Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Age-related changes in the activity of antioxidant and redox enzymes in rats

Cellular defense system, including glutathione, glutathione-related enzymes, and antioxidant and redox enzymes, may play crucial roles in the aging of aerobic organisms. To understand the physiological roles of these factors in the aging process, their levels were compared in the livers and brains of 5-week- and 9-month-old rats. GST activity was higher in livers and brains of 9-month-old rats than in those of 5-week-old rats, and brain catalase activity was about 2-fold higher. However, it was unchanged in the livers of the 9-month-old rats. gamma-Glutamylcysteine synthetase activity was about 2-fold higher in the brains of the older rats but again not in their livers. In contrast glutathione synthetase activity appeared to be lower in the livers of the older rats while GSH content did not change with age in livers and brains. Glutathione peroxidase activity was higher in 9-month-old rat brains, but lower in 9-month-old rat livers, while superoxide dismutase activity was higher in both tissues in the older rats. The activities of two redox enzymes, thiol-transferase and thioredoxin reductase, did not change with age, nor did that of glutathione reductase. These results indicate that levels of different cellular defense systems vary with age in an irregular manner.     

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.     

Modulation of antioxidant enzymes p21WAF1 and p53 expression during proliferation and differentiation of human melanoma cell lines

The activities of antioxidant enzymes, and the expression of p21(WAF1) and p53 proteins were studied at different times after subculture during proliferation and differentiation phases. Two human melanoma cell lines were used: IPC182, which is a non-differentiating cell line, and IGR221, which spontaneously differentiates at the end of the exponential growth phase, as evidenced by a marked increase of melanin content and tyrosinase activity. In the two cell lines, the slowing of proliferation coincided with an increase in the activity and amount of immunoreactive superoxide dismutases (SOD1 and SOD2), and a decrease of catalase and glutathione peroxidase activities, and of the glutathione content. The levels of p21WAF1 and p53 proteins were found to be lower in confluent than in proliferative cells. Several parameters were modified only during the differentiation phase of IGR221 cells; in these cells the increase of tyrosinase activity was highly correlated with the increase in SOD2, GST, glutathione reductase, and G6PD activities. The level of glutathione was found to be lower in differentiated IGR221 than in non-differentiated IPC182 cells. These results suggest that p21WAF1 and p53 proteins are not involved in the spontaneous differentiation process of melanoma cells, and that abnormal regulation of the cell cycle inhibition pathway occurred in these cells. The results sustain the hypothesis that alterations of antioxidant enzyme expression are involved in the control of proliferation and differentiation of melanoma cells. Alterations of SOD2 activity may be of particular importance, since variations are observed with both cell growth and cell differentiation.     

Differential responses from seven mammalian cell lines to the treatments of detoxifying enzyme inducers

Cell-based models have been used extensively in screening novel bioactive chemical entities. In this study, seven well-established mammalian cell lines, which have different origins, were utilized to compare their responses to the treatments of three detoxifying enzyme inducers, tert-butylhydroquinone (tBHQ), beta-naphthoflavone (beta-NF), and sulforaphane (SUL), which are potential chemopreventive compounds. The enzymatic activities of glutathione s-transferase (GST), NAD(P)H:quinone oxidoreductase (QR), aldehyde reductase (AR), and glutathione reductase (GR) were measured by kinetics methods using UV-Vis spectroscopy, and analyzed statistically by Student's t-test. Among these mammalian cell lines, the mouse hepatoma Hepa1c1c7 cells were the most robust and sensitive cells, which had higher basal as well as upregulated enzymatic activities. In human cell lines, the prostate LNCaP and hepatic HepG2 cells were also very responsive to the inducers. The results suggested that different cell lines responded differently to individual detoxifying gene inducer, and the selection of appropriate cell line is important for screening potential chemopreventive agents.     

Consequences of selenite supplementation on the growth and metabolism of cultures of canine mammary cells

Previous studies with cultures of canine mammary cells revealed differences in the degree of growth inhibition caused by selenite supplementation, with canine mammary tumor cell line 13 > 11 > non-neoplastic canine mammary cells. The present studies show this variation in growth retardation cannot be explained by selenium retention. Intracellular glutathione related inversely to the degree of growth inhibition resulting from the addition of selenite. Dimethyl selenide formation by S-9 preparations corresponded to the sensitivity of the culture to supplemental selenite. DL-buthionine-SR-sulfoximine, a specific inhibitor of glutathione biosynthesis, accentuated the growth inhibition and prevented the increase in intracellular glutathione caused by supplemental selenite. Treatment of canine mammary tumor cell line 13 cultures with DL-buthionine-SR-sulfoximine resulted in a persistent depletion of intracellular glutathione without affecting growth. Glutathione reductase activity, before and following selenite, was inversely related to the degree of growth inhibition, with canine mammary tumor cell line 13 > 11 > non-neoplastic canine mammary tumor cell line. Selenite addition increased the activity of gamma-glutamylcysteine synthetase in canine mammary tumor cell line 11 and non-neoplastic canine mammary cells, but not in canine mammary tumor cell line 13 cells. The present data suggest the differences in the growth inhibition caused by selenite among these mammary cells is related to glutathione regulation and ultimately to selenium detoxification.     

Activated Human Hepatic Stellate Cells Promote Growth of Human Hepatocellular Carcinoma in a Subcutaneous Xenograft Nude Mouse Model

Tumor cell microenvironment defines cancer development, also in hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are believed to be the key contributors to tumor microenvironment in HCC, yet their precise role in cancer progression is still unclear. The aim of this study was to determine the effect of human HSCs on progression of HCC using a subcutaneous xenograft nude mouse model. Nude mice were stratified to receive subcutaneous injections of human HCC cell line HepG2 and human HSC line LX-2 (HepG2 + LX-2), HepG2 alone, LX-2 alone, or phosphate-buffered saline. Tumor growth was assessed by measuring tumor size. After 30 days, final tumor size, weight, and histology were assessed. Compared with mice that were only injected HepG2 cells, mice injected with HepG2 + LX-2 exhibited more rapid tumor growth, increased tumor size and weight, higher tumor cell numbers due to increased proliferation and reduced apoptosis, increased fibrotic bands containing LX-2 cells, and increased tumor angiogenesis. In conclusion, HSCs play a significant role in promotion of HCC growth.     

Glutathione-related enzymes,glutathione and multidrug resistance

This review examines the hypothesis that glutathione and its associated enzymes contribute to the overall drug-resistance seen in multidrug resistant cell lines. Reports of 34 cell lines independently selected for resistance to MDR drugs are compared for evidence of consistent changes in activity of glutathione-related enzymes as well as for changes in glutathione content. The role of glutathione S-transferases in MDR is further analyzed by comparing changes in sensitivity to MDR drugs in cell lines selected for resistance to non-MDR drugs that have resulting increases in glutathione S-transferase activity. In addition, results of studies in which genes for glutathione S-transferase isozymes were transfected into drug-sensitive cells are reviewed. The role of the glutathione redox cycle is examined by comparing changes in elements of this cycle in MDR cell lines as well as by analyzing reports of the effects of glutathione depletion on MDR drug sensitivity. Overall, there is no consistent or compelling evidence that glutathione and its associated enzymes augment resistance in multidrug resistant cell lines.     

Expression of superoxide dismutases, catalase, and glutathione peroxidase in glioma cells

Four primary antioxidant enzymes were measured in both human and rat glioma cells. Both manganese-containing superoxide dismutase (MnSOD) and copper-zinc-containing superoxide dismutase (CuZnSOD) activities varied greatly among the different glioma cell lines. MnSOD was generally higher in human glioma cells than in rat glioma cells and relatively higher than in other tumor types. High levels of MnSOD in human glioma cells were due to the high levels of expression of MnSOD mRNA and protein. Heterogeneous expression of MnSOD was present in individual glioma cell lines and may be due to subpopulations or cells at different differentiation stages. Less difference in CuZnSOD, catalase, or glutathione peroxide was found between human and rat glioma cells. The human glioma cell lines showed large differences in sensitivity to the glutathione modulating drugs 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO). A good correlation was found between sensitivity to BCNU and the activities of catalase in these cell lines. Only one cell line was sensitive to BSO and this line had low CuZnSOD activity.     

Biological effects of a nano red elemental selenium.

A novel selenium form, nano red elemental selenium (Nano-Se) was prepared by adding bovine serum albumin to the redox system of selenite and glutathione. Nano-Se has a 7-fold lower acute toxicity than sodium selenite in mice (LD(50) 113 and 15 mg Se/kg body weight respectively). In Se-deficient rat, both Nano-Se and selenite can increase tissue selenium and GPx activity. The biological activities of Nano-Se and selenite were compared in terms of cell proliferation, enzyme induction and protection against free racial-mediated damage in human hepatoma HepG2 cells. Nano-Se and selenite are similarly cell growth inhibited and stimulated synthesis of glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and thioredoxin reductase (TR). When HepG2 cells were co-treated with selenium and glutathione, Nano-Se showed less pro-oxidative effects than selenite, as measured by cell growth. These results demonstrate that Nano-Se has a similar bioavailability in the rat and antioxidant effects on cells.     

Disruption of redox homeostasis and induction of apoptosis by suppression of glutathione synthetase expression in a mammalian cell line

The stable HepG2 transfectants anti-sensing expression of the glutathione synthetase (GS) gene exhibited delayed cell growth and increased reactive oxygen species (ROS) level. After the treatment with hydrogen peroxide, the intracellular ROS level was much higher in the stable transfectants than in the vector control cells. However, the GSH levels decreased more significantly in the stable transfectants than in the vector control cells, in the presence of hydrogen peroxide. Hydrogen peroxide-induced apoptosis of the stable transfectants was notably higher than that of the vector control cells. The GS anti-sense RNAs rendered the HepG2 cells more sensitive to growth arrest caused by glucose deprivation. They also sensitized the HepG2 cells to cadmium chloride (Cd) and nitric oxide (NO)-generating sodium nitroprusside (SNP). In brief, the results confirm that GS plays an important role in the defense of the human hepatoma cells against oxidative stress by reducing apoptosis and maintaining redox homeostasis.     

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.     

Use of the inhibition of enzymatic antioxidant systems in order to evaluate their physiological importance

Chemical inhibitors of the different antioxidant enzymes were systematically testet either on purified enzymes of after incubation with human fibroblasts in culture. Inhibition values were obtained for catalase with aminotriazole, for superoxide dismutase with diethyldithiocarbamate, for glutathione peroxidase with mercaptosuccinate, for glutathione reductase with bischloroethylnitrosourea and for glutathione synthesis with buthionine sulfoximine. Viability of cells incubated with these inhibitors was then tested under normal conditions and under high oxygen pressure; the data were correlated with the above-mentioned inhibitory values. Cell viability was particularly affected when the glutathione-related enzymes, especially glutathione peroxidase, were inhibited.     

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

Summary The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.