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Articles Cigarette Smoke Extract Induces Disruption of Structure and Function of Tubulin-Microtubule in Lung Epithelium Cells and in Vitro Amlan Das, Abhijit Bhattacharya, and Gopal Chakrabarti* Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, UniVersity of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB, India 700019 ReceiVed June 13, 2008
In the present study, we have investigated the effect of the aqueous extract of cigarette smoke (AECS) on tubulin-microtubule, a major cytoskeleton protein that maintains cellular morphology and participates in cell division. We found that treatment of AECS results in the loss of both structural and functional properties of tubulin-microtubule. Disruption of the microtubule network was observed in AECS-treated human lung epithelial (A549) cells and noncarcinoma human lung alveolar epithelium (L132) cells, in a dose and time-dependent manner. Tubulin-microtubule mediated important cellular properties, such as proliferation, migration, and maintenance of the cellular morphology, were affected by AECS in A549 cells. The aqueous extract of cigarette smoke (AECS) was also found to interfere the microtubule dynamics inside the cell and induce tubulin degradation. The structure of microtubules was also disrupted by AECS in the presence of protease inhibitors accompanied by a change of morphology of cells and loss of cell viability. In vitro, the functional properties of tubulin, such as the ability of polymerization, was inhibited by AECS in a dose and time-dependent manner, and it was accompanied by the loss of reactive cysteine residues, destabilization of the secondary structure, and quenching of intrinsic tryptophan fluorescence. Carbonyl content of tubulin was increased after treatment with AECS, indicating that one of the pathways of tubulin damage is protein oxidation. The damage of tubulin by AECS thus may be correlated with the pathogenesis of cigarette smoke induced disorders, which result in cellular apoptosis and tissue damage. Introduction Microtubules are dynamic cytoskeletal polymers composed of tubulin heterodimers (R and β), which are present in all eukaryotic cells and play an important role in the maintenance of cell shape and morphology. They also 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 (1–3). It was reported that tubulin is a potential target for the oxidative damage in the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD) (4, 5) and Parkinson’s disease (6). Cigarette smoking is one of the major lifestyle factors that adversely influences the health of human beings. According to the World Health Organization (WHO), by the year 2020 tobacco smoking will become the largest single health problem worldwide and will cause an estimated 8.4 million deaths annually (http://www5.who.int/tobacco/). Cigerette smoke is the main etiological factor in several disorders, which include respiratory disorders in the lung such as emphysema and bronchitis, collectively known as chronic obstructive pulmonary disease (COPD) (7–9), cardiovascular disorders including myo* To whom correspondence should be addressed. Gopal Chakrabarti, Dr. B.C.Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700 019 India. Tel: 91-33-2461-4983. Fax: 91-22-2461-4849. E-mail: :
[email protected].
cardial infarction and ischemic stroke (10, 11), gingival diseases (12), and various types of cancer (13). Cigarette smoke contains a large number of bioactive compounds, which reside in the hydorphobic, hydrophilic (11), and votatile fractions (12, 14) of cigarette smoke extracts (CS1), which are responsible for its pathogenesis. The hydrophobic tar fraction consists of numerous mutagens such as benzo[a]pyrenes, while the hydrophilic fractions of cigarette smoke comprise nicotine, metals, and a large number of oxidants and free radicals (11). There are a list of over 60 carcinogens in cigarette smoke, which are considered as major cancer-causing agents and include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, aromatic amines, aldehydes, volatile organic compounds, metals, and others (15, 16).The volatile fraction of cigarette smoke contains acrolein and acetaldehyde, which causes the disruption of the human gingival fibroblast cytoskeleton (12) and inhibition of pulmonary proliferation (14). Various bioactive compounds present in the acqueous extract of cigarette smoke (AECS) are known to cause severe lung, heart, and gingival damage by inducing chronic inflammatory and oxidative stress responses resulting in cellular apoptosis (10–12, 14, 17). 1 Abbreviation: AECS, aqueous extract of cigarette smoke; CS, cigarette smoke; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DNP, dintrophenyl hydrazone; MAPs, microtubule associated proteins; Pipes, piperazinedinethane-suofonate; DTNB, 5,5′-dithiobis(2-nitro-benzoicacid); PI, propidium iodide.
10.1021/tx8002142 CCC: $40.75 2009 American Chemical Society Published on Web 02/18/2009
Cigarette Smoke Damages Microtubules
Since tubulin is one of the major structural proteins participating in the maintenance of cellular integrity, in the present study we have investigated the effect of AECS on tubulin and microtubules in ex ViVo and in Vitro conditions. The intracellular status of the microtubule in the absence and presence of different doses of AECS extract was examined with the human lung epithelium cells (A549) and noncarcinoma human lung alveolar epithelium (L132) cells. In the in Vitro studies, structural and functional properties of purified tubulin were assayed in the absence and presence of different doses of AECS. The change of major functional properties of tubulin, e.g., assembly of tubulin, in the presence of AECS was monitored by light scattering assay and electron microscopic studies. Structural changes such as aggregation pattern, oxidation, quenching of tryptophan fluorescence, and loss of secondary structure were studied by SDS-PAGE, oxyblot, fluorescence spectroscopy, and circulardichroismspectroscopy,respectively.Tubulin-microtubulemediated important cellular events such as proliferation, migration, and maintenance of cellular morphology in the presence of different doses of AECS at different time intervals were studied with A549 cells, and the extent of microtubule depolymerization, degradation, and inhibition of temperature-dependent assembly were also studied under similar conditions. The results are presented in this article.
Experimental Procedures Material. Nutrient mixture F12 Ham(supplemented with 1 mM FBS, penicillin-streptomycin, and amphotericin B were purchased from HyClone, USA. Human lung epithelium cells (A549) and human lung aleveoler epithelium noncarcinoma cells (L132) were obtained from NCCS, Pune, India. Trypsin-Versene (1×) was purchased form Cambrex Bioscience, USA, FITC conjugated monoclonal anti R-Tubulin antibody (raised in mouse), GTP, PIPES, MgCl2, EGTA, DTNB, and protease inhibitor cocktail (containing AEBSF, aprotinin, bestatin hydrochloride, E-64-[N(trans-epoxysuccinyl)-L-4-guanidinobutylamide], EDTA, and leupeptin hemisulphate) were purchased from Sigma, USA. Oxyblot protein oxidation detection kit was purchased from Intergen, USA. Antimouse HP conjugated secondary antibody was purchased from GeNei, India. All other chemicals and reagents are of analytical grade and were purchased from Sisco Research Laboratories, India. Cell Culture. Human lung epithelial (A549) cells were maintained in nutrient mixture F12 Ham supplemented with 1 mM L-glutamine,10% FBS, 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. The human lung alveolar epithelium cell line (L132) was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 50 µg/ mL penicillin, 50 µg/mL streptomycin, and 2.5 µg/mL amphotericin B at 37 °C in a humidified atmosphere containing 5% CO2. Cells were grown in tissue culture flasks until they were 80% confluent before trypsinisation with 1× Trypsin-Versene and splitting. Preparation of an Aqueous Extract of Cigarette Smoke (AECS) Solution. An aqueous extract of cigarette smoke was prepared as described in ref 18. A commercial filter tipped cigarette (74 mm), was lit, and puffs of AECS were introduced in 1 mL of 50 mM Pipes buffer, pH 7.0, or 50 mM PBS, pH 7.2, placed in a 250 mL glass Erlenmeyer flask with a side arm. The resultant dark yellow colored solution was filtered though a 0.22 µm filter and is termed whole phase AECS solution. After filtration, the wavelength spectrum of the smoke solution was routinely taken from 200 to 500 nm, and the λmax was obtained at 264 nm with the corresponding absorbance 0.4 ( 0.05 of 500-fold dilution of the stock. The AECS was lyophilized, and approximately 21 ( 3.6 mg of powder was obtained per mL AECS solution (after subtracting dry weight of buffer). For all experiments, freshly prepared whole phase AECS solution was used. L-glutamine),
Chem. Res. Toxicol., Vol. 22, No. 3, 2009 447 Cell Treatment and in Vitro AECS Doses. Cultured A549 and L132 cells were grown to confluency (1 × 106 cells/mL) and treated with different doses of AECS (0%-5%) by volume (17) and incubated for different time intervals, unless otherwise stated. In the case of in vitro studies, doses of AECS were kept in between 0%-50% by volume as described in ref 18. Cell Viability Assay. Cell viability was determined by MTT assay (19). Cells were seeded in 96-well plates at 1 × 104 cells per well, allowed to grow to 70%-80% confluency. Treated cells with the mentioned AECS doses were incubated with MTT for 4 h at 37 °C, the medium was removed, and dye crystal formazan were solubilized in 150 µL of dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm. Detection of Hypoploidy and Apoptosis by Flowcytometry. Human lung epithelial (A549) cells (1 × 106 cells/mL) were incubated in the absence and presence of different doses of AECS. Then cells were fixed using chilled methanol, and nuclear DNA was labeled with propidium iodide (PI). Cell cycle phase distribution of nuclear DNA was determined on an FACS calibur (Becton Dickinson) fluorescence detector equipped with a 488 nm argon laser light source and a 623 nm band-pass filter (linear scale) using CellQuest software (Becton Dickinson). Apoptosis was measured using AnnexinV-PI double staining. Cultured A549 (1 × 106 cells/mL) cells were incubated in the absence and presence of different doses (0-5%) of AECS and PI and AnnexinV flours were added directly to the medium and then analyzed on the flow cytometer using Cell Quest software. A total of 10000 events were acquired, and the cells were properly gated for analysis. Sample Preparation for Confocal Microscopy. Aqueous extracts of CS-treated A549 (1 × 106 cells/mL) cells and L132 (1 × 106 cells/mL) cells were cultured and fixed for immunofluorescence microscopy against R-tubulin as described in ref 20 and incubated with mouse monoclonal FITC conjugated anti-R-tubulin (1:50 dilution) overnight at 4 °C. After incubation, cells were washed twice with PBS. Microtubules were observed using a Ziess LSM 510 Meta confocal microscope. Assembly of Spindle Microtubules after Cold Treatment in AECS Treated A549 cells. Cultured A549 (1 × 106 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 different doses (0%, 3%, and 5%) of AECS and incubated at 37 °C. Cells were then fixed at different time points (0, 30, and 60 min) with 2% (v/v) paraformaldehyde at room temperature for 20 min. The fixed cells were then processed with mouse monoclonal FITC conjugated anti-R-tubulin and DAPI (1:1000 dilution of 1 mg/mL stock solution) to visualize the spindle microtubules and DNA, respectively. Microtubules were observed using a Ziess LSM 510 Meta confocal microscope. Western Blotting of AECS-Treated A549 Cells against Tubulin. Protein (50 µg) from each sample of A549 (1 × 106 cells/ mL) cells treated with mentioned AECS doses was loaded in 10% SDS-PAGE, and Western blotting was performed using a polyvinylidene difluoride membrane. The membrane was incubated with antimouse R-tubulin antibody (1:5000 dilution), while horseradish peroxidase (goat antimouse IgG) was used as the secondary antibody (1:10000 dilution). Flowcytometric Analysis of Detection Intracellular Tubulin Level in AECS-Treated A549 Cells. For the investigation of intracellular status of tubulin in the cells, AECS-treated cells (1 × 106 cells/mL) with the mentioned AECS doses from each group were incubated with mouse monoclonal FITC conjugated anti-Rtubulin antibody (1:800 dilution) for 4 h at room temperature. Subsequently, cells were washed thoroughly and analyzed on a flowcytometer equipped with a 488 nm argon laser light source and a 515 nm band-pass filter for FITC-fluorescence. Purification of Tubulin from Goat Brains. Tubulin was purified from goat brains by two cycles of temperature-dependent assembly and disassembly in PEM buffer (50 mM PIPES, 1 mM EGTA, and 0.5 mM MgCl2 at pH 6.9), in the presence of 1 mM GTP, followed by two more cycles in 1 M glutamate buffer (21). The
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protein concentration was estimated by the method of Bradford (22) using bovine serum albumin as the standard. Spectrophotometric Measurement of the Carbonyl Group in AECS-Treated Tubulin. Protein carbonyl was measured by reaction with 2,4-dinitrophenyl hydrazine (DNPH) as mentioned (23). Tubulin (1 mg/mL) was incubated in the absence and presence of different doses of AECS for 1 h at 37 °C in a final volume of 200 µL. The DNPH-reacted protein was then dissolved in 6 M guanidium chloride solution, and the carbonyl content was measured at 390 nm against a reagent blank of 2 M HCl. The results were expressed as nmol of phenylhydrazones formed per mg protein using a molar extinction coefficient of 22,000 M-1 cm-1 at 390 nm. Oxyblot Analysis of AECS-Treated Tubulin. Tubulin (1 mg/ mL) was incubated in the absence and presence of different doses of CS extract for 1 h at 37 °C, in a final volume of 200 µL assay mixture. Oxyblot of AECS-treated tubulin was performed as described in ref 23, and the intensity of the blots produced by autoradiography was quantified by densitometric scanning. Inhibition of Purified Tubulin Assembly in Vitro by AECS. Tubulin (1.2 mg/mL) was mixed with different doses of AECS extract in 300 µL (final volume) of polymerization buffer (1 mM MgSO4, 1 mM EGTA, 1 mM GTP, and 1.0 M monosodium glutamate, pH 6.8), and the assembly reaction was initiated by incubating the sample at 37 °C. The rate and extent of the polymerization reaction were monitored by light scattering at 350 nm. Transmission Electron Microscopy. Tubulin (1.2 mg/mL) was polymerized at 37 °C in the absence and presence of different doses of AECS, for 1 h in a 300 µL mixture. Microtubules were then fixed in 0.5% prewarmed glutaraldehyde for 5 min. Each sample (10 µL) was loaded in carbon-coated electron microscope grids (300-mesh) for 20 s and blotted dry. The grids were subsequently negatively stained with 1% uranyl acetate and air-dried. The samples were viewed using a Philips Fei Technai G212 electron microscope. Images were taken at 63000× magnifications. Measurement of Reactive Cysteines of AECS-Treated Tubulin by DTNB Kinetics. Formation of thio-nitrobenzoate anion (TNB) by DTNB reaction with the free sulfhydryl groups was measured by monitoring absorbance at 412 nm, and the number of reactive cysteines was calculated using ε412 )13,600 M-1 cm-1 for TNB (24). Tubulin (10 µM) in 50 mM PEM buffer, was incubated with different doses of AECS in 200 µL final volume, at 37 °C for 15 min. After incubation, tubulin was diluted 10-fold to make the final tubulin concentration 1 µM, and reactive cysteines were calculated using excess (400 µM) DTNB. Similarly, tubulin (10 µM) in 50 mM PEM buffer was incubated with 25% of AECS in 200 µL final volume at 37 °C. The first aliquot was taken after 1 min of incubation; after that, samples were taken at 15 min intervals. Silver Staining of AECS-Treated Tubulin. Tubulin (1 mg/mL) was incubated in the absence and presence of different doses of AECS for 1 h at 37 °C, in a final volume of 200 µL, and 5 µg of each sample was separated in 8% SDS-PAGE, and silver staining was done as described (25). Measurement of Quenching of Trytophan Fluorescence. Tubulin (10 µM) in 50 mM PEM buffer was incubated with different doses of AECS in 200 µL final volume, at 37 °C for 30 min. After incubation, the whole mixture was passed twice though a Sephadex G-10 gel filtration column equilibrated with PEM buffer to remove all unbound smoke components. Tubulin concentration in each set was measured by the Bradford method (22), and final concentration was kept at 1 µM in all the samples. Tryptophan fluorescence was measured by using a Hitachi fluorescence spectrophotometer model F-3010. Excitation was at 295 nm, and a wavelength scan was done in between 310-400 nm. Circular Dichroism Studies. Circular dichroism studies were done on a Jasco J600 spectropolarimeter to investigate the effect of AECS on the secondary structure of tubulin. Secondary structure was monitored in the 200-260 nm wavelength regions using a cell of path length 0.1 cm. Tubulin (10 µM) in 20 mM NaPi buffer was incubated with different doses of AECS in 200 µL final volume,
Das et al. at 37 °C for 30 min, and the unbound smoke components were removed as described previously (as in the tryptophan fluorescence measurement). Tubulin concentration in each set was measured by the Bradford method (22), and the tubulin concentration was kept at 1 µM in all cases. All measurements were done at 25 °C. Statistical Analysis. Quantitative data are presented as the mean ( SE of at least thee independent experiments. 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
