Sep 20, 1996 - Glutathione and buthionine sulfoximine were purchased from Sigma. Chemical Co. (St Louis, MO); and acetonitrile and monohydrate sodium.
carc$$0112
Carcinogenesis vol.18 no.1 pp.37–42, 1997
Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells
Brad L.Upham1, Kyung-Sun Kang, Hye-Youn Cho and James E.Trosko
Oxidants are known to play a role in several stages of carcinogenesis, in particular as promoters and progressors (1–6). In addition, promoters that lack oxidizing properties might owe part of their carcinogenic activity to the induction of a cellular pro-oxidant state (3). For example, the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA*), induced an increase in the ratio of oxidized over reduced glutathione in mouse epidermal cells (7). Antioxidants, such as CuZn superoxide dismutase (SOD), inhibited the promoting activity of TPA in vivo and in vitro (8–10). Lipophylic and hydrophilic antioxidants, such as α-tocopherol, Trolox®, ascorbic acid and
2-o-octadecyl ascorbic acid, inhibited the induction of enzyme altered putative preneoplastic lesions in the livers of rats fed on a choline-deficient diet (11). The mechanism by which a pro-oxidative state causes tumor promotion is unknown. The onset of tumor promotion depends on a series of epigenetic events. A key epigenetic event in the clonal expansion of an initiated cell is the down-regulation of gap junctional intercellular communication (GJIC) (12–15). The down regulation of GJIC by tumor promoting agents, i.e. TPA, is often a consequence of an altered phosphorylation state of the gap junction protein (16–22). Therefore, the induction of signal transduction pathways involving kinases and phosphorylases could play an important role in the regulation of GJIC (20). Oxidative stress can also activate signal transduction pathways. For example, reactive oxygen species (ROS) are known to increase intracellular levels of Ca21 (23–25) and protein kinase C (PKC) activity (26). Oxidant induced increases in intracellular Ca21 has been implicated in phosphorylation of ribosomal protein (27) and activation of calmodulin dependent protein kinases (9). Exogenous and endogenous hydrogen peroxide also initiates the induction of mitogen activated protein kinase (MAPK), particularly the extracellular signal regulated kinase (ERK), and increased the level of mRNA expression of MAPK-dependent genes such as c-jun, c-fos and MAPK-phosphatase-1 (28,29). Lipid peroxidation is also known to activate various phospholipases (30), in which the lipid hydrolysis products such as inositol triphosphate (IP3) can alter Ca21 levels (31) and diacyl glycerol can activate protein kinases and acid sphingomyelinases (32). Ceramide, a hydrolysis product of sphingomyelin (SM) by acid sphingomyelinases, can also activate protein kinases (32). ROS have also been shown to activate the pleiotropic transcription factor NF-kB (32—35) and changes in antioxidant levels will activate the transcription factors c-myc, c-jun, c-fos and AP1 (35,36). The role of ROS in GJIC has not been extensively studied but antioxidants were shown to alleviate the phenobarbital and DDT-induced down regulation of GJIC (37) and paraquat generated oxygen radicals inhibited GJIC (38). Hydrogen peroxide (H2O2) also inhibits GJIC (39,40). Glutathione (GSH) peroxidase mimetics and antioxidants reversed the inhibitory effect of TPA on GJIC and prevented the hyperphosphorylation of GJIC proteins by TPA (41,42). In this study we looked at the inhibitory effect of H2O2 on GJIC and on the phosphorylation pattern of the gap junction protein connexin43 in WB-F344 rat liver epithelial cells. We also determined if GSH, the major reductant of H2O2, modified the inhibitory effect H2O2 had on GJIC.
*Abbreviations: TPA, 12-O-tetradecanoyl phorbol-13-acetate; SOD, superoxide dismutase; GJIC, gap junctional intercellular communication; PKC, protein kinase C; MAPK, mitogen activated protein kinase; ERK, extracellular signal regulated kinase; IP3, inositol triphosphate; SM, sphingomyelin; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; DEM, diethylmaleate; Cx43, connexin43.
Chemicals The following chemicals are from Bio-Rad Laboratories (Hercules, CA): sodium dodecyl sulfate (SDS), Tween 20, TRIS, glycine, acrylamide and TEMED. Glutathione and buthionine sulfoximine were purchased from Sigma Chemical Co. (St Louis, MO); and acetonitrile and monohydrate sodium
Department of Pediatrics and Human Development, Michigan State University, B240 Life Sciences, East Lansing, MI 48824, USA 1To
whom correspondence should addressed
Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 µM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox®, did not prevent the inhibition of GJIC by H2O2 , which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione-sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH. Introduction
© Oxford University Press
Materials and methods
37
B.L.Upham et al. Bioassay of GJIC The scrape loading/dye transfer (SL/DT) technique was adapted after the method of El-Fouly et al. (44) and described in detail by Upham et al. (45). The H2O2 was added to 2 ml of cell culture medium from a 100 mM stock solution, which was made up in de-ionized, distilled water. The culture medium was batch-treated with chelating resin for 4 h prior to the addition of H2O2 . Bioassay of cytotoxicity Cytotoxicity was determined by the neutral red uptake assay according to the method of Borenfreund and Puerner (46). WB-F344 cells were grown at the same conditions and confluency as those grown for the SL/DT assay. Following the H2O2 treatment, the cells were rinsed three times with PBS and then 2 ml of fresh growth medium containing 0.033% neutral red was added to the cells for 1 h. Prior to adding this neutral red solution to the cells, the neutral red was incubated with the D-medium for 2 h and the resulting precipitation was centrifuged at 2000 g and filtered through a 0.22 µ-Millipore syringe filter (Millipore, New Bedford, MA). The time required for the adequate uptake of neutral red into WBF344 cells was 1 h. The extracellular neutral red was rinsed off with PBS and the cells were lysed with 2 ml of an aqueous solution containing 1% acetic acid and 50% ethanol. The lysed cells were measured for neutral red at a wavelength of 540 nm and a background absorbance was measured at 690 nm.
Fig. 1. Dose–response of WB F344 rat liver epithelial cells to H2O2. Cells were treated with H2O2 for 1 h. Each value represents an average of three replicates 6 one SD.
Western blot analysis of connexin43 Cells were grown either in 25 cm2 flasks or 60-mm tissue culture plates from Corning (Corning, NY) to the same confluency as the SL/DT assay. The cells were treated with H2O2 in the same way as the SL/DT assay. The proteins were extracted with 20% SDS solution containing 1 mM phenylmethylsulfonyl fluoride (PMSF) which is a protease inhibitor. The protein content was determined with the DC assay kit (Bio-Rad Corp., Richmond, CA). The proteins were separated on 12.5% SDS–PAGE according to the method of Laemmli (47). The amount of protein loaded onto the gels was 15 µg. The proteins were electrophoretically transferred from the gel to PVDF membranes (Millipore Corp, Bedford, MA) according to the method of Matesic et al. (48). Connexin43 was detected with anti-connexin43 monoclonal antibodies (Zymed, South San Francisco, CA) for Figure 4 and with anti-connexin43 polyclonal antibodies (Zymed, South San Francisco, CA) for Figure 5 using the ECL detection kit (Amersham, Life Sci, Denver CO) according to the method of Kang et al. (22). GSH determination Cells were lysed with 2 ml of 0.05 N perchloric acid and the lysate was filtered through a 0.22 µm filter and used for the determination of GSH. The lysate was fractionated using an 150 mm34.6 mm adsorbosphere C18 5 µ MF-Plus HPLC column (Alltech Assoc., Deerfield, IL) and a mobile phase that consisted of 50 mM sodium phosphate, 0.05 mM 1-octanesulfonic acid, 2% acetonitrile, pH 5 2.70 and a flow rate of 1.0 ml/min. The HPLC system used was a 580 solvent delivery module from ESA (Chelmsford, MA). The GSH was detected with a Coulochem Model 5200 electrochemical detector (ESA, Chelmsford, MA) with applied potentials of 1400, 1900 and 1950 mV set for the screening, analytical and guard cell electrodes, respectively.
Fig. 2. Time of recovery from H2O2-induced inhibition of GJIC. Cells were treated for 1 h with 500 µM H2O2 after which the cells were rinsed with PBS and fresh H2O2-free media was added to the cells. GJIC was measured after the media was changed at the indicated times.
dihydrogen phosphate were purchased from EM Science (Gibstown, NJ). Sodium chloride, methanol, sodium 1-octanesulfonate and ammonium persulfate are from Columbus Chemical Industries (Columbus, WI), J.T.Baker, Inc. (Phillipsburg, NJ), ACROS (Pittsburgh, PA) and Life Technologies, Inc. (Gaithersburg, MD), respectively. Cell culture WB-F344 rat liver epithelial cell lines were obtained from Drs J.W.Grisham and M.S.Tsao of the University of North Carolina (Chapel Hill, NC) (43). Cells were cultured in 2 ml of D medium (Formula No. 78-5470EF, Gibco Laboratories, Grand Island, NY), supplemented with 5% fetal bovine serum (GIBCO Laboratories, Grand Island, NY). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells were grown in 35 mm tissue culture plates (Corning Inc., Corning, NY) and the culture medium was changed every other day. Bioassays were conducted with confluent cultures that were obtained after 2 to 3 days of growth.
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Results Cytotoxicity of H2O2 Hasler et al. (39) showed that a dose of 500 µM H2O2 maximally inhibited GJIC in WB F344 rat liver epithelial cells and was non-cytotoxic as determined by lactate dehydrogenase activity. Therefore, we also used a dose of 500 µM H2O2 for all experiments and determined cytotoxicity by an alternative technique using a neutral red uptake assay. The viable uptake of neutral red by WB-F344 cells treated with 500 µM H2O2 was 0.62 6 0.08 OD540–690 as compared with the uptake of neutral red in untreated cells (0.73 6 0.18 OD540–690). These results were not significantly different as determined by a Student’s paired t-test at P ø 0.05 and n 5 3. Dose–response H2O2 inhibited GJIC in a dose–response fashion (Figure 1). Maximal inhibition occurred at 400 µM. The concentration that inhibited at 0.50 of the fraction of the control was 200 µM (I50).
Inhibition of GJIC by H2O2 and glutathione
Table I. SL/DT results of cells treated with 500 µM H2O2 in the presence and absence of free radical scavengers Treatment
GJIC (Fraction of the control)
Control H2 O2 t-Butanol t-Butanol 1 H2O2 Propylgallate Propylgallate 1 H2O2 Trolox® Trolox® 1 H2O2
1.00 0.38 0.93 0.35 0.91 0.36 0.90 0.35
6 6 6 6 6 6 6 6
0.06 0.06 0.03 0.07 0.03 0.04 0.04 0.00
Concentrations of tertiary-butyl alcohol (t-butanol), propylgallate and Trolox® were 500, 750 and 750 µM, respectively. Each datum is reported as a fraction of the distance the dye traveled in the control in which any number less than 1.00 was inhibitory. Each value represents the average of at least three replicates 6 one standard deviation.
Table II. The effect of hydrogen peroxide on GJIC in GSH sufficient and deficient cells. Treatment
GJIC (Fraction of the control)
Control H2O2, 1 h H2O2, 1 h1 aminotriazole, 1h BSO, 1 h (0%↓ in GSH) BSO, 24 h (72%↓ in GSH) 1 aminotriazole, 1h DEM, 1 h (95%↓ in GSH) H2O2, 1 h 1 BSO, 1 h H2O2, 1 h 1 BSO, 24 h H2O2, 1 h 1 DEM, 1 h
1.00 0.31 0.32 1.01 0.99 1.06 0.32 1.01 1.06
6 6 6 6 6 6 6 6 6
0.08 0.05 0.04 0.06 0.03 0.05 0.05 0.04 0.20
Fig. 3. Photographs of scrape load/dye transfer of cells treated with and without 500 µM H2O2 and 100 µM BSO.
SL/DT results of cells treated with 500 µM H2O2 and depleted of GSH with either 100 µM BSO or 1 mM diethylmaleate (DEM). The concentration of aminotriazole was 20 mM. Each value represents the average of at least three replicates 6 one standard deviation. The values in parentheses are the reduction in GSH levels as compared with the control and was measured with the HPLC-system described in the Materials and methods section.
Effects of H2O2 on gap junctional intercellular communication WB-F344 rat liver epithelial cells fully recovered from inhibition of GJIC by 500 µM H2O2 4 h after the cell media containing H2O2 was replaced with H2O2-free media (Figure 2). Aqueous free radical scavengers such as t-butanol, Trolox® and propylgallate did not affect GJIC or change the level of inhibition of GJIC by H2O2 (Table I). Effects of intracellular glutathione on gap junctional intercellular communication Glutathione levels were measured using HPLC-electrochemical detection and were reported as a percentage of the GSH measured in the control cells. BSO and DEM depleted intracellular GSH by 72% and 95%, respectively (Table II). Photographic images of the scrape loading/dye transfer technique are shown in Figure 3. These results showed that H2O2 inhibited GJIC only in the GSH-sufficient cells and that depletion of intracellular GSH did not affect GJIC. These experiments, along with diethylmaleate (DEM) treated cells, were replicated in triplicate and the results were reported as the fraction the dye traveled in the control (Table II). The depletion of intracellular GSH had no effect on GJIC (Table II). H2O2 did not inhibit GJIC in cells depleted of GSH in either BSO or DEM-treated cells. In contrast, treatment of cells with BSO for 1 h did not deplete intracellular GSH and
Fig. 4. Western blot analysis of connexin 43 protein, which makes up gap junction proteins. The concentrations of TPA (lane 2) and H2O2 (lane 3) were 10 ηg/ml and 500 µM, respectively.
the inhibition of GJIC by H2O2 was not reversed (Table II). Aminotriazole did not affect GJIC or alter the level of H2O2induced inhibition of GJIC (Table II). Effects of H2O2 on the phosphorylation status of gap junctions Western blot analysis using antibodies specific to Cx43 was used to assess the phosphorylation status of the gap junction proteins (Figures 4 and 5). The blots showed three major 39
B.L.Upham et al.
Fig. 5. Western blot analysis of connexin43 protein extracted from cells treated with H2O2 (500 µM) in GSH-deficient or sufficient cells. BSO (500 µM) was used to deplete the cells of GSH to a level that was 76% less than the control. GSH was measured with the HPLC-system described in the Materials and methods section. This experiment was done in triplicate on two separate occasions in which the phosphorylation patterns were the same as that shown in this figure.
bands ranging from the unphosphorylated (P0) to the increased phosphorylated states (P1, P2). Cells treated with H2O2 displayed a mobility shift in the bands to the higher molecular weight P2-band (Figure 4, lane 3). The depletion of intracellular GSH by BSO did not alter the phosphorylation pattern of Cx43 as compared with the control (Figure 5, lane 3) but when these GSH-deficient cells were treated with H2O2, hyperphosphorylation of Cx43 was observed (Figure 5, lane 5). Anti-Cx43 polyclonal antibodies were used in the experiments of Figure 5 and the observed hyperphosphorylation pattern of H2O2 versus TPA treated cells were different (lanes 2 versus 7). H2O2 and TPA both caused the disappearance of the Po band but TPA caused a much stronger induction of the P3 band. These observations contrasted with those of Figure 4 in which anti-Cx43 monoclonal antibodies were used in these experiments and showed that the hyperphosphorylation pattern of H2O2 versus TPA treated cells were similar (lanes 2 versus 3). Discussion The carcinogenic effect of oxidant stress has focused primarily on the genotoxicity of ROS (49,50). However, active oxygen is known to play a significant role in the promotional phase of cancer (3,51). The promotional phase of cancer is a consequence of epigenetic events involving signal transduction and GJIC (52,53). Promotion is a reversible step in carcinogenesis, therefore the underlying mechanism of tumor promotion, such as the down regulation of GJIC, should also be reversible (21). Our results showed that H2O2, a known tumor promoter (6), reversibly inhibited GJIC (Figure 2) at a noncytotoxic dose. These observations also agree with the results of other tumor promoting reagents that were shown to reversibly inhibit GJIC (13,14). The generation of the extremely reactive hydroxyl radical (•OH) in Fenton-type reactions is often considered the central mechanism by which H2O2 induces oxidative damage in biological systems (54). However, in our system inhibition of GJIC by H2O2 was not a consequence of aqueous free radicals as determined by the lack of a scavenging effect by water soluble free radical scavengers (Table I). The OH radical is not the only free radical species scavenged by these antioxidants. For example, Trolox was shown to be an excellent scavenger of peroxynitrite radicals (55,56). This lack of a free 40
radical scavenging effect indicated that H2O2 was probably the major reactive oxygen species responsible in the ultimate down regulation of GJIC. The endogenous scavenging system of H2O2 involve the enzymes catalase and GSH-peroxidase (57,58). Catalase is located primarily in the peroxisomes and catalyzes the dismutation of H2O2 to water and O2 (59). GSH peroxidase is located primarily in the cytosolic fraction of the cell and catalyzes the reduction of H2O2 to water using GSH as the reductant (57,58). Hu and Cotgreave (40) showed that the depletion of intracellular GSH levels potentiated the inhibitory effect of TPA on GJIC. Therefore, we also explored the possibility that the down regulation of H2O2-scavenging systems would potentiate the inhibitory effect of H2O2 on GJIC. Aminotriazole is a specific inhibitor of catalase (60) and buthionine sulfoximine is a specific inhibitor of γ-glutamyl synthetase that catalyzes the rate limiting step of GSH biosynthesis (61). Aminotriazole did not alter the inhibitory effect of H2O2 on GJIC but the depletion of intracellular levels of GSH by BSO actually reversed H2O2-induced inhibition of GJIC (Table II). These results were not a consequence of BSO directly scavenging H2O2 because cells treated with BSO for only 1 h were not depleted of GSH and the inhibitory effect of H2O2 was not reversed in this situation (Table II). Intracellular levels of GSH was also depleted by the GSHconjugating compound diethyl maleate (DEM) and again the inhibitory effect of H2O2 on GJIC was reversed (Table II). These results suggest that GSH is an integral component of the mechanism of H2O2-induced inhibition of GJIC. The depletion of GSH alone, however, does not effect GJIC activity (Table II), which is consistent with the results of Hu and Cotgreave (40). The phosphorylation status of gap junction proteins play an important role in the gating of gap junction channels (20,62). Many exogenous and endogenous chemicals that regulate GJIC also alter the phosphorylation state of the connexins, which are the proteins that form the hexameric gap junction channels (20,22). For example, TPA inhibition of GJIC in F344-rat liver epithelial cells was correlated with the hyperphosphorylation of connexin43 (Cx43) as measured by mobility shifts of the western blot bands of Cx43 (48). TPA-induced hyperphosphorylation of Cx43 is a result of protein kinase C (PKC) activation, in which PKC is translocated from the cytosolic to the particulate fraction of the cell (63). Similarly, we showed that H2O2 also caused a shift in the Cx43 bands to a hyperphosphorylated state (Figure 4). However, H2O2 also induced hyperphosphorylation in GSH-deficient cells (Figure 5), which had normal GJIC activity (Figure 3) that indicated that hyperphosphorylation was different from that of TPA-treated cells. These results also demonstrated that the global hyperphosphorylation of gap junction proteins cannot always be correlated with the down regulation of GJIC. The kinase pathway involved in the H2O2-induced hyperphosphorylation of Cx43 was not determined but PKC probably was not involved. H2O2 is also known to induce various MAPKs such as ERK2, p38 and JNK1 (28). The activation of ERK2 by H2O2 was the most prevalent and was not through a Ras-independent pathway which rules out PKC-activation of Raf (28). However, the involvement of MAPKs in the gating of gap junctions is not known. H2O2 also induces other signal transductants which are known to modulate gap junction function. In particular, Ca21 is a well known regulator of GJIC (64). Again the
Inhibition of GJIC by H2O2 and glutathione
interactive roles of signal transductants, kinase systems and GJIC is not well understood (65). Various GSH-mimetics reversed the inhibitory effects of TPA on GJIC and prevented the hyperphosphorylation of Cx43 (41). Depletion of GSH by BSO also potentiated TPA-induced inhibition of GJIC in WB-F344 cells (40). These results suggest that GSH plays an integral role in protecting the cell from TPA-dependent down regulation of GJIC. Based on these observations, we expected that the depletion of intracellular GSH would also potentiate the GJIC-inhibitory effect of H2O2. However, the observed results were the opposite of the expected potentiating effect in which a 75–95% depletion of intracellular GSH actually prevented 500 µM H2O2 from inhibiting GJIC (Figure 3; Table II). Apparently, the inhibition of GJIC required the presence of GSH but the reason for this was not determined. Similar results were observed by Kuo et al. (66), who showed that methylmethane sulfonate, at a non-genotoxic level, induces c-jun mRNA in GSH-sufficient but not deficient NIH 3T3 cells. The underlying mechanism of GSH-dependent induction of c-jun was not determined in the NIH 3T3 cells. Possibly, the H2O2 induced-signal transduction system responsible for the gating of gap junctions or the induction of c-jun required reducing equivalents from GSH or the conjugation of GSH to a key signal transductant. Conclusion The inhibition of gap junctional intercellular communication by H2O2 was a non-genotoxic and reversible event that resulted from the activation of a glutathione-dependent signal transduction pathway(s). Acknowledgements We would like to acknowledge the United States Air Force grant No. USAFOSR 94–NL-196 and the NIEHS Superfund grant No. ES04911–06, for the funding of this research.
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