Oxidative stress-induced cytotoxicity in cervical cancer cells may be of toxicological interest. In the present study, the effects of exogenous H2O2 on cell growth and death in HeLa cervical cancer cells were investigated, and the anti-apoptotic effects of various caspase (pan-caspase, caspase-3, -8 or -9) inhibitors on H2O2-treated HeLa cells were also evaluated with regard to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, H2O2 inhibited the growth of HeLa cells with an IC50 value of ~75 μM at 24 h. H2O2 increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, which was accompanied by the activation of caspase-3 and the loss of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 induced necrosis in HeLa cells. Caspase inhibitors significantly prevented H2O2-induced HeLa cell death. H2O2 increased ROS including O2•− at 24 h and increased the activity of catalase in HeLa cells. H2O2 also increased the ROS level at 1 h, and several caspase inhibitors attenuated the increased level at 1 h but not at 6, 12 and 24 h. H2O2 decreased the GSH level in HeLa cells at 1 h, and several caspase inhibitors attenuated the decreased level of GSH at this time. H2O2 induced GSH depletion at 24 h. In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular increases in ROS levels and GSH depletion. Caspase inhibitors are suggested to suppress H2O2-induced oxidative stress to rescue HeLa cells at the early time point of 1 h.
Reactive oxygen species (ROS) are a group of oxygen moieties, which include hydrogen peroxide (H2O2), the superoxide anion (O2•−) and the hydroxyl radical (•OH). Conventional theory has regarded ROS as deleterious or harmful to cells (
Compared with other members of ROS, H2O2 plays a pivotal role since it is able to freely travel through biological membranes to a distance of several cell diameters and interacts with ferrous iron (Fenton chemistry) causing the formation of the very aggressive and short-lived •OH. Tissue concentrations of H2O2 for the period of inflammation have been likely to reach close to millimolar levels whereas tiny amounts of H2O2 generated by NADPH oxidase are assumed to take action only in microenvironments of the plasma membrane such as lipid rafts (
The mechanism of apoptosis generally involves two signaling pathways, the mitochondrial pathway and the cell death receptor pathway (
Cervical cancer is a major cause of cancer-related death in women worldwide, and the occurrence of this cancer is ascribed to changes in cancer-related genes as well as environmental events including viral infections. The carcinogenesis of cervical cancer has been known to be tightly linked to tissue inflammation mediated by ROS. Moreover, ROS influence genetic and epigenetic changes thereby modulating cellular proliferation and differentiation (
Human cervical adenocarcinoma HeLa cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in a humidified incubator containing 5% CO2 at 37°C. HeLa cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (both from Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and 1% penicillin-streptomycin (Gibco-BRL, Grand Island, NY, USA). Cells were routinely grown in 100-mm plastic tissue culture dishes (Nunc, Roskilde, Denmark) and harvested with a solution of trypsin-EDTA while in a logarithmic phase of growth.
H2O2 was purchased from Sigma-Aldrich Chemical Co. The pan-caspase inhibitor (Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), caspase-3 inhibitor (Z-DEVD-FMK; benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), caspase-8 inhibitor (Z-IETD-FMK; benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone) and caspase-9 inhibitor (Z-LEHD-FMK; benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA) and were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Co.). Based on a previous study (
Cell growth changes were determined by measuring the absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT; Sigma-Aldrich Chemical Co.) in living cells as described previously (
Cell cycle distribution and sub-G1 cell analysis were determined by propidium iodide (PI) (Sigma-Aldrich; Ex/Em = 488/617 nm) staining. In brief, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated amounts of H2O2 with or without 15 μM caspase inhibitors for 1, 6, 12 or 24 h. Total cells including floating cells were then washed with PBS and fixed in 70% (v/v) ethanol. Cells were washed again with PBS, and then incubated with PI (10 μg/ml) with simultaneous RNase treatment at 37°C for 30 min. Cellular DNA content was measured using a FACStar flow cytometer and analyzed using Lysis II and CellFit software (both from Becton-Dickinson, Franklin Lakes, NJ, USA).
Necrosis in cells treated with H2O2 was evaluated using the LDH kit (Sigma-Aldrich Chemical Co.). In brief, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated doses of H2O2 for 24 h. After treatment, the culture media were collected and centrifuged for 5 min at 1,500 rpm. Fifty microliters of the media supernatant was added to a fresh 96-well plate along with the LDH assay reagent and then incubated at room temperature for 30 min. The absorbance values were measured at 490 nm using a microplate reader (Synergy™ 2). LDH release was expressed as the percentage of extracellular LDH activity compared with the control cells.
Apoptotic cell death was determined by staining the cells with Annexin V-fluorescein isothiocyanate (FITC; Invitrogen Life Technologies, Camarillo, CA, USA; Ex/Em = 488/519 nm) as previously described (
MMP (ΔΨm) levels were measured by Rhodamine 123 fluorescent dye (Sigma-Aldrich Chemical Co.; Ex/Em = 485/535 nm). In brief, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated amounts of H2O2 with or without 15 μM caspase inhibitors for 24 h. Cells were washed twice with PBS and incubated with Rhodamine 123 (0.1 μg/ml) at 37°C for 30 min. Rhodamine 123 staining intensity was determined by a FACStar flow cytometer (Becton-Dickinson). Rhodamine 123-negative cells indicated the loss of MMP (ΔΨm) in the cells.
The change in caspase-3 and PARP in H2O2-treated cells was determined by western blotting. In brief, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated amounts of H2O2 for 24 h. The cells were then washed in PBS and suspended in five volumes of lysis buffer [20 mM HEPES. (pH 7.9), 20% (v/v) glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT and 1% (v/v) protease inhibitor cocktail]. The protein concentrations in the supernatant were determined using the Bradford method. Samples containing 10 μg total protein were resolved by 8 or 12.5% SDS-PAGE gels, transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting and then probed with anti-caspase-3, anti-PARP, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-LC3A/B (Cell Signaling Technology, Waltham, MA, USA) antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Amersham, Arlington Heights, IL, USA).
The activities of caspase-3 and -8 were assessed using the Caspase-3 and Caspase-8 Colorimetric Assay Kits (R&D Systems, Inc.) as previously used (
Intracellular ROS levels were detected by the fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Ex/Em = 495/529 nm; Invitrogen Molecular Probes, Eugene, OR, USA) at 1, 6, 12 or 24 h. H2DCFDA is poorly selective for the superoxide anion radical (O2•−). In contrast, dihydroethidium (DHE) (Invitrogen Molecular Probes; Ex/Em = 518/605 nm) is a fluorogenic probe that is highly selective for O2•− among ROS. In brief, 1×106 cells/ml in FACS tube (Becton-Dickinson) were treated with 100 μM H2O2 with or without 15 μM caspase inhibitors in the presence of 20 μM H2DCFDA or DHE. The fluorescence levels of DCF and DHE were evaluated using a FACStar flow cytometer at 1 h. DCF (ROS) and DHE (O2•−) levels were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software (Becton-Dickinson). In addition, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated amounts of H2O2 with or without 15 μM caspase inhibitors for 6, 12 and 24 h. Cells were incubated with 20 μM H2DCFDA or DHE at 37°C for 30 min. H2DCFDA or DHE fluorescence was assessed using a FACStar flow cytometer.
SOD enzyme activity was measured using the SOD assay kit-WST (Fluka Co., Milwaukee, WI, USA), and catalase enzyme activity was measured using a catalase assay kit from Sigma-Aldrich Chemical Co. In brief, 1×106 cells were incubated with 100 μM H2O2 for 24 h. The cells were then washed in PBS and suspended in 5 volumes of lysis buffer [20 mM HEPES (pH 7.9), 20% glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% NP-40, 0.5 mM DTT and 1% protease inhibitor cocktail (from Sigma)]. The protein concentration of the supernatant was determined by the Bradford method. Supernatant samples containing 100 μg total protein were used for determination of SOD and catalase enzyme activities. These were added to each well in 96-well microtiter plates (Nunc) with the appropriate working solutions (according to the manufacturer’s instructions) at 25°C for 30 min. The color changes were measured at 450 or 520 nm using a microplate reader (SpectraMax 340). The value for the experimental group was expressed as a percentage of the control group.
Cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA) (Invitrogen Molecular Probes; Ex/Em = 522/595 nm) at 1, 6, 12, or 24 h. In brief, 1×106 cells/ml in a FACS tube (Becton-Dickinson) were treated with 100 μM H2O2 with or without 15 μM caspase inhibitors in the presence of 5 μM CMFDA. The level of CMF fluorescence was evaluated using a FACStar flow cytometer at 1 h. CMF (GSH) levels were expressed as MFI, which were calculated by CellQuest software. In addition, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the indicated amounts of H2O2 with or without 15 μM caspase inhibitors for 6, 12 and 24 h. Cells were incubated with 5 μM CMFDA at 37°C for 30 min. CMF fluorescence was assessed using a FACStar flow cytometer. Negative CMF staining (GSH depleted) of cells was expressed as the percentage of (-) CMF cells.
The results represent the means of at least two independent experiments (means ± SD). The data were analyzed using InStat software (GraphPad Prism4; GraphPad Software, San Diego, CA, USA). The Student’s t-test or one-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s multiple comparison test was used for parametric data. The statistical significance was defined as p<0.05.
The effect of H2O2 on the growth of HeLa cells was examined at 24 h. Treatment with 50–250 μM H2O2 significantly decreased the viable (trypan blue-negative) cell number in the HeLa cells in a dose-dependent manner whereas H2O2 dose-dependently increased the number of dead (trypan blue-positive) cells (
Next, we aimed to ascertain whether the H2O2-induced cell death was through apoptosis or necrosis in HeLa cells. While 50 or 100 μM H2O2 significantly increased the percentages of sub-G1 cells in HeLa cells, 250 μM H2O2 did not increase the percentages of sub-G1 cells in these cells (
We investigated whether caspases are required for H2O2-induced apoptosis. Based on a previous study (
To assess the intracellular ROS levels in the H2O2-treated HeLa cells, H2DCFDA and DHE dyes were used. All the tested doses of H2O2 increased the ROS (DCF) level in the HeLa cells at 24 h (
To determine whether the levels of intracellular ROS and GSH in the H2O2-treated HeLa cells were altered by treatment with each caspase inhibitor, ROS and GSH levels in the HeLa cells were assessed at the early time point of 1 h and at the extended time point of 24 h (
Exogenous H2O2 was applied for inducing oxidative stress in HeLa cervical cancer cells. After exposure to H2O2 for 24 h, the IC50 value in the HeLa cells was ~75 μM based on MTT assays. H2O2 dose-dependently increased the number of dead cells and Annexin V-FITC-positive cells in the HeLa cells, suggesting that H2O2-induced HeLa cell death occurred via apoptosis. Evidently, H2O2 decreased the level of pro-caspase-3 and induced the cleavage of PARP proteins in the HeLa cells. The activity of caspase-3 was also increased in the H2O2-treated HeLa cells. However, 250 μM H2O2 did not significantly increase the percentages of sub-G1 cells in the HeLa cells, implying that the relatively higher dose of H2O2 fixed HeLa cells similar to ethanol or methanol. In addition, 100 or 250 μM H2O2 significantly induced LDH release in the HeLa cells at 24 h. Therefore, H2O2 appeared to provoke HeLa cell death via apoptosis as well as necrosis depending on its concentration. Moreover, autophagy appeared to be involved in H2O2-induced HeLa cell death since LC3-I was converted to LC3-II in these cells. Apoptosis is closely related to the collapse of MMP (ΔΨm) (
Treatment with the caspase inhibitors tested in this experiment significantly prevented HeLa cell death by H2O2, and Z-VAD showed a stronger effect on reducing apoptosis. In particular, although H2O2 slightly increased the activity of caspase-8, its inhibitor significantly prevented HeLa cell death by H2O2. Thus, a subtle change in the activity of caspase-8 seemed to strongly affect the pro-apoptotic pathway in H2O2-treated HeLa cells. These data suggest that the mitochondrial pathway and cell death receptor pathway are together necessary for the complete induction of apoptosis in H2O2-treated HeLa cells. However, Wu
The ROS level was significantly increased in HeLa cells treated with H2O2 at 24 h. Since H2O2 did not decrease the activity of SOD and increased the activity of catalase at 24 h, increases in ROS levels including O2•− were likely to occur via their strong generation rather than the lack of scavenging them. In addition, it is possible that exogenous H2O2 strongly generates O2•− via the damage of mitochondria, and both H2O2 and O2•− can be efficiently converted into the toxic •OH via the Fenton reaction to kill HeLa cells. However, H2O2 did not increase the O2•− (DHE) level in HeLa cells at 1 h, suggesting that it did not affect the mitochondrial respiratory transport chain and the activity of various oxidases to generate O2•− within this early time point. Moreover, caspase inhibitors showing the prevention of H2O2-induced cell death failed to significantly decrease the ROS level including O2•− at 6, 12 and 24 h. However, Z-VAD, caspase-3 and -8 inhibitors appeared to attenuate the increased ROS (DCF) level by H2O2 at 1 h. In addition, all of the caspase inhibitors decreased the basal level of ROS including O2•− in the HeLa control cells. It is conceivable that the reduced basal activity of caspase by their inhibitors improves the reliability of antioxidant-related enzymes to strongly scavenge basal intracellular ROS in HeLa cells. Therefore, the early suppression of H2O2-induced oxidative stress by caspase inhibitors seems to be crucial for the protection of HeLa cells against it. The exact role of each caspase inhibitor in preventing H2O2-induced HeLa cell death still needs to be defined further.
GSH is a main non-protein antioxidant in cells. Apoptotic effects are inversely comparable to the GSH content (
In conclusion, H2O2 inhibited the growth of HeLa cells via apoptosis and/or necrosis, which was accompanied by intracellular ROS increase and GSH depletion. The anti-apoptotic effect of caspase inhibitors on H2O2-induced HeLa cell death may result from the early suppression of H2O2-induced oxidative stress. The present data provide useful information for the understanding of the toxicological effect of exogenous H2O2 on HeLa cells.
This study was supported by the National Research Foundation of Korea (NRF), a grant funded by the Korean government (MSIP) (no. 2008-0062279), and supported by the Basic Science Research Program through the NRF funded by the Ministry of Education (2013006279).
reactive oxygen species
glutathione
lactate dehydrogenase
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketon
benzyloxycarbonyl-Asp- Glu-Val-Asp-fluoromethylketon
benzyloxycarbonyl- Ile-Glu-Thr-Asp-fluoromethylketon
be oxycarbonyl- Leu-Glu-His-Asp-fluoromethylketon
superoxide dismutase
mitochondrial membrane potential
fluorescein isothiocyanate
propidium iodide
2′,7′-dichlorodihydrofluorescein diacetate
dihydroethidium
5-chloromethylfluorescein diacetate
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Effects of H2O2 on the cell growth in HeLa cells. Exponentially growing cells were treated with the indicated concentrations of H2O2 for 24 h. (A) The graph shows the numbers of viable (trypan blue-negative) and dead (trypan blue-positive) cells in the HeLa cells. (B) The graph shows the cellular growth changes in HeLa cells as assessed by MTT assays. (C) The graph shows the cell cycle distribution in HeLa cells as measured by a FACStar flow cytometer. *p<0.05 compared with the control group.
Effects of H2O2 on cell death and MMP (ΔΨm) in HeLa cells. Exponentially growing cells were treated with the indicated concentrations of H2O2 for 24 h. (A) The graph shows the percentages of sub-G1 cells in the HeLa cells as measured by FACStar flow cytometer. (B) The graph shows the percentage of LDH release when compared with that in the control cells. (C) The graph shows the percentages of Annexin V-FITC-positive cells as measured by FACStar flow cytometer. (D) The graph shows the percentages of Rhodamine 123-negative [MMP (ΔΨm) loss] cells as measured by FACStar flow cytometer. (E) Western blot data of the levels of pro-caspase-3, PARP and LC3 in H2O2-treated HeLa cells. (F) The graphs show the activities of caspase-3 and -8 in the H2O2-treated HeLa cells. *p<0.05 compared with the control group. MMP, mitochondrial membrane potential; LDH, lactate dehydrogenase.
Effects of caspase inhibitors on apoptosis in H2O2-treated HeLa cells. Exponentially growing cells were treated with 100 μM H2O2 for the indicated times following 1 h of pre-incubation with 15 μM of a caspase inhibitor. (A and C) The graphs show the percentages of sub-G1 cells. (B and D) The graphs show the percentages of Annexin V-FITC-positive cells. (E) The graph shows the percentages of Rhodamine 123-negative [MMP (ΔΨm) loss] cells. *p<0.05 compared with the control group. #p<0.05 compared with cells treated with H2O2 only. Z-VAD; pan-caspase inhibitor, Z-DEVD; caspase 3 inhibitor, Z-IETD; caspase 8 inhibitor, Z-LEHD; caspase 9 inhibitor. MMP, mitochondrial membrane potential.
Effects of H2O2 on ROS and GSH levels in HeLa cells. Exponentially growing cells were treated with the indicated concentrations of H2O2 for 24 h. ROS and GSH levels in the HeLa cells were measured using a FACStar flow cytometer. (A) The graph indicates DCF (ROS) levels (%). (B) The graph indicates DHE (O2•−) levels (%). (C) The activities of catalase and SOD were measured as described in Materials and methods. The graphs show changes in catalase and SOD activities following exposure to 100 μM H2O2. (D) The graph indicates the (-) CMF (GSH-depleted) cells (%) in the HeLa cells when compared with the control cell group. *p<0.05 compared with the control group. ROS, reactive oxygen species; GSH, glutathione.
Effects of caspase inhibitors on ROS and GSH levels in H2O2-treated HeLa cells. Exponentially growing cells were treated with 100 μM H2O2 for 1 or 24 h following 1 h pre-incubation of 15 μM of a caspase inhibitor. ROS and GSH levels in HeLa cells were measured using a FACStar flow cytometer. (A and D) Graphs indicate DCF (ROS) levels (%) at (A) 1 h and (D) 24 h. (B and E) Graphs indicate DHE (O2•−) levels (%) at (B) 1 h and (E) 24 h. (C and F) The graphs indicate mean CMF (GSH) levels (%) at (C) 1 h and (-) CMF (GSH-depleted) cells (%) in HeLa cells compared with control cell group at (F) 24 h. *p<0.05 compared with the control group. #p<0.05 compared with cells treated with H2O2 only. ROS, reactive oxygen species; GSH, glutathione.