IL-6 inhibits apoptosis and retains oxidative DNA lesions in human gastric cancer AGS cells through up-regulation of anti-apoptotic gene mcl-1

Ming-Tsan Lin1, Chiung-Yao Juan1,2, King-Jen Chang1, Wei-Jao Chen1 and Min-Liang Kuo2,3

1 Department of Surgery, National Taiwan Hospital, Taipei and
2 Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apoptosis plays a critical role in maintaining genomic integrity by selectively removing the most heavily damaged cells from the population. Reactive oxygen species (ROS) and certain inflammatory cytokines are always elevated during the human carcinogenic process. However, the biological significance of the interplay between ROS and inflammatory cytokine remains elusive. This study demonstrates that interleukin-6 (IL-6) effectively protects gastric cancer cells from the apoptosis induced by hydrogen peroxide (H2O2). The cell death signaling JNK pathway elicited by H2O2 is also inhibited by IL-6. We further found that Mcl-1, but not other Bcl-2 family members, was up-regulated by IL-6, by a substantial level over 24 h. We further transfected a mcl-1 expression vector, pCMV-mcl-1, into the AGS cells, and successfully obtained several mcl-1-overexpressing clones. Flow cytometric analysis shows that these mcl-1-overexpressing AGS cells are more resistant to the apoptosis induced by H2O2 when compared with the neo control AGS cells. Consistently, the activation of the JNK pathway induced by H2O2 is also blocked in mcl-1-overexpressed cells. These results indicate that the anti-apoptotic effect of IL-6 is, at least in part, due to the up-regulation of mcl-1. To our surprise, either IL-6 exposure or mcl-1 overexpression fails to reduce the level of intracellular peroxides in the AGS cells triggered by H2O2. This study also determined the level of 8-hydroxydeoxyguanosine (8-OH-dGua), an indicator for oxidative DNA lesions in IL-6-treated or mcl-1-overexpressed AGS cells after treatment with H2O2. Notably, our results indicate that a majority of the 8-OH-dGua is efficiently removed in the AGS cells without IL-6 treatment, whereas only ~50% of the 8-OH-dGua was repaired in the IL-6-treated AGS cells after 24 h. Similarly, ~60–70% of the 8-OH-dGua also failed to repair and was retained in the genomic DNA of the mcl-1 transfectants. Results in this study provide a novel mechanism by which up-regulation of the Mcl-1 protein by IL-6 may enhance the susceptibility to H2O2-induced oxidative DNA lesions by overriding apoptosis.

Abbreviations: ROS, reactive oxygen species; IL-6, interleukin-6; H2O2, hydrogen peroxide; 8-OH-dGua, 8-hydroxydeoxyguanosine


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Programmed cell death or apoptosis profoundly influences a wide variety of physiological processes. The active physiological cell death selectively removes the most heavily damaged cells from tissues. Dysregulation of apoptosis has been implicated in several human diseases, ranging from cancer to autoimmunity, AIDS and neurological disorders (13). An accumulation of evidence has indicated that many tumor promoters exert their activities by inhibiting apoptosis (4,5). A supportive study has demonstrated that transformation of the colorectal epithelium into adenomas and carcinomas are associated with a progressive inhibition of apoptosis (6). The bcl-2 gene family is the key regulator of apoptosis and was found to have increased expression in a wide variety of human cancers (1,7,8). Bcl-2 and its family members have shown the ability to block apoptosis induced through different stimuli (911). Overexpression of the bcl-2 gene in transgenic mice leads to lymphomagenesis, implying that anti-apoptotic Bcl-2 protein expression could promote oncogenic potency (12). Our recent study and those of others have further demonstrated that bcl-2- or bcl-xL-transfected cells conferred resistance to apoptosis and enhanced the frequency of gene mutations in response to oxidative stress (13,14). However, which physiological factors might regulate these apoptosis-related genes and in turn facilitate the oncogenic process is largely unknown.

Chronic infection and inflammation have long been recognized as risk factors for a variety of human cancers. Gastric carcinogenesis is a typical infection and inflammation-associated pathological alterations in which Helicobacter pylori plays a critical role (15). Current knowledge of the detailed mechanisms underlying the interplay between the biological modulators and the lesions induced by H.pylori is still incomplete. For instance, reactive oxygen species (ROS) could be elevated and cause the oxidative DNA lesions, 8-hydroxydeoxyguanosine (8-OHdG) in gastric epithelial cells during H.pylori-elicited inflammation (16). The level of oxidative DNA lesions was significantly higher in patients with chronic atrophic gastritis or gastric cancer than in normal gastric tissues, indicating that a progressive accumulation of oxidative DNA lesions could play a major role in gastric carcinogenesis (17).

Cytokines have been proposed to play an important role in H.pylori-associated gastric inflammation and carcinogenesis, but the exact mechanism remains unclear. Several studies have indicated that infection with H.pylori induced the expression and production of various cytokines in the gastric mucosa, epithelial cells, or macrophages (18,19). Interleukin-6 (IL-6), but not other cytokines, has recently been reported to be induced by H.pylori infection in gastric epithelial cells (20). In addition, IL-6 has a strong activity in stimulating the growth of human gastric cancer cell lines (21). These findings suggest that IL-6 may have a potential role in the pathogenesis of gastric cancer.

To gain insight into the molecular mechanisms of gastric carcinogenesis, we designed experiments to investigate the effect of IL-6 on the ROS induced apoptosis and oxidative DNA lesions in human gastric cancer AGS cells. The results presented herein demonstrate that IL-6 could inhibit hydrogen peroxide-induced apoptosis and death signaling JNK pathway activation. The anti-apoptotic protein Mcl-1 was significantly up-regulated in AGS cells by IL-6. Transfection of cells with Mcl-1-expressing vectors resulted in resistance to apoptosis and blockage of death signaling JNK activation. Interestingly, we further found that a majority of the oxidative DNA lesions, i.e. 8-OH-dGua, was not repaired in IL-6-treated or mcl-1-overexpressing AGS cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and cell culture
Human gastric cancer AGS cells obtained from the American Type Culture Collection (Rockville, MD) were cultured in RPMI 1640 supplemented with fetal bovine serum (10%) and gentamicin sulfate (50 µg/ml). Cells were grown in humidified atmosphere in 5% CO2 and 37°C. Cell viability was determined using trypan blue exclusion in which 200 cells/culture were analyzed. All initial viabilities were >98%. Hydrogen peroxide, propidium iodide, catalase, proteinase K, ribonuclease A, nuclease P1 and alkaline phosphatase were purchased from Sigma Chemical (St Louis, MO). DCFH-DA was from Molecular Probes, Inc (Eugene, OR).

Establishment of mcl-1 overexpressing clones
AGS cells constitutively expressing human mcl-1were created by transfection of AGS cells with mcl-1 expression vector, pCMV-mcl-1, as described in our previous study (22). Transfection was performed using Lipofectamine reagent (Life Technologies, Gothenburg) or Transfectin (Promega Co., Madison, WI) according to the manufacturers' instruction. For selecting stable clones, G418 (400 µg/ml) was added to culture medium 48 h after transfection. Finally, several independent resistant clones were obtained and were subjected to determine Mcl-1 protein levels by immunoblotting.

Quantification of apoptosis by flow cytometry
Apoptosis was measured by flow cytometry, using a fluorescein isothiocyanate (FITC) conjugate of Annexin V and propidium iodide ( Oncogene Research Products, Cambridge, MA). Annexin V binds to early apoptotic, as well as late apoptotic/necrotic cells. Propidium iodide binds to late apoptotic/necrotic cells. Briefly, AGS cells (1 x 106 cells/60 mm dish) were exposed to 0–60 ng/ml IL-6 or 500 units/ml of catalase for 4 h and followed by treatment with 1 mM H2O2 for a further 24 h. Treated cells were removed with trypsin-EDTA, washed twice with 0.01 mol/L PBS, pH 7.4, centrifuged, and then treated as directed by the manufacturer.

Detection of peroxides by flow cytometry
AGS cells or mcl-1-overexpressing cells (1 x 106 cells/dish) were exposed to 20 ng/ml IL-6 for 24 h and followed by treatment with 1 mM H2O2, 20 ng/ml IL-6, 20 ng/ml IL-6 plus 1 mM H2O2, 500 units/ml catalase, or 500 units/ml catalase plus 1 mM H2O2 for a further 1 h. DCFH-DA, a sensitive fluorimetric probe of peroxides, was dissolved in ethanol, 10 µM DCFH-DA were added to the medium, and the cells were incubated for 30 min at 37°C. After the incubation, the medium was removed, and the cells were washed with PBS once and suspended in PBS. The cells were analyzed with a FACScan.

Immunoblotting
Cells were lysed in a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1% NP-40, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin and leupeptin, pH 7.4) for 20 min on ice. The lysates were centrifuged at 14 500 r.p.m. for 20 min at 4°C, and then protein concentrations were determined by using a commercial BCA kit (PIERCES Co.). A 50 µg sample of each lysate was subjected to a 12% SDS-polyacrylamide gel electrophoresis. Finally, proteins were then transferred to nitrocellulose paper and immunoblotted with antibodies indicated in the text. Detection was performed using the ECL (Amersham, Arlington Heights, IL).

Determination of 8-OH-dGua in DNA
DNA was isolated from AGS cells and mcl-1 transfectants by the phenol extraction procedure of Gupta (23). To avoid any additional oxidative damage to the DNA due to peroxide or quinone contaminants in phenol, high purity double distilled phenol was used for extractions. About 200–400 µg DNA were resuspended in 200 µl 20 mM sodium acetate (pH 4.8), and digested to nucleotides with 20 µg nuclease P1 at 70°C for 15 min. To adjust the pH, 20 µl of 1 M Tris–HCl (pH 7.4) were added to the nucleoside mixture, which then was treated with 1.5 units alkaline phosphatase and incubated at 37°C for 60 min. These hydrolyzed DNA solutions were then filtered using an Ultrafree Millipore filtration system (10 000 dalton cutoff). The HPLC conditions used in the present study have been described by Kolachana and in our previous study (13,24). Briefly, the amount of 8-OH-dGua present in the DNA was analyzed by flow-through electrochemical detection using an ESA model 5100 coulochem detector equipped with a 5011 high sensitivity analytical cell with the oxidation potentials of electrodes 1 and 2 adjusted to 0.1 and 0.35 V, respectively. A C18 HPLC column (15 x 4.6 mm) was utilized for separation of 8-OH-dGua. The mobile phase consisted of 10% methanol and 50 mM KH2PO4 buffer, pH 5.5, run isocratically at a flow rate of 1 ml/min.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
IL-6 prevents AGS cells from H2O2-induced apoptosis and JNK signaling pathway
To determine whether IL-6 affect H2O2-induced apoptosis, AGS cells (1 x 106 cells/60 mm dish) were pretreated with various concentrations of IL-6 for 30 min and then treated with 1 mM of H2O2 for an additional 6 h. Using a flow cytometric assay to quantitate the number of apoptotic cells as stained by Annexin-V, we observed IL-6 suppressed H2O2-induced apoptosis in a dose-dependent manner (Figure 1AGo). At a concentration of 60 ng/ml, IL-6 elicited a maximal protection. Notably, in the absence of H2O2, IL-6 did not promote or suppress cell survival (Figure 1AGo). In addition, the catalase (500 units/ml) treatment completely prevented H2O2-induced apoptosis in AGS cells (Figure 1AGo). The IL-6-mediated anti-apoptotic effect was also consistently demonstrated by the determination of the number of apoptotic nuclei by staining with the fluorescence dye Hoechst 33258 (Figure 1BGo, I–IV).




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Fig. 1. (A) Effect of IL-6 on H2O2-induced apoptosis in human gastric cancer AGS cells. AGS cells were exposed to 0–60 ng/ml IL-6 or 500 units/ml of catalase for 4 h and followed by treatment with 1 mM H2O2 for a further 24 h. Apoptotic cells were quantified by the flow cytometry analysis of annexin-V-stained samples, as described in Materials and methods. Data are the mean of three reproducible experiments. Bar, SD. The values indicated in the figure represent the percentage of apoptosis. (B) Nuclear morphological characteristics in AGS cells treated with IL-6 and/or H2O2. AGS cells were treated with buffer control (I), 1 mM H2O2 (II), 1 mM H2O2 and 20 ng/ml IL-6 (III), or with 20 ng/ml IL-6 alone (IV), and harvested at 12 h followed by Hoechst 33258 staining. The nuclear morphological changes of cells were detected by fluorescent microscope observation. Magnification: 40x.

 
Activation of the JNK signaling pathway is critical for H2O2-elicited apoptosis in a wide variety of cell systems (25,26). Thus we determined whether IL-6 suppressed H2O2-induced apoptosis through the inhibition of JNK activation. JNK activation was determined by detecting the level of phospho-JNK using a specific antibody (27). Consistent with the apoptosis assay data, IL-6 inhibited H2O2-elicited JNK activation in a dose-dependent manner (Figure 2AGo). Notably, IL-6 caused a strong inhibition on JNK activation with increasing concentrations of H2O2 up to 2 or 3 mM (Figure 2BGo). As expected, catalase, an antioxidant enzyme, effectively suppressed the H2O2-induced JNK activation (Figure 2BGo). These results suggest that IL-6 prevents H2O2-induced apoptosis through inhibiting the JNK pathway activation.




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Fig. 2. Effect of IL-6 on the phosphorylation of JNK by H2O2. (A) AGS cells were treated with varying concentrations of IL-6 (as indicated in the figure) for 4 h, and then treated with 1 mM H2O2 for a further 2 h. (B) AGS cells were pretreated 20 ng/ml IL-6 or 500 units/ml catalase for 4 h and then exposed to 2 or 3 mM H2O2 for a further 2 h. Cells were harvested and lysis for the subsequent SDS-polyacrylamide electrophoresis. Phospho-JNK (activated JNK) was detected by immunoblotting using a specific antibody (New England Biolabs, Inc., Beverly, MA) which recognized the phosphorylated Thr 183/Tyr185 residues of JNK.

 
Anti-apoptotic protein Mcl-1 is up-regulated by IL-6 in AGS cells
To investigate how IL-6 protects H2O2-induced cell death, we examined the expression of the Bcl-2 family of proteins in AGS cells after IL-6 treatment. Western blotting revealed that upon IL-6 stimulation, the Mcl-1 protein level became substantially elevated after 4 h, and maintained a maximal level over 24 h (Figure 3AGo). In contrast, the levels of other family members such as Bcl-2, Bcl-xL, and Bax were little affected. Whether the level of Mcl-1 would change with varying concentrations of IL-6 was further investigated. Upon a 10 ng/ml of IL-6 stimulation, the level of Mcl-1 increased significantly (Figure 3BGo). Expression of Mcl-1 was maximized at a concentration of 20 ng/ml (Figure 3BGo). Other Bcl-2 family protein levels were unaltered, even with a treatment of 80 ng/ml of IL-6.




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Fig. 3. IL-6 induces Mcl-1 protein expression in a time and dose-dependent manner. (A) AGS cells were exposed to 20 ng/ml of IL-6 for various periods of time as indicated. (B) AGS cells were treated with various concentrations of IL-6 for 8 h. After treatment, cell lysates were prepared and analysed by immunoblotting as described in Materials and methods. The antibodies including anti-MCL-1, anti-Bcl-2 and anti-Bcl-xL antibodies were obtained from Santa Cruz.

 
Overexpression of Mcl-1 in AGS cells confers resistance to apoptosis induced by H2O2
To further determine whether the anti-apoptotic effect of IL-6 is possibly due to the up-regulation of the Mcl-1 protein, a mcl-1 gene expression plasmid, pCMV-mcl-1, was transfected into AGS cells. After transfection, cells were cultured in medium containing 400 µg/ml of G418. Each colony that grew after 418 selection was picked and expended. western blotting analysis showed that the level of Mcl-1 protein in three stable clones (AGS/mcl-1-1, AGS/mcl-1-2, and AGS/mcl-1-5) was much higher than that in neo control cells (AGS/neo) (Figure 4AGo). The apoptosis sensitivity of these mcl-1 stable transfectants as well as neo control cells in response to H2O2 was then examined. Flow cytometry analysis of the apoptotic cells revealed that all mcl-1-overexpressed AGS clones were much less sensitive to H2O2 when compared with AGS/neo cells (Figure 4BGo). Trypan blue exclusion assay consistently confirmed that mcl-1-overexpressing AGS cells displayed more viability than the neo control AGS cells (data not shown).





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Fig. 4. Effect of Mcl-1 overexpression on H2O2-induced JNK activation and apoptosis. (A) Expression of Mcl-1 protein in several clonal cell lines by immunoblotting. (B) Sensitivity of Mcl-1 overexpressed cells and vector control cells to H2O2-induced apoptosis. Briefly, AGS/mcl-1-1, AGS/mcl-1-5, or AGS/neo cells were exposed to 1 mM H2O2 for various periods of time. The apoptotic cells were quantified by the flow cytometric analysis of Annexin-V-stained cell samples. Data are the mean of two highly reproducible experiments. Bar, SD. (C) Mcl-1 overexpression inhibits JNK activity elicited by H2O2. Mcl-1 transfectants and neo control cells were treated with 0, 1, 2, or 3 mM H2O2 for 2 h, after which cells were harvested and the cytosolic fraction analyzed for JNK1. The kinase activities were determined by immune complex kinase assay and the protein levels were determined by immunoblotting as described in Materials and methods. GST-c-jun was used as a substrate for JNK1.

 
We wondered if mcl-1 expression would also affect the JNK activity elicited by H2O2. A JNK kinase activity assay was thus performed in AGS/mcl-1-1 and AGS/neo cells following exposure to 1 mM or 2 mM of H2O2. The immuno-complex kinase assay demonstrates that H2O2-elicited JNK activity could be blocked in mcl-1-overexpressing cells but not in neo control cells (Figure 4CGo). This observation is correlated well with our above data that IL-6 treatment effectively suppressed H2O2-induced JNK phosphorylation (JNK activation) in AGS cells (Figure 2A and BGo).

These results suggest that up-regulation of Mcl-1protein in AGS cells can prevent H2O2-triggered JNK activation and the subsequent apoptosis.

IL-6 treatment or mcl-1 overexpression fails to inhibit H2O2-induced oxidative stress in AGS cells
Since a previous study has contended that the Bcl-2 family of protein may act as an antioxidant to protect cells from oxidative damage (28), we speculate that IL-6 exposure or Mcl-1 up-regulation prevented H2O2-induced JNK activation and apoptosis is mediated by neutralization of intracellular peroxides. To address this issue, we initially determined the intracellular peroxides level in AGS cells treated with IL-6 and H2O2 by using a dye DCFH-DA. Flow cytometric analysis shows that a similar peroxides level was observed in H2O2-treated AGS cells in the presence or absence of IL-6 (Figure 5AGo, I–IV). However, catalase treatment strongly abolished H2O2-elicited peroxides production in AGS cells (Figure 5AGo, V–VI). Under the same experimental conditions, we found that mcl-1-overexpressing cells and neo control cells generated a relatively equal peroxide level when exposed to H2O2 (Figure 5BGo). Catalase treatment almost abolished the H2O2-induced increase of intracellular peroxides in mcl-1-overexpressing or neo control cells (Figure 5BGo).




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Fig. 5. (A) Effect of IL-6 on H2O2-induced intracellular peroxides level. AGS cells were exposed to 20 ng/ml IL-6 for 24 h and followed by treatment with none (I), 1 mM H2O2 (II), 20 ng/ml IL-6 (III), 20 ng/ml IL-6 plus 1 mM H2O2 (IV), 500 units/ml catalase (V), or 500 units/ml catalase plus 1 mM H2O2 (VI) for a further 1 h. Intracellular peroxides level was quantified by DCFH fluorescence using a flow cytometer as described in Materials and methods. The data are representative of two independent and reproducible experiments. (B) Effect of Mcl-1 overexpression on H2O2-induced intracellular peroxides level. AGS/mcl-1-1 and neo control cells were exposed to 1 mM H2O2 with or without 500 units/ml catalase for 1 h. Intracellular peroxides were determined by DCFH fluorescent compound using a flow cytometer. Each value represents the mean ± SD of three different experiments.

 
The above data suggest that the inhibition of H2O2-induced apoptosis by IL-6 exposure or up-regulation of Mcl-1 protein is not mediated by decreasing the intracellular peroxides level.

Effect of IL-6 and Mcl-1 overexpression on H2O2-induced oxidative DNA lesion
The above data suggest that IL-6 treatment or Mcl-1 overexpression, although they prevented H2O2-induced apoptosis, failed to reduce the peroxide level within cells. This led us to suspect whether IL-6 treatment would affect the integrity of genomic DNA in surviving AGS cells following exposure to H2O2. To test this hypothesis, we pre-incubated AGS cells with or without 40 ng/ml of IL-6 for 24 h and then further treated the cell with1 mM H2O2 plus another 40 ng/ml IL-6 for different periods of time. The genomic DNA from each cell culture was subsequently extracted and then the extent of oxidative DNA damage, i.e. the formation of 8-OH-dGua, was analyzed using HPLC analysis system. Figure 6AGo indicates that cell treatment with 1 mM H2O2 for 30 min resulted in a 4.5-fold increase in 8-OH-dGua levels; however, the increase declined towards background levels after 3 h and remained constant through 24 h. A slight amount or no cytotoxicity was observed from exposure to 1 mM H2O2 for at least 6 h (data not shown), indicating that 8-OH-dGua formation in cells does not occur after cell death. Notably, IL-6 treatment did not affect the maximum 8-OH-dGua level in cells after 30 min exposure to H2O2 (Figure 6AGo). However, IL-6 retained over 65% of the level of 8-OH-dGua in AGS cells after 3 h of H2O2 treatment. Upon 24 h of H2O2 treatment, ~40–50% of the 8-OH-dGua was retained in the genomic DNA of IL-6-exposed AGS cells (Figure 6AGo). Again, catalase treatment completely inhibited H2O2-induced 8-OH-dGua formation in AGS cells (data not shown).




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Fig. 6. (A) Time course of H2O2-induced DNA 8-OH-dGua formation in AGS cells. AGS cells (3 x 106) were treated with 1 mM H2O2 or 1 mM H2O2 plus 20 ng/ml IL-6 for various periods of time as indicated. After treatment, DNA in each sample was extracted and 8-OH-dGua was determined by using HPLC as described in Materials and methods. (B) Time course of H2O2-induced DNA 8-OH-dGua formation in Mcl-1 overexpressing and neo control AGS cells. Briefly, both cells were treated with 1 mM H2O2 for various periods of time as indicated. Measurement of 8-OH-dGua was performed using HPLC. The data are mean ± SD, n = 4.

 
Furthermore, we determined the level of 8-OH-dGua in mcl-1-overexpressing cells and neo control cells treated with H2O2. Interestingly, the increased level of 8-OH-dGua induced by H2O2 in the neo control cells was rapidly and efficiently removed within 1 h (Figure 6BGo). In contrast, over 70% of 8-OH-dGua was not repaired in the mcl-1 transfectants treated with H2O2 for 24 h (Figure 6BGo).

The above results indicate that enhanced expression of anti-apoptotic Mcl-1 protein by IL-6 may interfere with certain cellular functions that possibly regulate and maintain the genomic integrity.


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
IL-6 is a pleotropic cytokine capable not only of inducing growth and differentiation but also of modulating cellular apoptosis in many cell types (2931). Our previous studies have demonstrated that IL-6 effectively protected human hepatoma cells (32), basal cell carcinoma cells (33) and cervical cancer cells (34) from apoptosis induced by a wide variety of stimuli, including TGF-ß1, retinoic acid, UV irradiation, and anticancer drugs. The data presented in this study show that IL-6 is also capable of preventing apoptosis of human gastric cancer AGS cells following exposure to H2O2. Based on these findings, we suggest that IL-6 is a fundamental survival factor for epithelial type of cancer cells. The anti-apoptotic effect of IL-6 is possibly attributed to the up-regulation of the Bcl-2 family of genes. In pro-B cells, IL-6 up-regulates the bcl-2 gene and provides a survival advantage (35). IL-6 has been found to prevent apoptosis in multiple myeloma cell lines by the coordinate expression of Bcl-XL and Mcl-1 mRNA and proteins (36,37). Our recent studies revealed that Mcl-1, but not other Bcl-2 family proteins, is dominantly elevated in many cancer cell systems in response to IL-6 (32,33). Here we found that Mcl-1 is also significantly increased in human gastric cancer cells by IL-6. These investigations strongly indicate that Mcl-1 is an important downstream effector responsible for IL-6 induced effects in human cancer cells.

Mcl-1, a member of the Bcl-2 family, was cloned from the ML-1 human myeloblastic leukemia cell line (38). The amino acid sequence of the carboxyl portion of Mcl-1 is very similar to that in the Bcl-2 protein (38). The intracellar distribution of the Mcl-1 protein overlaps that of Bcl-2 with a prominent mitochondrial membrane (38), suggesting that they share the same function in apoptosis regulation. Indeed, overexpression of Mcl-1 delays apoptosis induced by various inducing agents (39,40). Supportively, our current data have shown that overexpression of Mcl-1 in AGS cells confers resistance to H2O2-induced apoptosis. The H2O2-triggered the activation of the cell death pathway JNK is blocked in Mcl-1 overexpressing cells, implying that Mcl-1 protein may function at the upstream of JNK. A similar observation was made that Bcl-2 expression diminished the anticancer drug-induced JNK activation in cells (41). The mechanism of how Mcl-1 expression can block the H2O2-triggered JNK activation in AGS cells is completely unknown. Since either IL-6 treatment or mcl-1 expression does not affect the peroxides level in AGS cells, we suggest that the inhibition of H2O2-induced JNK activation by mcl-1 overexpression is not mediated by attenuating the intracellular peroxides level. Consistent with this finding, our previous study (13) demonstrated that bcl-2 overexpression failed to affect the benzene metabolites-elicited peroxides level in HL-60 cells. These findings, however, contradict that of other reports (28), which suggest that Bcl-2 countered apoptotic death via an antioxidant pathway operated at sites of free radical generation induced by dexamethasone. Possibly, this discrepancy is at least partially due to the different cell context or due to different modes of free radical generation by different stimuli.

8-OH-dGua is the most abundant product from oxidative damage to DNA by reactive oxygen species and induces G–T and A–C base substitutions (24). This fact suggests that formation of this hydroxylated base may contribute to the mutagenic and carcinogenic properties of ROS. Herein, we report that H2O2 increase the steady state level of 8-OH-dGua and peaks at 30 min, in the DNA of AGS cells. This oxidized base was effectively and rapidly repaired within 1 h. The removal of 8-OH-dGua is not obvious in IL-6-treated or mcl-1-transfected AGS cells. This finding suggests that the IL-6-mediated the increased Mcl-1 protein may attenuate certain repair enzyme activity, subsequently delaying oxidative DNA bases removal. It is believed that the retention of 8-OH-dGua in genomic DNA may facilitate the mutagenesis (42). The base excision repair enzyme has been found to be responsible for the removal of oxidative DNA lesions (43). Supportive of our findings, Liu et al. (44) recently observed that cyclobutane pyrimidine dimers induced by UV irradiation were efficiently removed in HL-60 cells, but were not repaired in bcl-2-overexpressing HL-60 cells. Their results and ours suggest that overexpression of the Bcl-2 family of proteins may affect nucleotide excision repair in cells in response to oxidative stress or UV irradiation.

To our best knowledge, this is the first time that IL-6 and its novel downstream effector, Mcl-1, was demonstrated to not only inhibit apoptosis but also abolish the repairing of oxidative DNA lesions in human gastric cancer cells when exposed to H2O2. Supportively, IL-6 and 8-OH-dGua have been found to be increased in patients with intestinal metaplasia and gastric cancers when compared to normal counterparts (17,20). Our studies therefore suggest that IL-6 may enhance the gastric carcinogenesis by both overriding apoptosis and attenuating the DNA repair process through up-regulation of the anti-apoptotic Mcl-1 protein. Under that premise, we believe that the interplay between ROS and IL-6 may occur during the oncogenic process in other human cancers.


    Notes
 
3 To whom correspondence should be addressed at: Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan Email: toxkml{at}ha.mc.ntu.edu.tw Back


    Acknowledgments
 
The authors would like to thank the National Science Council of the Republic of China for financially supporting this research under Contact No. NSC-89-2314-B002-434.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 15, 2001; revised August 21, 2001; accepted August 22, 2001.