Arsenic trioxide mediated cytotoxicity and oxidative stress, in breast and lung carcinoma cell lines Alice M. Walker, Jacqueline J. Stevens and Paul B. Tchounwou Molecular Toxicology Research Laboratory, Molecular and Cellular Biology Research Laboratory, NIHCenter for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 JR Lynch Street, Box 18540, Jackson, Mississippi, USA; email:
[email protected] ABSTRACT Arsenic is a metalloid that is commonly found in soil, water and air. It is an element that has no known physiological function but is present in the body as a result of environmental exposure. The primary source of human exposure is through drinking water and food. Arsenic acts on cells through a variety of mechanisms influencing numerous signal transduction pathways resulting in cellular effects such as apoptosis induction, growth inhibition and angiogenesis inhibition. Although arsenic has been reported to induce reactive oxygen species formation and oxidative stress in liver cells and hematopoetic cells, its effects on breast and lung cells are not well elucidated. The primary objective of this research is to evaluate the effects of arsenic cytotoxcity and to determine whether arsenic induces oxidative stress in breast and lung carcinoma cell lines. To achieve this goal, breast cancer (MCF-7) and lung cancer (A549) cells were cultured following standard protocols, and exposed to various doses of arsenic trioxide for 48 h. The 3-(4, 5 dimethylthiazoyl-2-yl) 2,5diphenyl-tetrazolium bromide (MTT) assay was performed to determine the cytotoxicity, and the thiobarbituric acid test was performed to evaluate the degree of lipid peroxidation and oxidative stress. Data obtained from the MTT assay indicated that arsenic significantly reduced the viability of MCF-7 and A549 cells. Upon 48 hr of exposure, the LD50 values from arsenic trioxide treatment were 11.5 and 14.1 µg/ml for A549 cells and MCF-7 cells, respectively. The result of the thiobarbituric acid test demonstrated that arsenic trioxide treatment resulted in a significant increase (p < 0.05) of malondialdehyde-MDA, indicating that oxidative stress may play a key role in arsenic-induced toxicity in the breast and lung cells. Key Words: Arsenic, cytotoxicity, MCF-7 cells, A549 cells, lipid peroxidation INTRODUCTION Arsenic is a metalloid that is ubiquitous in the environment. People may be exposed to arsenic in three ways; by ingestion of contaminated food and water, by inhalation of contaminated air, aerosols or particulates, and by dermal or skin contact. The toxicity of arsenic depends upon its chemical form; the organic forms being usually less harmful than the inorganic ones (1). Acute exposure, whether from ingested or inhaled arsenic, can damage many tissues and organ systems including the nervous system, respiratory system, cardiovascular system, gastrointestinal tract, and skin. Intense acute arsenic exposure can be fatal (2). Chronic exposure to arsenic has been associated with several adverse health effects including vascular, peripheral neuropathy, exacerbation of the complications of diabetes, cardiac arrhythmias, liver and kidney toxicity, anemia, and leukopenia, and several types of cancers (2). The major mechanism by which arsenic exerts its toxic effect is through impairment of cellular respiration by inhibition of various mitochrondrial enzymes, and phosphorylation. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions (3, 4). Arsenic in vitro reacts with protein sulfhydryl groups to inactivate enzymes, such as dihydrolipoyl dehydrogenase and thiolase, thereby producing inhibited oxidation of pyruvate and betaoxidation of fatty acids (5). Arsenical compounds have shown to induce oxidative stress in mammalians cells (3, 6). The objective of this study is to
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determine the cytotoxic effects of arsenic trioxide in breast and lung carcinoma cell lines and to assess whether arsenic toxicity is mediated via oxidative stress in these cell lines. To accomplish this objective, lipid peroxidation is used as an indicator of oxidative stress, and as a biomarker to ascertain cellular injury. It has been reported that the peroxidation of lipids in cell membranes can damage these biologic structures by disrupting fluidity and permeability. Lipid peroxidation can also adversely affect the function of membrane bound proteins such as enzymes and receptors (7). MATERIALS AND METHODS Cell Lines and Chemicals The breast cancer cell line (MCF-7) was generously provided by Dr Ernest Izevbigie, Department of Biology, Jackson State University; Jackson, MS. The human lung carcinoma cell line (A549), the F-12 K medium and trypan blue were purchased from American Type Culture collection (ATCC) (Manassa, VA). The RPMI 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin/fungizone, phosphate buffered saline (PBS) and trypsin versene were purchased from Invitrogen, (Grand Island, NY). BCA protein assay kit was obtained from Pierce (Rockford, IL). Arsenic trioxide (As2O3) was purchased from Fisher Scientific (Houston, TX) and the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reagent was obtained from Sigma Chemical Company (St. Louis, MO). The lipid peroxidation assay kit was purchased from EMD Bioscience (San Diego, CA). Cell Culture MCF-7 cells were maintained in RPMI 1640 supplemented with 10% FBS, and 1% penicillin (10,000 units/ml), streptomycin (10,000 µg/ml) and fungizone mixture. A549 cells were maintained in F12-K medium supplemented in 10 % FBS and 1% penicillin, streptomycin and fungizone mixture, as adherent cells. The cells were grown in a humidified incubator under an atmosphere of 95% air and 5% CO2 at 37ºC to sub-confluence (80-95%). The culture medium for each cell line was replaced every 48 hours. After growing to 80-95% confluence, the medium was aspirated off and the cell monolayer was washed three times with sterile phosphate buffered saline (PBS). The cell monolayer was treated with 1 mL trypsin versene per plate and incubated briefly at 37ºC. The cells were then viewed microscopically to ensure a complete cell detachment. Cells were re-suspended in RPMI 1640 complete medium for MCF-7 and F-12K complete medium for A549, stained with 4% trypan blue (1 to 2 minutes), and counted with a hemocytometer. The cells were seeded at a density of 5 x 105 cells in 13 x 100 mm tissue culture plates, prior to arsenic trioxide treatment. Cytotoxicity Assay MCF-7 and A549 cells were seeded in a 96-well plates with 5000 cells per well for a period of 24 hours at 37ºC in a 5% CO2 humidified incubator. The medium was removed and replaced with various doses of arsenic trioxide using deionized water as solvent. The plates were treated with arsenic trioxide at 0.78, 1.58, 3.125, 6.25, 12.5, 25, 50 µg/ml doses for 18, 24, and 48 hrs. The MTT assay was performed as previously described, and optical densities were read on a microtiter plate reader (Bio-Tek Instruments Inc) at a wavelength of 550 nm (8). A data analysis was performed to determine the chemical doses required to reduce cell viability by 50% (LD50s). Lipid Peroxidation Assay MCF-7 and A549 cell lines were seeded at a density of 3 x 105 cells in 13 x 100 mm tissue culture plates. The cells were then grown in a humidified incubator under an atmosphere of 95% and 5% CO2 at 37ºC, to 75% confluence. The medium was aspirated from the cell monolayer and treated with 4 ml of
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various doses of arsenic trioxide (0, 4, 6, 8, 10 µg/ml). The experiment was carried out in triplicates. The control was grown in the absence of As2O3. After 48 hr exposure time, the treatments were removed and the cells were removed from the plate by scraping. The cells were collected and resuspended in 1 ml of PBS, and were washed three times with PBS pH 7.4. The cell lysis was by homogenization (Omni GLH homogenizer) and sonication (Branson sonifier). The lipid peroxidation assay was performed according to the manufacture protocol (Calbiochem-EMD Biosciences, Inc.). Optical densities were read at 586 nm on a Varian Cary 300 Bio UV-Visible spectrophotometer. Statistical Analysis The absorbance values obtained per treatment were converted to percentages of cell viability. Regression analysis was performed on cell viability data and the resulting equation was used to compute the lethal dose required to produce a 50% reduction (LD50) in cell viability. Statistical analysis for differences in mean levels of MDA was done using student’s t-test for comparing two sample sets, and ANOVA for multiple sample sets. P-values less than 0.05 were considered statistically significant. RESULTS Data obtained from the bioassay with MCF-7 cells revealed a strong dose response with the cytotoxicity of arsenic trioxide. The mean percentages of cell viability were 100% ± 0.8, 85% ± 5%, 72% ± 7%, 72% ± 3%, 5.4 ± 0.2%, 5.5% ± 0.3% and 6.3% ± 0.7% (18 hr); 100% ± 6%, 97% ± %5, 93% ± 0.3%, 110% ± 1%, 40% ± 2%, 6% ±.03% and 6% ±0.2% (24 hr); and 100% ± 1%, 85% ± 6%, 72% ± 13%, 72% ± 4%, 5.4% ± 4%, 5.5% ± 7% and 6.2% ± 11 % (48 hr) for 0, 1.56, 3.125, 6.25, 12.5, 25 and 50 µg/ml of arsenic trioxide, respectively (Fig. 1). A time-dependent response was also observed. Arsenic trioxide doses required to reduce cell viability by 50% were computed to be 21.5, 18.4 and 15.3 µg/ml for 18, 24 and 48 hrs of exposure, respectively.
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Figure 1: Cytotoxic effect of arsenic trioxide on MCF-7 cells Tests with A549 cells also revealed a strong dose response with cytotoxicity of arsenic trioxide. The mean percentages of cell viability were 100% ± 6%, 122% ± 8%, 131%± 1, 146%± 6%, 80% ± 2%, 27% ± 5% and 27%± 5% (18 hr); 100% ± 5%, 136% ± 13%, 149% ± 8%, 115% ± 3%, 49% ± 9%, 4% ± 5% and 4% ± 3% (24 hr); and 100% ± 6%, 95% ± 8%, 90% ± 5%, 71% ± 12%, 27% ± 4%, 2% ± 4%, and 2%
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±1% for 0, 1.56, 3.125, 6.25, 12.5, 25 and 50 µg/ml of arsenic trioxide. Computed LD50 values of 32.24, 25.76 and 11.5 µg/ml were obtained for 18, 24, and 48 hrs of exposure, respectively.
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Figure 2: Cytotoxic effect of arsenic trioxide on A549 cells Lipid Peroxidation
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Figure 3 shows the results from the lipid peroxidation assay of arsenic trioxide toxicity to MCF-7 and A549 cells. The MDA levels in MCF-7 cells were 0.50±0.0, 0.75±0.35, 0.50±0.71, 1.50±0.71, 1.25±0.35 and 1.25±0.35 uM in 0, 2, 4, 6, 8, and 10µg/ml arsenic trioxide, respectively. The MDA levels in A549 cells were 0.25±0.35, 0.25±0.35, 2.50±0.71, 4.0±0.0, 2.5±0.0 and 2.5±0.0 in 0, 2, 4, 6, 8, and 10µg/ml arsenic trioxide, respectively.
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Figure 3: MDA levels in MCF-7 and A549 cells exposed to arsenic trioxide.
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DISCUSSION Cytotoxicity is a tool for ascertaining the negative effect of a chemical compounds such as arsenic trioxide in biological systems. Toxicity is dependent upon the chemical species, the route of exposure, and other factors (9). In this study, it was demonstrated that arsenic trioxide is acutely toxic to both breast carcinoma cells (MCF-7) and lung carcinoma cells (A549). There was a time and dose-dependent response with regard to arsenic trioxide toxicity to the two cell lines. A biphasic response was obtained showing a slight increase in cell viability within the dose range of 0-0.8 µg/ml in MCF-7 and dose range of 0-0.4 µg/ml in A549, followed by a gradual decline. The median lethal doses (LD50s) of arsenic trioxide for MCF-7 cells were 21.5, 18.4, 15.3 µg/ml upon 18, 24, and 48 hr treatment, respectively. LD50 values for A549 cells were 32.24, 25.79 and 11.5 µg/ml upon 18, 24, 48 hr treatment, respectively. This study demonstrated that cytotoxicity differs in human cell lines, indicating that some cell types are more sensitive than others (9, 10). The cytotoxicity occurred at higher levels of exposure whereas proliferation occurred at lower doses. Chow et al. reported that the arsenic trioxide exhibited inhibitory effects on the proliferation of MCF-7 cells in a dose and time dependent manner, and found the LD50 to be 8, 1.8 and 1.2 µM upon 1-, 2-, and 3day treatments respectively (11). Chow et al. also pointed out that arsenic was capable of reducing cell survival in MCF-7 cells via the suppression of the estrogen induced growth stimulatory effects in MCF-7 cells (11). Studies in our laboratory have revealed that arsenic trioxide is acutely toxic to human carcinoma cells (HepG2). Upon 48 hr exposure, the LD50 value was 8.55±0.58 µg/mL (4). This study indicated that arsenic trioxide is less toxic in breast and lung carcinoma compared to the liver carcinoma cells. Graham-Evans et. al (10) studied the cytotoxicity effect of arsenic trioxide on several established human cell lines such as keratinocytes (HaCat), dermal fibroblast (CRL 1904), monocytes (THP1/A23187), and melanocytes (1675). These authors reported that arsenic was toxic at high doses to keratinocytes (6 µg/ml), fibroblasts (1.5 µg/ml), monocytes (0.19 µg/ml) and toxic at lower doses in melanocytes (0.19 µg/ml) (10). Malondialdehyde (MDA) has been used to evaluate lipid peroxidation and DNA damage caused by exogenous free radicals or endogenous reactive oxygen species. In this study, we investigated the effect of oxidative stress in breast and lung cancer cell lines treated with arsenic trioxide by evaluating MDA production. We found a dose-dependent increase in MDA production in both cell lines, with increasing doses of arsenic trioxide. In the MCF-7 cell line, there was an increase of MDA production from 0 to 6 µg/ml (2.5 µM), followed by a decrease in production that reaches 1.5µM at both 8 and 10 µg/ml. We believe that this decrease is related to the decrease in the number of viable cells at higher levels of arsenic trioxide exposure. In the A549 cell line, our findings revealed a gradual increase of MDA production to 4 µM at the 6 µg/ml dose, and a 50% reduction (2 µM) at both 8 and 10 µg/ml doses. The lung carcinoma cell line (A549) showed a higher level of MDA production (4 µM at 6 µg/ml) compared to the breast carcinoma cell line at the same dose (2 µM at 6 µg/ml). This is indicative that the lung cells appear to be more sensitive to arsenic-induced oxidative stress than the breast cells. It has been reported that because of hormonal activity, the breast is a constant target for oxidative stress, a process that occurs in the breast ductal system where it is surrounded by adipose tissues (12). Acknowledgements: This research supported by a grant from the National Institutes of Health (Grant NO.1G2RR13459), through the NCRR-RCMI Center for Environmental Health at Jackson State University (JSU). We thank Dr. Abdul K. Mohamed, Dean of the College of Science, Engineering and Technology at JSU for his support of this research.
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