a), showed an antiproliferative activity in the drug sensitive cell line that led to cell accumulation in G2/M phase. In addition, repression of cdk1, a G2/M transition key regulator, as well as induction of p21 were observed at the mRNA level. Programmed cell death (apoptosis) was induced in early time treatments and was accompanied by p53 mRNA induction. The antiproliferative and proapoptotic properties of 3a make this CDK inhibitor an interesting candidate for further investigations.

We studied the effect of age on the response of aortic rings to injury produced by three days' incubation, and the mechanism of this response. Five-mm rings of the thoracic aorta isolated from Wistar rats were incubated or not in culture medium. Isometric contraction evoked by agonists (norepinephrine or serotonin) or high [K(+)](e) was determined in the presence and absence of endothelium. Experiments were repeated in the presence of propranolol (0.3 microM), polymixin B (36 microM), pyrrolidine dithiocarbamate (50 microM) or glutathione (3 mM). Inductible NO-synthase and cyclo-oxygenase-2 mRNA were determined by real-time PCR, and glutathione-related enzymes and catalase activity by spectrophotometry. Incubation reduced the isometric contraction evoked by agonists but not by high [K(+)](e). The reduction in agonist-evoked contraction was greater in rings from adult (norepinephrine Emax-80%) than in young (-40%) rats. The removal of the endothelium had no effect. The reduction in norepinephrine-evoked contraction was not due to endotoxin contamination, beta-adrenoceptor-mediated dilation or any change in ring structure (no fibrosis or edema). Inductible NO-synthase (but not cyclo-oxygenase-2) mRNA increased on incubation. N(G)-nitro-L-arginine methyl ester partially restored contractility in rings from adult animals, further addition of an anti-oxidant restored norepinephrine-evoked contraction. Catalase fell with age and glutathione reductase increased upon incubation in rings from young donors only. In conclusion, incubation of the aorta produces a specific reduction in agonist-evoked contraction that involves induction of smooth muscle cell oxidative stress and iNOS. The reaction is greater in rings from older animals.

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

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.