Vitamin C Protects Against Hydrogen Peroxide-Induced Inhibition of

Chapter 24

Downloaded by UNIV LAVAL on July 14, 2016 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch024

Vitamin C Protects Against Hydrogen Peroxide-Induced Inhibition of Gap-Junction Intercellular Communication through the Blocking Phosphorylation of Connexin-43 and ERK1/2 in Rat Liver Epithelial Cells Ki Won Lee and Hyong Joo Lee Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Seoul 152-741, South Korea

Antioxidants are known to protect cells from oxidative D N A damage, but the effect of antioxidants on the tumor-promotion process has not yet been clarified. The present study found that vitamin C exhibited antioxidant effects similar to those of butylated hydroxyanisole (BHA) using 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and superoxide radical-scavenging assays. Vitamin C protected against the hydrogen peroxide (H O )-induced inhibition of GJIC, which is a tumor-promotion process, while B H A had no recovery effect, instead inhibiting synergistically. Vitamin C blocked H O -induced phosphorylation of connexin-43 and extracellular-signal-regulated protein kinase 1/2, which are critical regulators of GJIC. These findings suggest that antioxidants exert different effects on reactive oxygen species-mediated tumor promotion. 2

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© 2007 American Chemical Society Shahidi and Ho; Antioxidant Measurement and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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374 Numerous antioxidative substances have been found to exhibit potential cancer chemopreventive activities, but the definition of the appropriate biomarkers to quantify their chemopreventive effects remains subjective (7). A precise understanding of the underlying biochemical and molecular mechanisms is the first step to identifying such biomarkers, and is essential for the successful implementation of chemopreventive strategies. Cellular enzymes and structural proteins, membranes, simple and complex sugars, and D N A and R N A are all susceptible to oxidative damage that leads to tumor initiation. Minimizing the exposure to diverse environmental carcinogens is one strategy for preventing the majority of human cancers, but the complete avoidance of exposure to etiologic factors that can initiate cancer may be unrealistic (2). Therefore, recent chemopreventive strategies have focused more on identifying substances possessing antipromoting or antiprogressive activities that can suppress the transformation of initiated or precancerous cells into malignant ones, rather than on searching for anti-initiators (2). Oxidation involves the withdrawal of energy by oxygen from reduced carbon-based molecules. The paradox is that this process of free-radical oxidation is both deleterious and life sustaining because it is coupled to electron transport in the mitochondria of living cells. The oxygen used by cells to generate energy represents a source of oxygen radicals and reactive oxygen systems. The action of carcinogens is often accompanied by oxidation reactions acting on D N A . Therefore, investigations of the carcinogenic effects of oxidative stress have primarily focused on genotoxicity, but reactive oxygen species (ROS) are also known to play a significant role in the promotional stage of carcinogenesis. In particular, several oxidants and free-radical generators are tumor promoters, and ROS - including the superoxide anion and hydrogen peroxide ( H 0 ) - are strongly associated with carcinogenic processes. 2

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Cell-to-cell communication through gap-junction channels (GJIC) is essential for maintaining homeostasis via the modulation of cell proliferation and differentiation in multicellular organisms (3). Inhibition of GJIC is strongly related to carcinogenicit and particularly to tumor promotion. Normal, contactinhibited fibroblasts and epithelial cells have functional GJIC, while most - if not all - tumor cells have dysfunctional homologous or heterologous GJIC. Cancer cells are characterized by a lack of growth control, inability to terminally differentiate or perform apoptosis under normal conditions, and an extended or immortalized life span. Chemical tumor promoters, growth factors, and hormones have been shown to inhibit GJIC. Several oncogenes have been shown to downregulate GJIC function, while antioncogene drugs and antitumorpromoting natural and synthetic chemicals have been shown to reinstate GJIC

Shahidi and Ho; Antioxidant Measurement and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

375 and growth control with loss of tumorigenicity. Therefore, one hypothetically rational approach to identify antitumor-promoting chemopreventive drugs and anticarcinogenic treatments is to prevent the downregulation of GJIC by the tumor promoters and thereby restore GJIC in neoplastic cells (4,5). The tumorpromoting effects of H 0 are supported by the formation of colonies in soft agar, appearance of foci in monolayer cultures, and disruption of GJIC (6). Since the inhibition of GJIC is strongly related to carcinogenic processes, particularly the tumor-promotion process, enhancers of GJIC are also anticipated to prevent cancer. Dietary or pharmaceutical augmentation of the endogenous antioxidant defense capacity has been considered a plausible way to prevent ROS-mediated carcinogenicity. Although antioxidants are known to protect cells from oxidative stress by scavenging free radicals and quenching lipid-peroxidation chain reactions (which may cause D N A damage) and thereby block the initiation of carcinogenesis, the effect of antioxidants on the tumor-promotion process remains to be clarified. Furthermore, most free-radical scavengers act in reversible oxidation-reduction reactions, and some antioxidants can act both as antioxidants and prooxidants depending on their structure and the reaction conditions. Although antioxidant capacity can be evaluated using chemical methods that are easy to execute and exhibit high reproducibility, such methods do not represent what happens in living cells. Therefore, assays using in vitro living cells may be useful for assessing the antioxidant activity of compounds. The present study investigated the effects of vitamin C and butylated hydroxyanisole (BHA) (Figure 1) on ROS-mediated carcinogeneis in vitro.


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Vitamin C

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Butylated hydroxyanisole

Figure 1. Chemical structures of vitamin C (ascorbic acid) and BHA (butylated hydroxyanisole).


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Materials and Methods Chemicals Vitamin C, B H A , ammonium phosphate monobasic ( N H H P 0 ) , 2,2diphenyl-l-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), dimethyl sulfoxide (DMSO), H 0 , 12-0tetradecanoylphorbol-13-acetate (TPA), lucifer yellow, xylenol orange, ammonium ferrous sulfate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), acrylamide, and Tris-HCl were obtained from Sigma Chemical (St. Louis, M O ) . Triton X-100 was obtained from A M R E S C O (Solon, OH). Antibodies of E R K and p-ERK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-connexin-43 (antiCx43) antibody was obtained from Zymed Laboratories (San Francisco, C A ) . A l l other chemicals used were of analytical or H P L C grade (Fisher, Springfield, NJ). 4

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DPPH Radical-Scavenging Activity Assay The D P P H radical-scavenging activities of vitamin C and B H A were measured using the method described by Brand-Williams et al. with minor modifications (7,8). D P P H radical was dissolved in 80% aqueous methanol. Varying concentrations of vitamin C and B H A (0.1 mL) were added, individually, to 2.9 mL of the D P P H radical solution. The mixture was then shaken vigorously and allowed to stand at 23°C in the dark for 30 min, at which time the decrease in absorbance at 517 nm was measured using a spectrophotometer (Hitachi, Japan).

A B T S Radical-Scavenging Activity Assay A method developed by van den Berg et al. was used, with slight modification, to assess the A B T S radical-scavenging activities of vitamin C and B H A (8,9). A A P H (1.0 mM) was mixed with 2.5 m M A B T S as diammonium salt in a phosphate-buffered saline (PBS) solution (100 m M potassium phosphate buffer, pH 7.4, containing 150 m M NaCl). The mixture was heated in a 68 °C water bath for 13 min, and the concentration of the resulting blue-green A B T S radical solution was adjusted to an absorbance of 0.650 ± 0.020 at 734 nm. The solution with varying concentrations of vitamin C and B H A (20 pL) was added, individually to 980 pL of the resulting blue-green A B T S radical solution. The mixture was incubated in the dark in a 37 °C water bath for 10 min, at which time the decrease in absorbance at 734 nm was measured.


377 Superoxide-Anion Radical-Scavenging Activity Assay HL-60 human promyelocytic leukemia cells (HL-60 cells) were suspended at a density of l x l O cells/ml in RPMI 1640 medium containing 10% fetal bovine serum and 1.3% D M S O , and incubated for 7 days at 37 °C in an incubator containing 5% C 0 . Cells were harvested by centrifugation, washed with PBS, and then resuspended in PBS at a density of l x l O cells/mL. After preincubation with varying concentrations of vitamin C and B H A , respectively, for 15 min, T P A (8 pM) and cytochrome c (60 pM) were added to the reaction mixture. After an additional incubation at 37 °C for 30 min, superoxide-anion generation was determined by measuring the absorbance of reduced cytochrome cat 550 nm. 6

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G J I C Bioassay WB-F344 rat liver epithelial cells (WB-F344 cells) were kindly provided by Dr. J. E. Trosko at Michigan State University (USA). WB-F344 cells were cultured in Dulbecco's modified Eagle's medium (GIBCO BRL), supplemented with 10% fetal bovine serum (GIBCO B R L ) and penicillin/streptomycin (GIBCO B R L ) , in a 37 °C humidified incubator (Forma Scientific, Mariotta, OH) containing 5% C 0 and 95% air. The GJIC was measured using the scrapeloading/dye-transfer technique (10). Briefly, WB-F344 cells were preincubated with noncytotoxic doses of the test compounds and 500 p M H 0 . After incubation, the cells were washed twice with 2 ml of PBS. Lucifer yellow was added to the washed cells, and three scrapes were made with a surgical steelbladed scalpel at low light intensities. Each scrape traversed a large group of confluent cells. After 3 min of incubation, the cells were washed four times with 2 ml of PBS and then fixed with 2 ml of a 4% formalin solution. The number of communicating cells indicated by the dye was counted under an inverted fluorescence microscope (Olympus 1x70, Okaya, Japan). 2

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Cell Viability Cell viability was measured using the M T T assay as described previously (77). Briefly, cells were cultured in 96-well plates at 10,000 cells/well in media for 24 h. Each well was filled with fresh media containing varying amounts of each compound, and the cells were incubated for an additional 24 h at 37 °C, followed by treatment with M T T for 4 h. The medium was removed and D M S O was added to dissolve the blue formazan residue. The optical density at 570 nm of each well was then measured using a microplate reader (Molecular Devices,


378 Sunnyvale, CA). A l l data are from at least three replications for each prepared sample.


Western Blot Analysis Western blot analysis was performed for Cx43, ERK1/2, and p - E R K l / 2 . Briefly, sample cells were resuspended into 4x sample buffer (8% SDS, 20% glycerin, 250 m M Tris-HCl, pH 7.5, 0.2% bromophenol blue, 40 m M DTT), heated for 5 min, and separated by 12.5% SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to a 0.45-pm polyvinylidene fluoride transfer membrane (Gelman Laboratory, Ann Arbor, MI). Blots were blocked with Tween-20 Tris-buffered saline (25 m M Tris-HCl, pH 7.5, 150 m M NaCl, 0.05% Tween-20) containing 5% skim milk, and then probed with antibodies according to the manufacturer's instructions and with HRP-goat antimouse IgG (Zymed Laboratories, San Francisco, C A ) coupled to peroxidase. Blots were developed using an enhanced chemiluminescence system (Amersham Biosciences, U K ) .

Statistical Analysis Data in the figures are presented as mean ± SD values. Statistical significance was assessed using one-way analysis of variance followed by twotailed Dunnett's /-test, with the level of significance set at /?

Vitamin C Protects Against Hydrogen Peroxide-Induced Inhibition of

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