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Effect of Natural and Synthetic Estrogens on A549 Lung Cancer Cells: Correlation of Chemical Structures with Cytotoxic Effects Silvana Andreescu,† Omowunmi A. Sadik,*,† and Dennis W. McGee‡ Departments of Chemistry and Biological Sciences, State University of New YorksBinghamton, P.O. Box 6000, Binghamton, New York 13902 Received September 17, 2004
A series of synthetic (nonylphenol, diethylstilbestrol, and bisphenol A) and natural (quercetin, resveratrol, and genistein) phenolic estrogens were investigated for their ability to affect the viability and proliferation of A549 lung cancer cells. To assess and distinguish the cytotoxic effect of individual estrogens, we used both the MTT tetrazolium spectrophotometric method and the fluorescence assay, while the induction of the cell specific apoptotic process was examined by fluorescence microscopy after treatment of cells with SYTO 24 green fluorescent dye. A systematic study of interferences for both fluorescence and MTT methods is presented. The results showed that both natural and synthetic estrogens decreased the viability and proliferation of A549 lung cancer cells in a dose-dependent manner but at different sensitivities. Nonylphenol appeared very different as compared to the other estrogens, acting by inducing the higher inactivation rate of the cells within a very short time. The cytotoxic effect of the estrogens was directly related to their structural and conformational characteristics including chain length, number, and position of hydroxyl groups and degree of saturation.
Introduction Evidence suggests that exposure to endocrine disrupting chemicals (EDCs) may result in disruption of endocrine and reproductive systems by interfering with normal hormone-regulated physiological processes in human and wildlife organisms (1-5). EDCs consist of synthetic and naturally occurring estrogens. Their estrogenic effects have been associated with a variety of mechanisms including cell cycle arrest (6, 7), druginduced DNA damage (8, 9), binding to estrogen receptors (1, 2, 10), inhibition of intracellular enzymes involved with cell proliferation such as tyrosine kinase or glucose6-phosphate dehydrogenase (G6PD) (6, 10-12), and antioxidant or prooxidant activity (13-15). However, despite a number of works in the field, no adequate mechanism exists to explain the effect of both synthetic and natural estrogens on cancerous cells. This could be attributed to (i) the complexity of the system involving many possible mechanisms as well as (ii) the experimental methodologies used that have been mainly restricted to a single target analyte. The existing information suggests that depending on the cell line, the quantification method, the dose, and the nature of the EDCs, these chemicals may stimulate or inhibit the growth of cancerous cells (3, 4, 16, 17). Among the cancerous cell lines, human lung cancer cells such as the A549 adenocarcinoma cells are of enormous interest. Lung cancer is one of the leading causes of cancer mortality all over the world, and at present, no successful anticancer drug exists to efficiently * To whom correspondence should be addressed. Fax: 607-777-4478. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Biological Sciences.
prevent and reduce the progression of lung cancer. Therefore, there is an urgent need to develop new strategies aimed at better understanding the molecular mechanism by which potential anticancer chemicals act and to further provide the basis for more effective treatments. Several studies suggest that the cytotoxic effect of EDCs, mainly polyphenols (PPh), may be strongly related to their chemical structure (7, 15-21). These natural estrogens are characterized by a phenyl side chain and a variable number of hydroxyl moieties and have some structural similarity with the natural estrogen, e.g., estradiol and other steroid hormones (1, 2, 5). For synthetic estrogens, a structure-activity relationship has been less studied. In this case, the chemical structure is not similar to the natural steroid hormones, and consequently, it has been suggested that their estrogenic effects were totally unexpected (1). Most works in this direction have been carried out to investigate the antioxidant and/or prooxidant activity of PPhs (13-15, 18), and relatively few studies have focused on cancerous cells (7, 17, 19). It has been reported that the presence and position of hydroxyl groups (particularly on the B ring) play a crucial role in the antioxidant activity; the most potent possesses between two and four hydroxyl groups (13). When these groups are methylated, the activity of the PPhs could be diminished or completely absent (1315, 18). A possible link was established between (i) the anticancer activity of several phenolic acids of both natural and synthetic origins in different human cancer cell lines, (ii) their chemical and conformational characteristics, and (iii) the antioxidant and prooxidant activities (17). Another study suggested that the presence of two additional OH groups might hamper the entrance of a naturally occurring estrogen, resveratrol, into the
10.1021/tx0497393 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/21/2005
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Figure 1. Chemical structures of tested EDCs.
cell (19). Generally, it was found that (i) the spatial arrangement of substituents is a major determinant (13), (ii) an increase in the OH number leads to a greater cytotoxicity, (iii) the presence of a double bond in the side chain also increased the cytotoxic effect, while (iv) the effect of the length and degree of saturation was variable and strongly dependent on the cell type (7, 16). However, the existing information does not allow us to predict if a particular compound possesses estrogenic activity or not based on its chemical structure (16). These chemicals can act as multidentate ligands that could bind simultaneously with different affinity to the cell at more than one point, while the complementarities between protein and EDCs are dictated by conformational flexibility and/or hydrophobicity in both components (13, 18). Studying the interactions between the two components requires the use of a reliable experimental methodology, free of interferences and able to identify at least one site of action in this complex system. Further studies are necessary to elucidate the effect of these chemicals on cancerous cells and establish their mechanism of action and a possible structure-activity relationship. In this context, the overall goals of this work are to (i) study the effect of natural and synthetic estrogens on A549 lung cancerous cells, (ii) identify a possible relation between the chemical structure and the cytotoxic effect, and (iii) investigate the basic interactions between phenolic estrogens and A549 lung cancerous cells in an attempt to establish their mechanism of action. The chemical structures of the natural and synthetic estrogens used in this study are presented in Figure 1. The effect of selected EDCs was studied using two distinct experimental protocols: the MTT spectrophotometric assay and a fluorescent-labeled DNA proliferation assay (fluorescence) assay. The performance of the two methods in studying these interactions were also compared. To our knowledge, this is the first report studying the effect of both synthetic and natural estrogens on A549 lung cancer cell with the ultimate goal of establishing a possible structure-activity relationship.
Material and Methods Reagents and Stock Solutions. Three natural estrogens, quercetin (Aldrich), genistein (Sigma), and resveratrol (Sigma), were tested and compared with three synthetic xenoestrogens,
bisphenol A (Aldrich), diethylstilbestrol (Sigma), and nonylphenol (Aldrich). Phenol was obtained from Sigma while catechol and resorcinol were from Aldrich. Because of their poor solubility, stock solutions of 5 × 10-3 M phenolic estrogens were freshly prepared in methanol. The final concentration of methanol in the medium did not exceed 1% (v/v), and this was found to have no effect on cell proliferation. Cell Culture. The lung adenocarcinoma A549 cell line (CCL185; American Type Culture Collection, Manassas, VA) was cultured in 75 cm2 cell culture flasks in tissue culture medium (TCM) containing Ham’s F12 nutrient (Sigma-Aldrich, St. Louis, MO) with 1.5 g/L sodium bicarbonate supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/ streptomycin (Fisher Scientific, Pittsburgh, PA), and 2 mM L-glutamine (Fisher Scientific, Atlanta, GA). The FBS was heat inactivated according to the procedure described by the producer, HyClone Laboratories, Inc. (South Logan, UT). The culture was incubated at 37 °C with a 5% CO2 in air atmosphere. Once a week, the cells were harvested through trypsinization with trypsin-EDTA (Sigma-Aldrich) and upon proper dilution were cultivated to a future growth in fresh medium. Cultures grown in a conventional 96 well plate were used for both the MTT and the fluorescence assays. Studies of the degradation of EDC by UV-vis spectrometry were carried out with cells grown in a 24 well plate. Before plating, the number of cells was determined using the Trypan Blue staining assay. Instruments. Fluorescence measurements were carried out using a multiwell plate fluorimeter CytoFluor II Fluorescence Reader set at an excitation wavelength of 485 nm and an emission of 530 nm. A dual ultramicroplate reader, model ELX808, BioTEK Instruments Inc., was used to measure the absorbance (A570-650 nm) for cytotoxicity measurement using the MTT method. Cytotoxicity Measurements. For the purpose of studying the effect of EDC on cancerous cells, cells were plated at a density of 2.5 × 104 cells per well in 100 µL of TCM. For these experiments, three similar plates containing TCM and/or cells were prepared simultaneously following the same experimental procedure. Studies of the effect of EDC on the growth of the A459 lung cancer cells were carried out by adding 2 µL of EDC solution (at the corresponding concentration) to each well containing cells grown for 1 day (at about 70-80% confluence). The plates were incubated at 37 °C under 5% CO2 atmosphere, and then, after 1, 2, and 3 days of incubation, cell viability was tested using the two methods, MTT and fluorescence assay (22). In the absence of EDC, assays that contained the same amount of methanol in which the EDC was added served as a control. Cytotoxicity data are expressed as percentages of the residual response using 100% sample in the absence of EDC but containing the same amount of methanol.
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MTT Assay. The cytotoxic activities of EDC on A549 cancerous cells were first determined using the standard microplate colorimetric MTT assay. MTT is a yellow tetrazolium salt: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Treatment of cells with MTT results in a dark blue formazan product generated via the reduction of MTT by mitochondrial dehydrogenases in living cells (23, 24). MTT was dissolved in phosphate-buffered saline (PBS) to obtain a stock solution at a concentration of 5 mg mL-1 and filtered. Five microliters of the stock solution was added to each well containing cells in 50 µL of medium, and the plates were incubated for 4 h under regular growth conditions. Two hundred microliters of acidified 2-propanol was then added to each well and mixed in order to dissolve the precipitate and to ensure color development. Next, the absorbance at 570 nm was measured on a dual microplate reader with the absorbance at 650 nm subtracted to account for the plastic well and cellular debris. To avoid the interferences generated by the presence of phytoestrogens with MTT (22, 25, 26), the effect of EDC on cancer cells was studied by washing the cells before adding the MTT reagent (22). For these experiments, after the supernatant (TCM containing EDC) was removed, the cells were washed twice with fresh medium. Finally, 50 µL of TCM was added to each well. Results are expressed as % of control and represent the percentage of residual activity (Ra) referring to a sample without EDC and containing the same amount of methanol in which the EDC was added and are calculated as
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Figure 2. Results obtained with the MTT assay showing the effect of QRC, GEN, and RES on A549 lung cancer cells (2.5 × 104 cells/well ) 100 µL) after 2 days of incubation. absence of cells, F0 is the fluorescence value corresponding to a well with cells in TCM with the same amount of methanol in which the EDC was added, and F0b is the background fluorescence in wells with TCM and methanol only. Results are presented as the average ( SD from at least four independent experiments in five different wells. Apoptotic cells were visualized using a Olympus fluorescence microscope at a magnification of 200×.
Results and Discussions As Ra ) × 100 A0 where As is the absorbance value obtained for a sample containing cells in the presence of a given concentration of EDC and A0 is the absorbance value corresponding to a well with cells in TCM containing the same amount of methanol in which the EDC was added. The MTT results represent the means ( SD from tests carried out in five wells with cells from at least three independent experiments using cells from different cultures. Fluorescence Assay. The effect of EDC on cells was also determined by measuring the level of DNA synthesis after staining the cells with a fluorescent dye. For these tests, a similar experimental procedure was used to study the cytotoxicity (i.e., EDC concentrations and incubation times) as for the MTT method. The level of DNA was determined by measuring the fluorescence intensity of the cells containing 5 µL per well solution of a cell permeant chromophore SYTO 24 green fluorescent nucleic acid stain (diluted 1/100 in PBS). For these experiments, the final volume of cell solution per each well was 100 µL. After 20 min of incubation with the cells, the fluorescent dye is incorporated into the cells and binds to DNA, exhibiting bright, green fluorescence. The corresponding fluorescence is measured in a fluorescence multiwell microplate reader or by fluorescence microscopy. The fluorescent reagent (5 mM solution in DMSO) was from Molecular Probes (Eugene, OR). Background fluorescence was considered for wells containing TCM with EDC in the absence of cells. The percentage of residual response was expressed considering a control sample containing untreated cells exposed to the same amount of methanol in which the EDC was added. Results are presented as percentages of residual activity by considering as 100% control a sample containing cells with the same amount of methanol but in the absence of EDC. The percentage of residual activity was calculated as
Ra )
Fs - Fsb F0 - F0b
× 100
where Fs is the fluorescence determined for a sample containing cells in the presence of EDC, Fsb is the background fluorescence determined in wells with the same amount of EDC but in the
Induction of cytotoxicity by inhibiting cell growth is the result of a complex mechanism involving various parameters including binding to estrogen receptors, enzyme inhibition, and antioxidant or prooxidant activity (8-12). Therefore, to ensure consistent results regarding the effect of natural and synthetic estrogens on cancerous cells, a reliable and accurate quantification method of their cytotoxic effect must be used. In the present work, we used two different techniques, each monitoring a different target analyte: The MTT measures the mitochondrial enzyme activity in living cells, while the fluorescence assay measures the level on DNA synthesis. On the other hand, to avoid any “false positive results”, we need to ensure that each method is interference free. Effect of Natural Estrogens (QRC, RES, and GEN) on A549 Lung Cancer Cells. The treatment of A549 lung cancer cells with QRC, RES, and GEN was found to exert a cytotoxic effect in a dose-dependent manner for concentrations ranging from 10 to 100 µM (Figure 2) but at a different sensitivity between these three natural estrogens tested. We found that up to 24 h of contact was necessary for all three natural EDCs to induce any measurable inhibitory effect. The higher inactivation rate of the cells was recorded after 2 days of treatment with QRC, while RES and GEN induced comparable effects. Incubating the cells with natural estrogens for a longer time (e.g., 3 days) did not result in enhanced cytotoxicity. The lowest residual activity was around 40.5 ((10)% after 2 days of incubation, in the case of QRC, and we did not observe a complete inhibition of the cells with QRC, GEN, or RES. The degree of inactivation followed the same variation in a dose- and timedependent manner for similar concentrations of EDC using both MTT and fluorescence assays. Effect of Synthetic Estrogens (NPh, DES, and BPhA) on A549 Lung Cancer Cells. Using the MTT assay, treatment of A549 lung cancer cells with the three synthetic estrogens DES, BPhA, and NPh induced inhibition of cell proliferation in a dose-dependent manner for concentrations ranging from 0 to 100 µM (Figure 3).
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Figure 4. Comparison between the MTT and the fluorescence assays when studying the effect of 50 µM QRC, GEN, RES, BPhA, DES, and NPh on A549 lung cancer cells (2.5 × 104 cells/ well ) 100 µL) after 2 days of incubation. Results are the means of n ) 5 wells from three independent experiments.
Figure 3. Effect of BPhA, DES, and NPh on A549 lung cancer cells (2.5 × 104 cells/well ) 100 µL) after 1 (A) and 2 (B) days of incubation using the MTT assay. Results are the means of n ) 5 wells from three independent experiments.
The effect was strongly dependent upon the type and the concentration of each EDC tested as well as the incubation time. Surprisingly, we found that NPh and DES produced a strong inhibition of cell proliferation. The higher inactivation rate of the cells was recorded with NPh, which is remarkable, considering the difference in its structural characteristics in comparison with natural estrogens that are well-known for their cytotoxic effects. In addition, the toxic effect of NPh seemed to be extremely strong; a concentration of 100 µM was found to almost completely inhibit the cells with only 4 ( 1% residual activities. This effect was not recorded with any of the natural estrogens tested. For instance, for the same concentration, the lower residual activity obtained after treatment with QRC under identical experimental conditions was approximately 40%. For DES, the inactivation of the cells was similar for concentrations ranging between 10 and 50 µM (Ra of around 41%) but higher for 100 µM (Ra of around 18%). BPhA seemed to have a lesser effect on cell proliferation, with a weak effect for concentrations of 10 and 25 µM after 2 days of incubation. For 50 and 100 µM, an Ra of around 65% was recorded, suggesting a slight inhibition at higher concentrations. This effect was very similar with that recorded for the natural estrogens GEN and RES. All of the results discussed so far were obtained using the MTT assay. Comparison between the MTT and Fluorescence Assays. Study of Interferences. Several works have suggested possible interferences from a direct interaction of certain phytoestrogens and antioxidants with the MTT formazan (25, 26). These findings have also been confirmed in our experiments for all of the natural estrogens tested. In a recent paper, we studied the exact nature of
these interferences using UV/vis spectroscopy (22). Our results showed that after 4 h of incubation of phenolic estrogens with the MTT reagents a new product appeared, characterized by a broad peak between 500 and 700 nm with a maximum at ∼680 nm. This peak was found in the region where the formazan product generated by the conversion of MTT by living cells absorbs. This chemical conversion of the MTT induced by the presence of estrogens is responsible for the interferences observed when studying the effect of EDC on cancerous cells using the MTT method. Hence, to avoid any direct contact of the MTT with the EDC, cells were washed twice using fresh TCM before the addition of MTT, thus removing the interferences. As opposed to the MTT experiments conducted under similar experimental conditions, no interferences were observed using the fluorescence assay when natural estrogens were tested. In this case, the residual activity of the A549 cells after exposure to 50 µM QRC, GEN, and RES determined using the fluorescence assay followed a similar trend as the MTT assay (Figure 4). However, the values for the fluorescence assay are slightly higher and the SD is broad. A similar variation was registered for the synthetic estrogen, BPhA. An interesting aspect noticed in these experiments is that for the other synthetic estrogens, NPh and DES, the results obtained using the MTT and fluorescence assays appeared contradictory. Thus, while the Ra obtained using the MTT assay decreased, suggesting an inhibition of cell growth, the fluorescence assay indicated an increase in the Ra (Figure 4). This may suggest an increase in DNA synthesis and, consequently, a possible enhancement of cell proliferation. These tests demonstrate strong interferences when the fluorescence assay is used. To clarify and eliminate the exact nature of these interferences, we washed the cells using fresh TCM before adding the SYTO 24 dye. For these experiments, background fluorescence was considered for wells containing only TCM without cells. All other parameters including EDCs and cells concentration and incubation time were similar. No significant differences were obtained between control samples containing only TCM in the presence and in the absence of EDC, suggesting that these interferences cannot be attributed to the presence of EDC in the medium. Washing the cells induced a decrease in the residual activity, but this was not in the same concentration range as with the MTT
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Table 1. Comparison between the Results Obtained Using the MTT and Fluorescence Assays after 2 Days of Treatment of 2.5 × 104 A549 Cells/Well with 50 µM NPh, DES, and BPhAa EDC
fluorescence (%) washing the cells
fluorescence (%)
MTT (%)
control NPh DES BphA
98.2 ( 14.4 58.9 ( 9.3 96.8 ( 22 67.7 ( 32
100 ( 9 151 ( 50 101.6 ( 27 86.8 ( 32
100 ( 8.4 14.8 ( 7 40.5 ( 1.6 84.7 ( 13.2
a Average ( SD of values obtained from n ) 5 experiments; percent of control by considering 100% a sample with cells in the absence of EDC.
assay. Table 1 compares the results obtained using MTT and fluorescence assays with or without washing the cells after 2 days of treatments of A549 lung cancer cells (2.5 × 104 cells/well ) 100 µL) with 50 µM NPh, DES, and BPhA. The results suggest that washing the cells before adding the fluorescent dye is not efficient to completely eliminate the interferences and, also, these are not a result of a direct interaction of the dye with the EDCs. Consequently, the fluorescence results reflect a possible artifact when measuring the DNA level. During cell death by apoptosis, even though the DNA is damaged, the nucleic acid fragments may still be present in the medium and may bind to the fluorescent dye thus generating enhanced fluorescence. In the NPhtreated culture, the cells may be lysed, releasing the DNA into the entire medium, and its fluorescence is actually measured upon binding to the fluorescent dye. These results confirm the need to use different assays to evaluate the effect of these chemicals on cells. Such confirmatory experiments are needed to avoid any “false positive results”. On the other hand, the fact that each method measures a different target analyte, it is important to take into account the specific detection principle as well as their limitations. In this case, the fluorescence assay may be less applicable due to high background fluorescence and interferences in the case of NPh and DES. Further confirmation of the MTT and fluorescence results may be obtained by microscopic examination of the cells exposed to these chemicals upon binding to the fluorescent dye. This technique is also very important to confirm the induction of the cell specific apoptotic process. Consequently, the cells were analyzed using fluorescence microscopy after treatment with the EDC by staining with SYTO 24 green fluorescent dye (Figure 5). In the presence of both natural (Figure 5B-D) and synthetic (Figure 5E-G) estrogens, the cultures showed fewer cell numbers along with high numbers of cells containing nuclei with condensed chromatin characteristic of apoptotic cells. Alternatively, the control cultures (Figure 5A) showed large nuclei with evenly distributed chromatin. The apoptotic process appears more intense in the case of NPh (Figure 5F). In conclusion, the visual examination of the cells by means of fluorescence microscopy confirmed the reduced number of cells in the wells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the apparent increase of cell proliferation suggested by the fluorescent assay does not reflect the real change in cell activity after exposure to EDC. Unusual Toxicity of Nonylphenol. NPh was accidentally found to possess estrogenic activity by Soto et al., when this chemical leached from the plasticware used
during cell culture (27). Several studies demonstrated that alkylphenols affect cell viability and function via a mechanism involving the inhibition of Ca2+ pumps (28, 29). For instance, Hughes et al. showed that alkylphenols such NPh dramatically decrease the viability of TM4 cells by inhibiting Ca2+ ATPase (SERCA proteins), which removes cytoplasmic Ca2+ or testis endoplasmic reticulum Ca2+ pumps, one of the most abundant Ca2+ transporters in eukaryotic cells (28). NPh, DES, and other synthetic alkylphenols were found to inhibit the rate constant of both the fast and the slow phases of inositol-1,4,5 (IP3)triphosphate sensitive Ca2+ and the extent of Ca2+ release (29). In the same study, the DNA laddering experiments established that the alkylphenols NPh, BPhA, and octylphenol induced cell death by apoptosis and not by necrosis, with NPh as the most potent. Other studies using NPh but with other cell lines reported an increase in cell proliferation at lower concentrations (27, 30). For instance, NPh was shown to induce cell proliferation and progesterone receptor in human estrogen sensitive MCF-7 breast cancer cells and in animal models (27). NPh was also shown to enhance carcinogenesis in BALB/3T3 cells in a dose-dependent manner at concentrations of 5-40 µM, in a similar way as 3-tert-butyl-4-hydroxyanisole, a phenolic antioxidant well-known to induce and enhance carcinogenesis (30). In our experiments, treatment of A549 lung cancer cells with over 50 µM NPh was found to induce higher inactivation rate in comparison with natural phytoestrogens. The cells were nearly unaffected at 10 and 25 µM but were completely inhibited at 100 µM NPh. To establish if this inactivation process at 100 µM was an immediate effect arising from the simple contact of the cells with NPh, we measured the cytotoxicity after lower incubation times. The results obtained using the MTT assay showed that after only 3 h of incubation, a concentration of 100 µM NPh induced a strong inhibition of the cells, with a Ra of 17 ((-4)%. Considering several literature data (28-30), we expected that the apoptotic process should only occur at high concentrations of NPh while lower concentrations should induce an enhancement of cell proliferation. To confirm this hypothesis, we tested concentrations of NPh ranging from 0.5 to 100 µM (Figure 6). Results showed that after 2 days of incubation, the cells were almost unaffected at concentrations below 10 µM NPh. These results have also been confirmed by fluorescence spectroscopy (Figure 7). As can be seen, a sample of A549 cells treated for 2 days with 10 µM NPh appeared almost unaffected but completely apoptotic when 100 µM was used. Correlation of Cytotoxic Effect to Structure of the Estrogens and Mechanism of Action. Despite a number of papers in the field, the information about the mode of action and a possible correlation of structureactivity among different estrogens is still limited. The results obtained in this work established that all natural and synthetic estrogens tested decreased the viability and proliferation of A549 lung cancer cells, while fluorescence microscopy suggested the induction of a cell specific apoptotic process. In addition, we have demonstrated that the cytotoxic effect was very much dependent upon the nature, type, and concentration of EDCs. Using the experimental results obtained, we attempted to (i) assess and distinguish the toxicity of individual estrogens on A549 lung cancer cells in relation to their chemical structure and (ii) establish the extent to which a change
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Figure 5. Fluorescence microscopy showing induction of apoptosis by 50 µM natural and synthetic estrogens in A549 lung cancer cells after 2 days of incubation (magnification, 200×). (A) Control cells treated with the same amount of methanol in which the EDC was added and (B) QRC, (C) GEN, (D) RES, (E) BPhA, (F) NPh, and (G) DES.
Figure 6. Effect of various concentrations of NPh after 2 days of incubation with 2.5 × 104 cells/well A549 lung cancer cells. Results are the means of n ) 5 wells from three independent experiments.
in conformation and the presence of substituents affect the cytotoxic effect on cancerous cells. In this case, we compared and summarized the results obtained using the MTT and fluorescence assays for both natural and synthetic estrogens tested to their chemical formula. Table 2 shows the comparative results obtained under identical experimental conditions after 2 days of treatment of A549 lung cancer cells (2.5 × 104 cells/well) with 50 µM phenolic estrogens.
Our results established that treatment of A549 lung cancer with the environmental xenoestrogens NPh, DES, and BPhA produced inhibition in a concentration-dependent manner. However, from a structural perspective, their chemical structure and conformation present very distinct characteristics as compared to natural estrogens. The most surprising result was obtained for NPh, which has a long carbon chain (9 × C), a single phenyl ring, and is strongly hydrophobic. More remarkably, under the experimental conditions, this compound showed a much higher cytotoxicity than any of the natural estrogens tested that are well-known for their anticancer activities. As confirmed by fluorescence microscopy, the treatment of cells with 50 or 100 µM NPh engendered a typical apoptotic process of basically all of the cells present in the medium, while at lower concentrations the cells were almost unaffected. We attempted to correlate the cytotoxic effect of NPh with its unique chemical characteristic. To assess the involvement of the phenyl ring in the cytotoxic effect, we tested simple phenolic compounds including phenol, catechol, and resorcinol. These are different by the number and the position of the OH groups. To study if the toxic effect is due to the hydrophobic chain, we also tested 1-tetradecanol, which contains a long C (10 × C) chain and a single hydroxyl group. After 2 days of incubation of the cells with these chemicals, the results
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Figure 7. Fluorescence microscopy showing induction of apoptosis by 0, 10, 50, and 100 µM NPh in A549 lung cancer cells after 2 days of incubation (magnification, 200×). Table 2. Structural Correlation with Cell Toxicity Using MTT and Fluorescence Assays for Selected Estrogens and Related Chemicals structural characteristics PPhs control quercetin resveratrol genistein bisphenol A diethylstilbestrol nonylphenol 1-tetradecanol phenol catechol resorcinol
OH 5 3 3 2 2
catechol C ring CdC aliphatic +
1 1 1 2 2
1
2
1 1
3 6 9 10
1
MTT (%)a
fluorescence (%)a
100 ( 9 100 ( 8.4 40.5 ( 10.4 43.23 ( 7.1 54.3 ( 10 69.3 ( 5.2 56.8 ( 12.4 82.8 ( 22 84.7 ( 13.2 86.8 ( 32 40.5 ( 1.6 101.6 ( 27 14.8 ( 7 100.3 ( 2.9 97 ( 2.4 66.3 ( 4.5 92.8 ( 1
151 ( 50 ND ND ND ND
a Percent of control by considering 100% a sample in the absence of the tested chemical. Average ( SD of values obtained from four independent experiments (in n ) 5 different wells) with cells from at least three different cultures; ND, not determined.
showed that neither 1-tetradecanol nor phenol or resorcinol induced any decrease in cell proliferation (Table 2). However, an inhibition of 33% was recorded for catechol. Among the natural estrogens tested, QRC appeared to induce the higher level of inactivation, while RES and GEN showed comparable effects. Following the decrease in cell growth over time, the results indicate that inhibition is not an immediate result of the simple contact of the chemical with the cells; at least 24 h is necessary in order to record any inactivation effect. The higher effect was recorded after 2 days of incubation. This can be explained by the fact that induction of cell growth inhibition could be related to a very complex mechanism
inside the cells. In that case, the mechanism first requires the penetration of the cell membrane and subsequent binding to the specific site of action: DNA, receptors, and enzymes. This may be or may not be the case with NPh, which acts much more rapidly possibly by destroying the membrane. QRC possesses five hydroxyl groups, as compared to only three for RES and GEN. In addition, it also contains a catechol ring in the molecule, which has often been related to a higher antioxidant activity (13-19). The effect of a catechol ring is more evident when comparing phenol and catechol: An inhibition of 33% was recorded for catechol while phenol showed no effect. This feature was also confirmed by the absence of inhibition in the case of resorcinol. This could be explained by a faster onestep oxidation of catechol to the quinone. This transformation may also occur for mono- and o-diphenolic estrogens and can be catalyzed by intracellular enzymes belonging to the oxidase family (31). On the other hand, RES possesses a double CdC bond, which basically confers more flexibility to the molecule. RES presents some structural similarities with the synthetic estrogen BPhA. As compared to RES, BPhA also presents two aryl rings but has a more rigid structure due to the absence of the CdC bond. In this case, the degree of inactivation could be more important for RES while the same concentration of BPhA had a very low cytotoxic effect. RES and DES also present a very close structural resemblance, the only difference being the nature of substituents: RES possesses one additional OH while DES has four extra aliphatic carbons, which may induce steric constraints and thus a lower cytotoxic effect. Indeed, the MTT values for DES as compared to RES support this hypothesis with a Ra of 54.3% for RES, as compared to 40.5% for DES.
Estrogens on A549 Lung Cancer Cells
Conclusion In this work, we have studied the cytotoxic effect of natural and synthetic estrogens on the viability and proliferation of A549 lung cancer cells in relation to their chemical structure and spatial arrangement of the molecule. The results have been achieved and compared using two different cell culture techniques including MTT spectrophotometric and fluorescence assays. The results obtained in this study demonstrated that both natural and synthetic estrogens inhibited the proliferation of A549 lung cancer cells. The cytotoxic effect was very much dependent upon the dose, the nature, and the type of EDCs as well as the incubation time. Surprisingly, despite very distinct structural characteristics, a higher degree of inhibition was recorded for NPh, which possesses a long carbon chain and only one phenolic ring. Study of interferences demonstrated that both methods present interferences when studying the interactions of estrogens with cancerous cells. However, using a procedure involving cell washing before adding the formazan compound eliminates this unwanted effect for the MTT assay. The fluorescence method showed no interferences and a good correlation with the MTT assay for natural estrogens and BPhA. On the contrary, for NPh and DES, the interferences were very strong, leading to contradictory results, while the use of a washing procedure was not effective to solve this problem. Nevertheless, fluorescence microscopy supported the MTT results and confirms the presence of fewer numbers of cells per wells and a cell specific apoptotic process. These results highlight the need to perform different assays in order to evaluate the effect of these chemicals on cells viability and proliferation and to avoid any false positive results. The fluorescence assay is very easy to perform but could be characterized by relatively high background fluorescence, and also, the SD was important. The MTT method is versatile and quantitative, even though some EDCs interfered with the MTT reagent. On the other hand, microscopic examination using a fluorescent dye is a very important technique to confirm cell numbers and show the presence of apoptotic cells, as presented in this work. We compared the results obtained with the MTT method and related these findings with the chemical structure of the EDCs in an attempt to explain their mechanism of action in lung cancer cells. Our study suggests that natural and synthetic estrogens can affect cell growth through a separate mechanism, involving a specific target analyte, and also, each compound may have different effects and implications. In conclusion, this study has provided future insights into the type, quantity, and specific influence of both natural and synthetic estrogens on A 549 lung cancer cell and may be useful to establish the connection between these chemicals and lung cancer.
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