Arsenate V induced glutathione efflux from human erythrocytes

ObjectiveThe objective of the present study was to investigate if arsenate V exposure results in glutathione efflux from human erythrocytes.ProcedureThe changes in intracellular and extracellular nonprotein sulfhydryl and glutathione levels were determined in arsenate (V) exposed erythrocytes. Presence of any cellular membrane damage was assessed by lactate dehydrogenase activity measurement in the supernatant.ResultsWhen erythrocytes were exposed to 10 mM of arsenate (V) for 4 h, the intracellular NPSH level decreased to 0.28 ± 0025 μmol/ml erythrocyte. In contrast, extracellular nonprotein thiol level was increased to 0.180 ± 0.010 μmol/ml erythrocyte in 4 h. Extracellular glutathione levels reached to 0.028 ± 0.001, 0.052 ± 0.002, and 0.054 ± 0.004 μmol/ml erythrocyte with 1, 5, and 10 mM of arsenate (V), respectively. Utilization of MK571 a multi drug resistance-associated protein 1 inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process.ConclusionThe results of the present study indicate that erythrocytes efflux glutathione when exposed to arsenate (V).     

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas-mediated apoptosis. Here, we describe the induction of the Fas-dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV-IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV-IgG-mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.     

Epidermal growth factor receptor down-regulation induced by UVA in human keratinocytes does not require the receptor kinase activity

Activation of the epidermal growth factor (EGF) receptor by EGF, its ligand, results in receptor internalization and down-regulation, which requires receptor kinase activity, phosphorylation, and ubiquitination. In contrast, we have found here in human HaCaT keratinocytes that exposure to UVA induces EGF receptor internalization and down-regulation without receptor phosphorylation and ubiquitination. The presence of the receptor kinase activity inhibitor AG1478 increased UVA-induced receptor down-regulation, whereas it inhibited EGF-induced receptor down-regulation. These observations demonstrate that, in contrast to EGF, receptor kinase activity is not required for receptor down-regulation by UVA. Concurrent with receptor down-regulation, caspases were activated by UVA exposure. The presence of caspase inhibitors blocked receptor down-regulation in a pattern similar to poly(ADP)-ribose polymerase cleavage. Much more receptor down-regulation was observed after UVA exposure in apoptotic detached cells in which caspase is activated completely. These results indicate that UVA-induced receptor down-regulation is dependent on caspase activation. Similar to UVA, both UVB and UVC induced receptor down-regulation, in which receptor kinase activity is not required, whereas caspase activation is involved. Inhibition of EGF receptor down-regulation increased receptor activation and activation of its downstream survival signaling ERK and AKT after UVA exposure. Preventing the activation of each of these pathways enhanced apoptosis induced by UVA. These findings suggest that EGF receptor down-regulation by UVA may play an important role in the execution of the cell suicide program by attenuating its anti-apoptotic function and thereby preventing cell transformation and tumorigenesis in vivo.     

Polyphenol (-)-epigallocatechin gallate inhibits apoptosis induced by irradiation in human HaCaT keratinocytes

Green tea is a rich source of polyphenols, and (-)-epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. In the present study, we investigated the effect of EGCG on apoptosis induced by irradiation in the human keratinocytic cell line HaCaT. Irradiation by gamma-ray induced apoptosis with concomitant cleavage of caspase-3 and its in vivo substrate poly(ADP-ribose) polymerase. Treatment of cells with EGCG inhibited irradiation-induced apoptosis as detected by Hoechst staining and internucleosomal cleavage of DNA, and prevented the cleavage of these proteins by irradiation. We also found that the treatment of cells with EGCG alone suppressed cell growth and induced apoptosis in these cells. Our results suggest that EGCG inhibits irradiation-induced apoptosis by inactivating the caspase pathway in HaCaT cells. Our study also indicates that EGCG has a dual effect on the survival of these keratinocytes.     

Factors influencing the inhibition of biliary glutathione efflux induced by biliary obstruction

The aim of this study was to investigate mechanisms responsible for the inhibition of biliary glutathione efflux in rats with secondary biliary cirrhosis. Rats were studied after bile duct obstruction for 28 days. The biliary secretion of reduced glutathione (GSH), oxidised glutathione (GSSG) and cysteine were completely inhibited in biliary obstructed rats. Hepatic gamma glutamyltranspeptidase (gamma-GT) activity increased significantly, but following its inhibition by acivicin administration GSH, GSSG and cysteine were still absent in bile. Biliary obstruction resulted in a significant increase of the permeability of the paracellular pathway, as shown by the higher bile/plasma ratio and hepatic clearance of [14C]sucrose. GSH and GSSG were, however, significantly lower in the carotid artery and hepatic vein of obstructed animals and the arteriovenous difference across the liver was reduced. The concentration of GSH was significantly reduced and that of GSSG increased in the liver of obstructed rats. Biliary obstruction induced an increase in the hepatic concentration of cysteine and an inhibition of both gamma glutamylcysteine synthetase and methionine adenosyl transferase activities. Dichlorofluorescein (DCF) and the GSSG/GSH ratio and thiobarbituric acid reactive substances (TBARS) concentration, markers of reactive oxygen species production and lipid peroxidation, respectively, were significantly increased. Our data indicate that increased degradation or blood reflux of glutathione do not participate in the disruption of its secretion into bile and support the view that impairment of glutathione synthesis and oxidative stress could contribute to the decline in biliary glutathione output.     

Juglans regia L. protects against UVB induced apoptosis in human epidermal keratinocytes

The present study was aimed to investigate the photoprotective effect of the male flower of J. regia L. (MEJR) against ultraviolet-B induced apoptosis in human skin cells. Human skin epidermal keratinocytes were pretreated with the MEJR (80 µg/ml, has been selected after MTT assay), prior to 30 min UVB-irradiation at a dose of 20 mJ/cm². Mitochondrial membrane potential was evaluated using Rhodamine-123 staining; the % apoptosis by Hoechst staining and acridine orange staining; DNA damage was measured by comet assay. The levels of p53, Bax, Bcl-xL, Bcl-2, Cytochrome c, Caspase-9 and Caspase-3 expression in HaCaT cells were analyzed by western blotting and RT-PCR. Pretreatment with MEJR 80 µg/ml prior to UVB-irradiation significantly prevents apoptotic characteristics, DNA damage and loss of mitochondrial membrane potential. Thus, MEJR protects UVB-mediated human skin cells, by modulating the expression of apoptotic markers and UVB-induced DNA damage in HaCaT cells.     

Role of glutathione in apoptosis induced by radiation as determined by 1H MR spectra of cultured tumor cells

The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.     

Role of glutathione in selenite binding by human plasma

The erythrocyte-mediated reduction of selenite has been reproduced by the addition of reduced glutathione to plasma at levels comparable to those present in the erythrocyte. The reaction has been followed by chromatography and ultraviolet (UV) absorption spectroscopy (in the absence of plasma). The first detectable compound, selenium diglutathione, is very unstable in physiological conditions. The product of the reaction does not contain glutathione and is able to react and incorporate selenium into plasma proteins without the participation of hemoglobin or glutathione reductase. A saturable low molecular weight compound is also able to bind selenium, which may be relevant in the initial distribution and excretion of selenium after selenite administration.     

2-deoxy-d-ribose induces apoptosis by inhibiting the synthesis and increasing the efflux of glutathione

Oxidative stress is caused by imbalance between the production of reactive oxygen species (ROS) and biological system ability to readily detoxify the reactive intermediates or repair the resulting damage. 2-deoxy-D-ribose (dRib) is known to induce apoptosis by provoking an oxidative stress by depleting glutathione (GSH). In this paper, we elucidate the mechanisms underlying GSH depletion in response to dRib treatment. We demonstrated that the observed GSH depletion is not only due to inhibition of synthesis, by inhibiting gamma-glutamyl-cysteine synthetase, but also due to its increased efflux, by the activity of multidrug resistance associated proteins transporters. We conclude that dRib interferes with GSH homeostasis and that likely cellular oxidative stress is a consequence of GSH depletion. Various GSH fates, such as direct oxidation, lack of synthesis or of storage, characterize different kinds of oxidative stress. In the light of our observations we conclude that dRib does not induce GSH oxidation but interferes with GSH synthesis and storage. Lack of GSH allows accumulation of ROS and cells, disarmed against oxidative insults, undergo apoptosis.     

UVA/B exposure promotes the biosynthesis of dehydroretinol in cultured human keratinocytes

Retinol and its metabolites modulate epithelial differentiation and serve as cellular UV sensors through changes in retinoid status. Of note is the dehydroretinol family which may serve functions distinct from parental retinol. This study focuses on the metabolism of this family and its potential participation in the response of normal epidermal human keratinocytes to UV irradiation. There were three findings. First, keratinocytes contain two pools of dehydroretinyl esters, one of which is shielded from UVB-, but not from UVA-induced decomposition. Second, using a novel in vitro assay we demonstrated that both UVA and UVB promote dehydroretinol biosynthesis in keratinocytes, but only UVB exposure promotes retinoid ester accretion by enhancing the activity of at least one acyl transferase. Finally, dehydroretinol sufficiency reduces UVA/B driven apoptosis more effectively than retinol sufficiency. This may in part be due to differences in the expression of Fas ligand, which we found to be upregulated by retinoic acid, but not dehydroretinoic acid. These observations implicate a role of dehydroretinol and its metabolites in UVA/B adaptation. Thus, the keratinocyte response to UV is jointly shaped by both the retinoids and dehydroretinoids.     

Stellate cell apoptosis by a soluble mediator from immortalized human hepatocytes

Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.     

Delayed and sustained activation of extracellular signal-regulated kinase in human keratinocytes by UVA: implications in carcinogenesis

Exposure to the sun's UV radiation appears to be the most important environmental factor involved in the development of skin cancer. UVA is the major portion of UV radiation in sunlight and is considered to be a human carcinogen. In this study, we have investigated the delayed and sustained activation of ERK MAPK by UVA exposure. In parallel, a delayed Ras activation with a similar time course was observed after UVA exposure. The activated Ras was found to be localized in endomembranes such as the Golgi apparatus instead of plasma membranes. Expression of dominant negative Ras (N17Ras) abolished ERK activation by UVA. The presence of AG1478, an epidermal growth factor (EGF) receptor (EGFR) kinase inhibitor, had no effect on ERK or Ras activation, indicating that EGFR kinase activity is not involved in ERK activation by UVA. In contrast, protein kinase C (PKC) depletion by chronic 12-O-tetradecanoylphorbol-13-acetate treatment nearly abolished UVA-induced ERK and Ras activation. The presence of the Ca(2+)-dependent-PKC inhibitor Go6976 had a similar effect. These findings suggest that ERK activation by UVA is mediated by PKC in a Ras-dependent pathway. In addition, a gradual increase in intracellular calcium level after UVA exposure was detected by flow cytometry. The presence of the PLC inhibitor U73122 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) blocked both ERK and Ras activation, suggesting that both PLC and calcium are required for ERK activation. Our findings demonstrated that, different from UVC and UVB, UVA-induced delayed and sustained ERK activation is EGFR kinase activity-independent, but PLC/calcium/PKC-mediated. The delayed and sustained ERK activation provides a survival signal to human HaCaT keratinocytes, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure.     

Potentiation of arsenic trioxide-induced apoptosis by 8-bromo-7-methoxychrysin in human leukemia cells involves depletion of intracellular reduced glutathione

The novel chrysin analog 8-bromo-7-methoxychrysin (BrMC) has been reported to induce apoptosis of various cancer cell lines. Arsenic trioxide (ATO) treatment induces clinical remission in acute promyelocytic leukemia patients. The combination of ATO with other agents has been shown to improve therapeutic effectiveness in vitro and in vivo. In this report, the mechanism of apoptosis induced by treatment with ATO alone or in combination with BrMC was studied in U937, HL-60, and Jurkat cells. Our results demonstrated that BrMC cooperated with ATO to induce apoptosis in human leukemia cells. This co-treatment caused mitochondrial transmembrane potential dissipation and stimulated the mitochondrial apoptotic pathway, as evidenced by cytochrome c release, down-regulation of X-linked inhibitor of apoptosis (XIAP) and Bcl-XL, and up-regulation of Bax. BrMC alone or in combination with ATO, decreased Akt phosphorylation as well as intracellular reduced glutathione (GSH) content. The thiol antioxidant N-acetylcysteine and exogenous GSH restored GSH content and attenuated apoptosis induced by co-treatment with ATO plus BrMC. In contrast, the non-thiol antioxidant butylated hydroxyanisole and mannitol failed to do so. These findings suggest that GSH depletion explains at least in part the potentiation of ATO-induced apoptosis by BrMC.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

p38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.     

Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine

The intracellular effects of GSH (reduced glutathione) and BSO (buthionine sulfoximine) treatment on glutathione content were investigated with immunogold labeling in individual cellular compartments of Cucurbita pepo L. seedlings. Generally, GSH treatment led to increased levels of glutathione in roots and leaves (up to 3.5-fold in nuclei), whereas BSO treatment significantly decreased glutathione content in all organs. Transmission electron microscopy revealed that glutathione levels in mitochondria, which showed the highest glutathione labeling density of all compartments, remained generally unaffected by both treatments. Since glutathione within mitochondria is involved in the regulation of cell death, these results indicate that high and stable levels of glutathione in mitochondria play an important role in cell survival strategies. BSO treatment significantly decreased glutathione levels (1) in roots by about 78% in plastids and 60.8% in the cytosol and (2) in cotyledons by about 55% in the cytosol and 38.6% in plastids. After a short recovery period, glutathione levels were significantly increased in plastids and the cytosol of root tip cells (up to 3.7-fold) and back to control values in cotyledons. These results indicate that plastids, either alone or together with the cytosol, are the main center of glutathione synthesis in leaves as well as in roots. After GSH treatment for 24 h, severe ultrastructural damage related to increased levels of glutathione was found in roots, in all organelles except mitochondria. Possible negative effects of GSH treatment leading to the observed ultrastructural damage are discussed.     

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.     

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.