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Chem. Res. Toxicol. 2010, 23, 1054–1066
1,4-Benzoquinone (PBQ) Induced Toxicity in Lung Epithelial Cells Is Mediated by the Disruption of the Microtubule Network and Activation of Caspase-3 Amlan Das, Subhendu Chakrabarty, Diptiman Choudhury, and Gopal Chakrabarti* Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, UniVersity of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB, India 700019 ReceiVed February 5, 2010
Parabenzoquinone (1,4-benzoquinone) (PBQ) is a bioactve quinone present in cigarette smoke and diesel smoke, which causes severe genotoxic effects both in Vitro and in ViVo. In the previous study, we showed that the microtubules are one of the major targets of cigarette smoke-induced damage of lung epithelium cells. In the present study, we have investigated the effect of PBQ on cellular microtubules using human type II lung epithelial cells (A549) and also on purified tubulin. Cell viability experiments using A549 cells indicated a very low IC50 value (∼7.5 µM) for PBQ. PBQ inhibited cell cycle progression and induced apoptosis of A549 cells. PBQ also induced the contraction and shrinkage of the A549 cells in a time- and concentration-dependent manner, which is proved to be a direct effect of the damage of the microtubule cytoskeleton network, and that was demonstrated by a immunofluorescence study. PBQ also inhibited the assembly of tubulin in lung cells and a in cell free system (IC50 ∼5 µM). Treatment with PBQ resulted in the degradation of tubulin in lung cells without affecting the actin network, and this was confirmed by a Western blot experiment. Upregulation of pro-apoptotic proteins such as p53 and Bax and downregulation of antiapoptotic protein Bcl-2 were observed in PBQ-treated A549 cells. Simultaneously, loss of mitochondrial membrane potential and activation of caspase-3 were also observed in the PBQ treated lung epithelium cells. Fluorescence and circular dichroism studies demonstrated that the denaturation of tubulin in a cell free system was caused by PBQ. However, in the presence of N-acetyl cysteine (NAC), damage of the microtubule network in A549 cells by PBQ was prevented, which led to a significant increase in the viability of A549 cells. These results suggest that microtubule damage is one of the key mechanisms of PBQ induced cytotoxity in lung cells. Introduction 1
Parabenzoquinone (1,4-benzoquinone) (PBQ ) is a nonaromatic six-membered ring compound derived form the oxidation of 1,4-hydroquinone (1). It is a product of benzene metabolism (1), reported to be present in the cigarette smoke (2, 3) as well as in diesel smoke (4) and can cause adverse effects in eyes, skin, and the respiratory tract. This bio-active quinone was reported to cause severe genotoxic effects both in Vitro and in ViVo by forming adducts with the DNA bases (5-7) and inducing apoptosis in cultured mammalian cell lines by generating reactive oxygen species (8, 9). Parabenzoquinone was known to react with the cellular nucleophiles such as free thiol groups of proteins, glutathione (GSH), and N-acetylcysteine (NAC), forming covalently linked quinine-thiol complexes known as Michael adducts (10). It is an active component of cigarette smoke, which was previously reported to kill lung epithelium cells and block the activation of T-lymphocytes (2, 3). Damage of lung tissue is a very common phenomenon in cigarette smokers, which is characterized by a disease called emphysema (11). Emphysematous lung damage is generally observed in * To whom correspondence should be addressed. Tel: 91-33-2461-5445. Fax: 91-22-2461-4849. E-mail:
[email protected]. 1 Abbreviations: AECS, aqueous extract of cigarette smoke; CS, cigarette smoke; COPD, chronic obstructive pulmonary disease; PBQ, para-benzoquinone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; PI, propidium iodide; MMP, mitochondrial membrane potential; MAPs, microtubule associated proteins; Pipes, piperazinedinethane-suofonate.
patients suffering from chronic obstructive pulmonary disease (COPD), which is one of the leading causes of global mortality and morbidity (12). Hence, PBQ may play an important role in the severe damage of lung cells as in COPD. Tubulin is a heterodimeric protein composed of R and β subunits, and is present in all eukaryotic cells. The tubulin dimers polymerize end to end to form protofilaments, which then bundle in hollow cylindrical filaments known as microtubules. Microtubules are the dynamic cytoskeletal structures, which play important roles in the maintenance of cell shape and morphology. They participate in various cellular processes such as cell signaling, cell motility, organelle transport and maintenance of cell polarity, and separation of the duplicated centrosomes in cell division (13-15). The disruption of cellular microtubules leads to apoptosis of the mammalian cells (16-18). Dynamic microtubules are a potential target for the oxidative damage in the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD) (19, 20), Parkinson’s disease (21), and cigarette smoke induced atherosclerosis (22). In the previous study, we reported tubulin as a potential target for the aqueous extract of cigarette smoke (AECS), and we demonstrated that damage of the microtubule cytoskeleton is one of the mechanisms of AECS induced apoptosis of lung epithelial cells (23). In this article, we have investigated the mechanism of action of PBQ, a component of cigarette smoke and diesel smoke, leading to extensive damage of lung epithelial cells, a major event occurring during emphysema. The cytotoxic
10.1021/tx1000442 2010 American Chemical Society Published on Web 05/25/2010
Parabenzoquinone Perturbs Microtubules Network
effects of PBQ were examined with human lung epithelium cells (A549). There are previous reports about the loss of polymerization activity of tubulin in the presence PBQ (24, 25). Hence, microtubules, polymers of tubulin, might be a likely target of PBQ in the lung epithelial cells. Simultaneously, the involvement of mitochondria-dependent apoptosis, via the activation of caspase-3 was also investigated in the PBQ treated lung epithelial cells. To investigate the loss of the functional and structural properties of tubulin in the presence of PBQ in a cellfree system, purified tubulin from a goat brain was used. Various functional and structural properties, such as polymerization activity, colchicine binding ability, change in tryptophan fluorescence due to gradual unfolding of the protein structure, and loss of secondary structure of the purified tubulin were studied in the presence of PBQ. Again, cell viability, cell cycle progression, and status of the microtubule network were investigated in PBQ treated A549 cells, which were preincubated with N-acetyl-cysteine (NAC). The results are presented in this article.
Experimental Procedures Materials. Nutrient mixture F12 HAM (supplemented with 1 mM L-glutamine), FBS, penicillin-streptomycin, and amphotericin B were purchased from HyClone. Human lung epithelium cell line (A549) was obtained from National Centre for Cell Science, Pune, India. Trypsin-versene (1×) was purchased form Cambrex Bioscience, FITC conjugated monoclonal anti-R-tubulin antibody (raised in mouse), mouse monoclonal anti-R-tubulin antibody, mouse monoclonal anti-p53 antibody, mouse monoclonal anti-Bax antibody, GTP, PIPES, MgCl2, EGTA, and DAPI were purchased from Sigma. Mouse monoclonal anti-Bcl-2 antibody and rabbit monoclonal anti-β-actin antibody were obtained from Santa Cruz, CA. Antimouse rhodamine-conjugated secondary antibody, antimouse HP-conjugated secondary antibody, and antirabbit HPconjugated secondary antibody were purchased from GeNei, India. All other chemicals and reagents were of analytical grade and were purchased from Sisco Research Laboratories, India. Cells Culture. Human type II lung epithelium cells (A549) were maintained in Nutrient mixture HAM’s F12 supplemented with 1 mM L-glutamine, 10% fetal bovine serum, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 2.5 µg/mL amphotericin B. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in tissue culture flasks until they were 80% confluent before trypsinization with 1× trypsin-versene and splitting. The morphology of normal and treated cells was observed by an Olympus inverted microscope model CKX41. Cell Treatment. Cultured A549 cells were grown to confluency (1 × 106 cells/mL) in a 35 mm Petri plate (Nunc, Denmark), treated with different concentrations of PBQ (0-25 µM), and incubated for 24 h, unless otherwise stated. To investigate the antagonistic effect of N-acetyl cysteine (NAC) against PBQ induced damage in the A549 cells, cultured A549 cells were preincubated with 500 µM NAC for 4 h. The old media were discarded, fresh media containing varying concentrations of PBQ (0-25 µM) were added to the cells, and subsequently, they were incubated for 24 h. Cell Proliferation Inhibition Assay (MTT Assay). To assess cell viability, MTT assay was performed following the protocol discussed in ref 23. Briefly, cultured A549 cells were plated in 96well culture plates (1× 104 cells per well), treated with different concentrations of PBQ (0-50 µM), and incubated for 24 h. MTT (2 mg/mL) was dissolved in PBS and filter sterilized, and then 50 µL of the prepared solution was added to each well. This was incubated until a purple precipitate was visible. The absorbance was measured on an ELISA reader (MultiskanEX, Lab systems, Helsinki, Finland) at a test wavelength of 570 nm and a reference wavelength of 650 nm. Cell Cycle Analysis by Flow Cytometry. Cultured A549 cells were grown at a density of 106 cells/mL and incubated in the
Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1055 presence of PBQ (0-10 µM) for 24 h. After treatment, the cells were harvested, fixed in ice cold methanol for at least 30 min in 4 °C, and incubated for 4 h at 37 °C in a PBS solution containing 1 mg/mL RNase A. Then nuclear DNA was labeled with propidium iodide (PI). Cell cycle analysis was performed using a Becton Dickinson FACS Caliber flow cytometer, and the data were analyzed using the Cell Quest program from Becton Dickinson. Flow Cytometric Analysis for Apoptotic and Necrotic Cells. Cultured A549 cells were grown at a density of 106 cells/mL and incubated in the presence of PBQ (0-10 µM) for 0 h-24 h. At different time intervals, cells were stained for 15 min at room temperature in the dark with fluorescein isothiocyanate (FITC)conjugated annexin V (1 µg/mL) and PI (0.5 µg/mL) in a Ca2+enriched binding buffer and analyzed by a two-color flow cytometric assay. Annexin V and PI emissions were detected in the FL1 and FL2 channels of a FACS Caliber flow cytometer, using emission filters of 525 and 575 nm, respectively. Apoptosis analysis was performed using Becton Dickinson FACS Caliber, and the data were analyzed using the Cell Quest program from Becton Dickinson. Detection of Mitochondrial Membrane Potential (MMP) by JC-1 Staining. Mitochondrial membrane potential changes in PBQ treated A549 cells were detected using the fluorescent probe JC-1 (Sigma, USA), a lipophilic cationic dye which accumulates in the living mitochondria, as described in refs 26 and 27. At low MMP, the dye exists as a monomer and emits green fluorescence, but with the increase in the MMP, JC-1 forms J-aggregates. Dye aggregation leads to a shift in fluorescence emission from green to red. For flow cytometric analysis, A549 cells treated with PBQ (0-10 µM) were incubated for 15 min with 1 µg/mL JC-1 in culture medium at 37 °C. Green fluorescence and red fluorescence were detected by FL-1 and FL-2 filters, respectively, using Becton Dickinson FACS Caliber. Preparation of the Cytosolic Extract and Detection of Cytochrome c Release in the PBQ Treated A549 Cells by Western Blotting. Release of the cytochrome c in apoptotic cells was detected by Western blotting, using mouse monoclonal anticytochrome c antibody (clone 6H2.B4, BD Pharmingen, San Diego, CA). Cultured A549 cells were grown to confluency in a 90 mm Petri plate and then treated with PBQ (0-10 µM) for 24 h. Cells were then trypsinized in ice chilled PBS and centrifuged at 600g for 10 min. The pellet was again washed in chilled PBS, and centrifuged at 600g for 10 min. The pellet was then incubated in 200 µL 1× Extraction Buffer A (isotonic solution, 10 mM HEPES, pH 7.5, containing 200 mM mannitol, 70 mM sucrose, and 1 mM EGTA) at 4 °C for 15 min. The whole cell lysates were transferred in a glass homogenizer and homogenized using 30 strokes. The homogenate was centrifuged at 600g for 10 min at 2-8 °C, and the supernatant was collected in a fresh tube. It was then centrifuged at 11000g for 10 min at 2-8 °C, and the supernatant was collected. The cytosolic part resides in the supernatant. Total protein was estimated from each set by using the Bradford reagent (28), and 30 µg was loaded in each well for the Western blot. Sample Preparation for Confocal Microscopy. Cultured A549 cells were grown at a density of 106 cells/mL and incubated in the presence of PBQ (0-25 µM) for 24 h. Subsequently, cells were washed twice by PBS, fixed by 2% para-formaldehyde, and incubated with permeable solution (0.1% Na-citrate, 0.1% Triton) for 1 h. Nonspecific binding sites were blocked by incubating the cells with 5% BSA. Cells were then incubated with mouse monoclonal anti-R-tubulin antibody (1:200 dilutions) followed by anti-mouse rhodamine-conjugated IgG antibody (1:150 dilutions) or FITC-conjugated mouse monoclonal anti-R-tubulin antibody (1: 50 dilutions), and DAPI (1 µg/mL). After incubation, cells were washed with PBS and viewed under a Ziess LSM 510 Meta confocal microscope. Following the same protocol, fluorescence images of the expressions of p53 (1:50 dilutions), Bax (1:50 dilutions), and Bcl-2 (1:50 dilutions), in the control and PBQ (10 µM) treated A549 cells were taken by using the a Ziess LSM 510 Meta confocal microscope. Assay of Caspase-3 Activity by Fluorimetric Analysis. Activity of caspase-3 in the PBQ untreated and treated A549 cells was
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Figure 1. Inhibition of proliferation and cell cycle progression, and induction of apoptosis in A549 upon treatment with PBQ. (A) Loss of viability of A549 cells with increasing concentration of PBQ (0-50 µM), when incubated for 24 h (described in Experimental Procedures). Cell viability was assessed by MTT assay. Data are represented as the mean ( SEM [* P < 0.05 vs control (PBQ untreated cell), where n ) 4]. (B) Cell cycle progression study using PBQ treated A549 cells. Cultured cells (1 × 106 cells/mL) were treated with PBQ (0-10 µM) for 24 h. The distribution of the cell cycle was analyzed by a flow cytometer, and the percentage of distribution in distinct phases of the cell cycle was determined using ModFIT software (Becton Dickinson). The sub G0/G1, G0/G1, S, and G2/M phases of the cell cycle were represented as a, b, c and d, respectively. Data are represented as the mean ( SEM, where n ) 3. (C) Time-dependent induction of apoptosis in PBQ treated A549 cells. Cultured A549 cells were treated with PBQ (10 µM), and apoptosis was monitored by annexin-V-FITC and PI double staining, after 0, 6, 12, and 24 h of incubation. Apoptotic cells were analyzed flow cytometrically, and a dot plot representation of annexin-V-FITC-fluorescence (x-axis) vs PI-fluorescence (yaxis) has been displayed. The percentage of early apoptotic cells in the lower right quadrant (annexin V-FITC positive and PI negative cells), as well as late apoptotic cells located in the upper right quadrant (annexin V-FITC positive and PI positive cells), is shown. The figure represents the best of three independent experiments. (D) Confocal images of the nucleus stained with DAPI of A549 cells treated with 0 µM and 10 µM PBQ. Data represents the best of three independent experiments.
assessed by using Caspase-3 In Situ Assay Kit, Fluorescein (Chemicon International, APT 403). In this assay, a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase-3 (FAM-DEVD-FMK) was used, which produced a green fluorescence at around 520 nm, after being excited at 488 nm. Cultured A549 cells were grown at a density of 106 cells/mL and incubated in the presence of PBQ (0-10 µM) for 24 h. Cells were centrifuged at 300g for 10 min, and the pellets were suspended in 200 µL of PBS. FAM-DEVD-FMK (10 µL) was added to each sample as recommended in the kit, and incubated at 37 °C for 1 h in dark condition. Cells were again centrifuged at 300g for 10 min, and the pellets were washed with 1× wash buffer provided in the kit. After washing, the final pellets were resuspended in 300 µL of PBS, and fluorescence was measured using an excitation wavelength of 490 nm and an emission wavelength of 520 nm by a Jasco F 6300 spectrofluorimeter.
Assembly of Microtubules after Cold Treatment in PBQ Treated A549 Cells. Assembly of the cold depolymerized microtubules of A549 cells in the presence of PBQ (0-10 µM) was observed by immunofluorescence against R-tubulin using a Ziess LSM 510 Meta confocal microscope. Cultured A549 (1 × 10 6 cells/mL) cells were grown on glass coverslips for 24 h and then incubated at 4 °C for 6 h. After cold treatment, the cold medium was replaced with warm medium containing the indicated doses of the above-mentioned PBQ, and subsequently, the samples were incubated at 37 °C. Cells were then fixed at different time points (0 and 60 min) with 2% (v/v) paraformaldehyde at room temperature for 20 min. The fixed cells were then processed according to the method discussed previously to visualize the reassembled microtubules and DNA using a Ziess LSM 510 Meta confocal microscope.
Parabenzoquinone Perturbs Microtubules Network
Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1057 Fluorescence spectroscopic studies were done to detect the changes in the intrinsic tryptophan fluorescence of tubulin (1 µM) in the presence of different concentrations of PBQ (0-20 µM) by monitoring the emission spectra between 310-400 nm, after exciting at 295 nm, after incubation at room temperature for 45 min. Tryptophan fluorescence was measured by using a Hitachi fluorescence spectrophotometer model F-7000. To determine whether PBQ was interfering with colchicine binding of tubulin, the fluorescence of the tubulin (3 µM) and colchicine (5 µM) complex in the presence of various concentrations of PBQ (0-20 µM) was monitored by exciting at 350 nm, and a wavelength scan was done in between 310 and 400 nm. Statistical Analysis. Data are presented as the mean of at least three independent experiments along with standard error of the mean (SEM). Statistical analysis of data was done by Student’s t test, by using MS Excel, and two measurements were statistically significant if the corresponding p value was
