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Cell Biology International (2006) 30, 332–337 (Printed in Great Britain)
Hydrogen peroxide-induced apoptosis in human gastric carcinoma MGC803 cells
Yubin Maoab, Gang Songa, Qiufeng Caia, Min Liua, Haohong Luoa, Mingxin Shia, Gaoliang Ouyanga* and Shideng Baoa
aKey Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, China
bPathophysiology Department, Medical College, Xiamen University, Xiamen 361005, China


Abstract

Hydrogen peroxide (H2O2), a representative ROS, has been used to study the apoptosis of cancer cells to oxidative stress. In this study, we exploited the cellular and molecular mechanisms involved in H2O2-induced apoptosis in human gastric carcinoma MGC803 cells. Exposure of cells to H2O2 might cause significant viability loss and the increase in apoptotic rate. Treatment with 0.4mmol/L H2O2 up-regulated Bax but down-regulated Bcl-2 in a time-dependent manner, while Bcl-xL expression remained unchanged. Our results also showed that the levels of Fas and Fas-L were increased, the pro-caspase-3 and pro-caspase-9 were down-regulated in H2O2-treated MGC803 cells. Under H2O2 stress, we found that the protein p53 also participated in MGC803 cells apoptosis. Taken together, the present study indicated that Fas-mediated cell surface death receptor pathway and mitochondria-mediated pathway may participate in regulating the MGC803 cells apoptosis under oxidative stress.


Keywords: Hydrogen peroxide, Apoptosis, Human gastric carcinoma cells.

*Corresponding author. Tel./fax: +86 592 218 6091.


1 Introduction

Oxidative damage, mediated by reactive oxygen species (ROS), has been implicated as a major cause of cellular injuries in a vast variety of clinical abnormalities including cancer, diabetes, aging, cardiovascular disease, and neurodegenerative disorders (Gorman et al., 1997; Datta et al., 2002; Kannan and Jain, 2000; Von Harsdorf et al., 1999; Imlay and Linn, 1988). Recent studies have indicated that ROS and the resultant oxidative stress readily damage biological molecules, ultimately induce cell death either by apoptosis or necrosis (Kannan and Jain, 2000). Numerous chemical and physiological oxidative stress factors are found to cause apoptosis, each has its own specific pathway that leads to activation of apoptotic process. H2O2, a representative ROS, can induce apoptosis in many different types of cells (Barbouti et al., 2002; Datta et al., 2002; Zuliani et al., 2005). H2O2 is generated from nearly all sources of oxidative stress and can diffuse freely in and out of the cells and tissues. Apart from generating hydroxyl radicals leading to DNA damage (Gorman et al., 1997), H2O2 also alters intracellular redox state, changes mitochondrial membrane potential and releases cytochrome c from the mitochondria into the cytosol (Li et al., 2000; Clement et al., 1998), and changes the homeostasis of ions such as calcium and iron (Gorman et al., 1997; Lu and Tian, 2005). Moreover, H2O2 is found to trigger the activation of multiple signaling pathways, thereby to modulate the expression of a great number of genes (Kang et al., 2002; Jiang et al., 1999). Fas/apo1/CD95, a well-characterized death receptor at the cell surface, has been demonstrated to participate in apoptosis pathway induced by oxidative stress (Suhara et al., 1998; Cappello et al., 2002). It is well known that ROS plays an important role in triggering the mitochondria-mediated apoptosis pathway, in which caspases being the central components. In addition, it is also well established that mitochondria is the main site of the generation of oxygen radicals, such as superoxide anion, hydroxyl radical, singlet oxygen and H2O2 (Kannan and Jain, 2000). p53, an important transcription factor, has been shown to participate in apoptosis (Kannan and Jain, 2000; Hickman et al., 2002; Gottlieb and Oren, 1998). H2O2 damages DNA by free radical generation. Once DNA is damaged, wild type p53 and other DNA damage checkpoint proteins act to arrest the cell cycle for the repair of damaged DNA. If the damage is too extensive to be repaired, the cells undergo apoptosis. p53 is known to regulate many genes, such as Fas, members of the Bcl-2 families (Datta et al., 2002; Jiang et al., 1999; Cappello et al., 2002; Hickman et al., 2002). Both death receptor and mitochondria-dependent apoptotic signaling pathways are known to regulate the program cell death, however, the detailed molecular mechanism of apoptosis induced by H2O2 is not clearly known.

Gastric carcinoma is one of the most common causes of malignancy-related death in China (Gottlieb and Oren, 1998). However, its pathogenesis is not completely understood and there are few effective therapies in gastric carcinoma prevention and treatment (Jiang et al., 2003, 2004; Wang et al., 2002; Hampton and Orrenius, 1997). Currently, inducing cancer cell into apoptosis is one of the important therapeutic intervention approaches in cancer, therefore, it is crucial to reveal the molecular mechanism of apoptosis in gastric carcinoma cells.

In this study, MGC803, a human gastric carcinoma cell line, was exploited to investigate the effects of H2O2 on the human gastric carcinoma cells and the underlying molecular mechanisms.

2 Materials and methods

2.1 Cell culture and treatment

Human gastric carcinoma cells, MGC803, were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Science, Chinese Academy of Science. MGC803 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen), with the addition of 10% heat-inactivated fetal bovine serum (Si-Ji-Qing Biotechnology Co., Hangzhou, China), penicillin (100U/mL), kanamycin (100μg/mL) and streptomycin (100μg/mL), at 37°C with 5% CO2 in atmosphere. MGC803 cells were exposed to H2O2 at different concentrations for different periods of time. H2O2 was diluted freshly before use, and the concentration (C) was measured by UV spectrophotometer using the following formula: C (mmol/L)=A240×1000/394 (Spitz et al., 1987).

2.2 Cell viability assay

Approximately 8×103 cells were seeded into each well of the 96-well plates. After 24h, H2O2 was added to corresponding wells. At each incubation period, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well in 100μL (0.5mg/mL in PBS). The plates were incubated for 4h at 37°C until purple formazan crystal developed. Subsequently, the MTT-containing medium was removed and 100μL DMSO solution (containing 90% DMSO, 10% 0.1M Glycine–NaOH, pH 10) was added to each well and incubated at room temperature for 30min. The amount of MTT formazan was quantitated at the absorbance A590 with A630 as reference filter using Microplate Reader (Bio-Rad) (Datta et al., 2002). Cell viability was shown by comparing the viability of treated cells with that of untreated control cells.

2.3 Cell morphological observation

Following the treatment with 0.4mmol/L H2O2 for 48h, MGC803 cells were washed three times with PBS, cell morphology was observed under phase contrast microscope. For fluorescent staining, the cells were then fixed with 4% paraformaldehyde for 10min at room temperature, washed with PBS, and incubated with 10μg/mL Hoechst 33258 (Calbiochem, San Diego, CA) for 10min in the dark (Jiang et al., 2004). Cell morphology was observed under fluorescence microscope (Leica DM IRB).

2.4 Flow-cytometric analysis for apoptotic cell rate

MGC803 cells, both untreated and treated with H2O2, were harvested, rinsed in PBS, resuspended and fixed with 70% ethanol at 4°C overnight. Following RNase A (20μg/mL) digestion at 37°C for 30min, the cells were stained with 50μg/mL propidium iodide at 4°C for 30min in the dark. Cell cycle distribution at different phase was analyzed with FACS flow cytometry (Beckman Coulter). More than 10,000 events were acquired for analysis using WinMDI 2.9 software pack.

2.5 Western blot

The H2O2-treated and -untreated cells were harvested, washed twice with PBS and lysed in lysis buffer (20mmol/L Tris–HCl, 100mmol/L NaCl, 20mmol/L KCl, 1.5mmol/L MgCl2, 50mmol/L β-GPA, 10mmol/L NaF, 0.5% NP-40, plus proteinase inhibitors and phosphates inhibitors) for 10min and centrifuged at 12,000g for 10min at 4°C. The total protein, as determined by Bio-Rad protein assay, was mixed with 4× loading buffer, and pre-heated at 95°C for 10min. The samples were then loaded onto SDS-PAGE gel. The proteins were transferred onto PVDF membrane for 1h using semi-dry transfer system (Bio-Rad). The membrane was blocked in 0.25% gelatin for 1.5h at room temperature, and then incubated for 1.5h with the following primary antibodies: anti-p53 and anti-phospho-p53 (Ser15) polyclonal antibodies (Calbiochem), anti-Bax, Bcl-xL, Bcl-2, Fas, Fas-L, caspase-3 and caspase-9 polyclonal antibodies (Santa Cruz), and anti-Actin monoclonal antibody (Sigma). Following the hybridization with primary antibody, the membrane was washed with TBST (Tris-buffered saline containing Tween-20) three times, followed by incubation with HRP-labeled secondary antibody (anti-rabbit IgG, anti-mouse lgG, Santa Cruz) for 1h at room temperature and washed with TBST three times. Final detection was performed with ECL (Amersham, Pharmacia Biotech). For repeated hybridization with other probes, the blot was stripped in buffer (62.5mmol/L Tris–HCl pH 6.8, 2% SDS, 200mmol/L 2-mercaptoethanol) at 50°C for 30min.

2.6 Statistical analysis

All data were expressed as mean±S.D. and evaluated by using the t-test with one-way analysis of variance (ANOVA). Differences were considered as statistically significant when P<0.05.

3 Results

3.1 Effect of H2O2 on MGC803 cell viability

As shown in Fig. 1, H2O2 significantly inhibited the viability of MGC803 cells at the concentrations of 0.6, 0.8 and 1.0mmol/L for 12, 24 and 48h, respectively (P<0.05). The inhibitory effect of H2O2 on MGC803 cells was dose- and time-dependent. At the concentration of 0.4mmol/L, H2O2 significantly inhibited the viability after exposing for 24 and 48h, but for 12h, the viability did not change obviously.


Fig. 1

Effects of H2O2 on MGC803 cell viability. Survival curves of cells following exposure to varying concentrations of H2O2 for 12, 24, and 48h. The viable cells were quantified by MTT assay. Each point represents the mean±S.D.


3.2 Effect of H2O2 on MGC803 cell morphology

After exposing to 0.4mmol/L H2O2 for 48h, morphological changes indicative of apoptotic cells, cell shrinkage and fragmentation, were observed in MGC803 cells, in comparison to the untreated cells (Fig. 2A, C). By Hoechst 33258 staining, the H2O2-treated cells exhibited nuclear condensation and fragmentation under fluorescence microscope (Fig. 2B, D).


Fig. 2

Cell morphology of MGC803 cells (A, C) and the cells treated with 0.4mmol/L H2O2 for 48h (B, D). In comparison to the untreated cells (A), H2O2-treated MGC803 cells (B) demonstrate typical appearance of apoptotic cells under phase contrast microscope. By staining with Hoechst 33258 H2O2-treated MGC803 cells (D) exhibit nuclear condensation and fragmentation under fluorescence microscope (A, B original magnification ×100; C, D original magnification ×400).


3.3 Effect of H2O2 on the proportion of apoptotic MGC803 cells

Using flow cytometry, increase in the apoptotic percentage from 6.4%, 11.3% to 23.2% was obtained following the treatment with H2O2 at the concentrations of 0.4, 0.6 and 0.8mmol/L for 24h (Figs. 3 and 4).


Fig. 3

Apoptosis rate of H2O2-treated MGC803 cells with flow cytometry. With PI staining, approximately 6.4%, 11.3% to 23.2% of H2O2-treated cells die via apoptosis following exposure to H2O2 at 0.4, 0.6, 0.8mmol/L for 24h.


Fig. 4

Demonstration of apoptosis in MGC803 cells by flow cytometry analysis. Sub-G1 peaks increased in correlation with the increase in H2O2 concentration (a, control; b, 0.2mmol/L; c, 0.4mmol/L; d, 0.6mmol/L; and e, 0.8mmol/L) following 24h exposure.



3.4 Effect of H2O2 on the expression of apoptosis-related Bcl-2 family

To investigate the molecular mechanism of H2O2-induced apoptosis in MGC803 cells, the expression of apoptosis-related Bcl-2 family with 0.4mmol/L H2O2 was examined. Bax, a pro-apoptotic factor, was up-regulated whereas Bcl-2, an anti-apoptotic factor, was down-regulated in a time-dependent manner, but the expression of Bcl-xL, another anti-apoptotic factor, remained unchanged. Therefore, the ratios of Bcl-2:Bax and Bcl-xL:Bax were all decreased in a time-dependent manner (Fig. 5).


Fig. 5

Expression of apoptosis-related Bcl-2 family in MGC803 cells after treated with 0.4mmol/L H2O2. Bcl-2, an anti-apoptotic factor, lowered as early as 1h, meanwhile, Bax, a pro-apoptotic factor, increased. The changes in the expression of both factors occurred in a time-dependent manner. Bcl-xL, an anti-apoptotic factor, did not change obviously.


3.5 Expression of Fas, Fas-L, pro-caspase-9 and pro-caspase-3 in H2O2-treated MGC803 cells

As shown in Fig. 6, Fas and Fas-L were increased as early as 1h following 0.4mmol/L H2O2 treatment. Meanwhile, the expression of pro-caspase-3 and pro-caspase-9 was down-regulated in H2O2-treated cells (Fig. 7).


Fig. 6

Expression of death receptor signals Fas and Fas-L in MGC803 cells after treated with 0.4mmol/L H2O2. The level of Fas and Fas-L increased as early as 1h. The up-regulation of Fas and Fas-L induced by H2O2 occurred in a time-dependent manner.


Fig. 7

Expression of pro-caspase-3 and pro-caspase-9 in MGC803 cells after treated with 0.4mmol/L H2O2. H2O2 could down-regulate these two pro-caspases.



3.6 Expression of p53 in H2O2-treated MGC803 cells

In order to understand precisely the molecular mechanism of H2O2-induced apoptosis in MGC803 cells, the expression of p53 was determined following H2O2 treatment either at different concentrations for 6h or at the concentration of 0.4mmol/L for different periods. No change was found in the total amount of p53 (Fig. 8A, B). However, the level of phosphorylated p53 (p-p53) was increased in a concentration-dependent manner. When treated with 0.4mmol/L H2O2, the up-regulation of p-p53 occurred as early as 1h and reached the peak at 2h.


Fig. 8

Expression of p53 and phosphorylated p53 in MGC803 cells treated with different H2O2 concentrations for 6h and (A), and at the concentration of 0.4mmol/L for various exposure periods (B). The total p53 amount did not change obviously. However, the phosphorylation of p53 (p-p53) level was increased and the increase of p-p53 was concentration-dependent. When treated with 0.4mmol/L H2O2, the up-regulation of p-p53 occurred as early as 1h and reaching the peak at 2h.


4 Discussion

It is generally accepted that oxidative stress plays an important role in the apoptosis of carcinomas. H2O2 mediated apoptosis has been studied as a model for the ROS-induced apoptosis in several experimental situations (Datta et al., 2002; Zuliani et al., 2005). Exogenously added H2O2 can freely diffuse across cell membranes. Low level of such diffusible ROS will trigger apoptosis, whereas, high level of it causes necrosis (Suhara et al., 1998; Hampton and Orrenius, 1997).

In our case, H2O2 treatment leaded to a concentration- and time-dependent decrease in the viability in MGC803 cells, and induced cell death mainly via apoptosis. However, numerous studies have revealed that the signal pathways governing apoptosis in mammalian cells are complex, and the pro- and anti-apoptotic signals regulating cell survival vary according to the type of cells (Von Harsdorf et al., 1999; Cory and Adams, 2002; Reed, 2002).

Apoptosis can be initiated by extracellular and intracellular signals that trigger a complex machinery of pro-apoptotic proteases and mitochondrial changes. It is the integration of multiple survival and death signals that determine whether a cell is to survive or undergo apoptosis. Many reports indicate that members of the Bcl-2 family are the mediators of cell survival and apoptosis (Kannan and Jain, 2000). Cell survival is enhanced when the expression of the anti-apoptotic factor Bcl-2 is relatively high, while the pro-apoptotic factor Bax is low. In agreement with a previous report (Jiang et al., 2004), H2O2 up-regulated Bax and down-regulated Bcl-2 in the current study. The time-dependent decrease in the ratios of Bcl-2:Bax and Bcl-xL:Bax showed that the Bcl-2 family proteins participated in H2O2-induced apoptosis in MGC803 cells.

The interaction between the anti-apoptotic and pro-apoptotic Bcl-2 family members can alter the permeability of mitochondrial membrane and release cytochrome c or activate caspase cascade (Budihardjo et al., 1999; Sun et al., 2002). In response to death inducing signals from cell surface receptors or from mitochondria, caspase cascade is a central mechanism promoting apoptosis. Membrane-dependent stimuli such as Fas/Fas-L interaction may trigger caspase-8 activation, which eventually leads to caspase-3 activation (Denning et al., 2002; Cappello et al., 2002; Dunkern et al., 2003). Fas is a type I membrane protein belonging to the TNF and NGF receptor family, which mediates a death signal (Denning et al., 2002). Similar to another report (Fujita et al., 2002), our data showed that the levels of Fas and Fas-L in H2O2-treated MGC803 cells were increased, which suggest that Fas-mediated death signal was involved.

It is known that released cytochrome c binds to Apaf-1 and participates in the activation of caspase-9 (the initiator). The activated caspase-9 activates caspase-3 (the effector), resulting in the onset of apoptosis. The increases in caspase-3 and caspase-9 are synchronized with the increase in Bax expression and the decrease in Bcl-2 (Tanabe et al., 1998). Our data showed that pro-caspase-3 and pro-caspase-9 were down-regulated by H2O2 treatment, thus activated the proteolysis. These indicated that both Fas-mediated death receptor pathway and mitochondria-dependent signal pathway were likely to be involved in the H2O2-induced apoptosis.

p53, a tumor suppressor gene, has been reported to induce the up-regulation of pro-apoptotic Bax and down-regulation of anti-apoptotic Bcl-2 (Wang et al., 2002; Moll and Zaika, 2001). However, the role of p53 in ROS-induced cell death has been shown to be consistent and seems to be cell type relevant. In many cases, ROS-mediated DNA damage has been shown to induce apoptosis through the accumulation of p53 (Datta et al., 2002; Jiang et al., 1999; Wang et al., 2002). p53 induces the transcription of death receptor Fas (Gottlieb and Oren, 1998), since there is a p53-response element located within the first intron of the Fas gene. In lymphoblastoid cells, apoptosis occurs by activating the p53-dependent Fas receptor-driven pathway (Dunkern et al., 2003). In ventricular myocyte or Jurkat T-cell, p53 activates the mitochondrial death pathway and provokes apoptosis (Gao et al., 2001; Barbouti et al., 2002; Regula and Kirshenbaum, 2001). Under H2O2 stress, p53 protein was activated via serine phosphorylation. Our results suggested that the protein p53 played an important role in H2O2-induced apoptosis in MGC803 cells.

Taken together, we concluded that H2O2 induced the cell death of MGC803 by apoptosis. Under oxidative stress, both death receptor pathway and mitochondria-dependent pathway participated in the regulation of the apoptotic program in human gastric carcinoma cells.

Acknowledgements

We thank X.S. Cao for critical reading of the manuscript. This study was supported by National Natural Science Foundation of China (No. 30400239, No. 30370307), Innovation Foundation of Fujian Province for Young Scientists (No. 2003J017), and Science Research Foundation of Xiamen University (No. 2003XDYY31).

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Received 24 May 2005/30 September 2005; accepted 20 December 2005

doi:10.1016/j.cellbi.2005.12.008


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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