Department of Pediatrics and
1 Cancer Center, University of Minnesota, School of Medicine, Box 610, UMHC, 420 Delaware Street SE, Minneapolis, MN 55455, USA
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; B[a]PDE, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BSA, bovine serum albumin; CCSP, Clara cell secretory protein; DMSO, dimethyl sulfoxide; MAPK, mitogen activated protein kinase; PI-3K, phosphatidylinositol-3 kinase; RT, room temperature; SAE, small airway epithelial; SAPK, stress activated protein kinase.
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Introduction |
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B[a]P-induced lung tumorigenesis in A/J mice is prevented by various naturally occurring agents that can detoxify B[a]PDE or block DNA adducts in the initiation phase (4). However, only a handful of agents including myo-inositol prevent or attenuate B[a]P-induced lung tumorigenesis in the post-initiation phase (46). Given the slow and multi-step process of lung tumorigenesis and addictive nature of smoking, any non-toxic substances that can inhibit development of lung cancer in the post-initiation phase deserve consideration as potential chemopreventive agents. Such agents may decrease lung tumorigenesis in high risk populations including current and ex-smokers and those with extensive environmental exposure.
Myo-inositol and inositol phosphates (IP) are present abundantly in mammalian cells (7,8). Lower inositol phosphates such as IP-3 function as intracellular messengers and prevent carcinogen-induced tumorigenesis in various in vivo and in vitro models (9). In addition to its chemopreventive action in the post-initiation phase (5,6), myo-inositol is present abundantly in regular dietary components (7,8), absorbed extremely well from the GI tract (7), and appears to be safe at high doses (10,11). Therefore, myo-inositol is a good candidate as a chemopreventive agent for lung cancer. However, it is pivotal to understand mechanisms of B[a]PDE carcinogenesis and myo-inositol chemopreventive activity in normal and cancerous human lung cells. Unfortunately, such information has been scarce partly due to the difficulty in culturing untransformed human lung cells.
Recently, small airway epithelial (SAE) cells obtained from healthy adult volunteers by bronchoalveolar lavage (BAL) became available. These untransformed SAE cells retain the features of basal cells in serum-free medium and can be maintained for a few passages (12) but differentiate into non-ciliated epithelial cells in serum-supplemented cultures. Thus these cells can serve as an in vitro model of normal human airway epithelium for evaluating the effects of B[a]PDE and myo-inositol. In this study, we evaluated the effects of B[a]PDE and myo-inositol on SAE and A549 human lung cancer cells. The hypothesis being tested is that physiologically achievable doses of B[a]PDE alter SAE cell growth and/or differentiation and that myo-inositol can reverse B[a]PDE effects in the post-initiation phase as observed in A/J mice (46). We also addressed mechanisms of B[a]PDE and myo-inositol actions by using several inhibitors of signal transduction pathways.
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Materials and methods |
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Effects of B[a]PDE on SAE cell differentiation
These experiments were designed to determine whether non-toxic doses of B[a]PDE affect serum and Ca2+-induced SAE cell differentiation. Near-confluent SAE cells were treated for 1 h with a non-toxic dose of B[a]PDE (1 nM) or solvent (DMSO; 0.5%), the medium was changed to serum-supplemented or high Ca (1 mM) serum-free medium, and then the cells were kept in culture for 4 days. Secondary to slow growth of SAE cells in serum-supplemented culture, no further change of medium was required. At the end of culture, changes in cell number, cytology (cell differentiation) and CCSP expression (14) were assessed. We also determined whether myo-inositol altered the effects of B[a]PDE on serum or Ca2+-induced SAE cell differentiation. In these experiments myo-inositol (1100 µM) was added into the medium, when the medium was changed immediately after B[a]PDE treatment. After 4 days of culture, the same parameters stated above were measured.
Mode of action of B[a]PDE and myo-inositol
These experiments were designed to determine how B[a]PDE inhibited serum-induced SAE cell differentiation and how myo-inositol prevented B[a]PDE action in the post-initiation phase. We tested the effects of inhibitors of phosphatidylinositol-3 kinase (PI-3K), protein kinase C (PKC), mitogen activated protein kinase (MAPK) kinase 1 (MEK1) and p38 MAPK on the actions of B[a]PDE and myo-inositol (1520). These inhibitors were added to confluent SAE cell cultures 1 h before B[a]PDE (1 nM) treatment. After the B[a]PDE treatment, medium was changed to serum-supplemented medium and the cells were cultured for 4 days with or without myo-inositol (10100 µM). In other experiments, these inhibitors were added to the serum-supplemented medium after B[a]PDE treatment with or without myo-inositol (10100 µM). The same parameters studied in the previous experiment were measured.
Reagents
B[a]PDE was obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository, dissolved in DMSO, aliquoted, and kept at 20°C until the time of the experiments. Right before the experiment, B[a]PDE was further diluted with DMSO and the final concentration of DMSO in the SAE cell cultures was adjusted as 0.5%. Wortmannin (a PI-3K inhibitor) was obtained from Sigma (St Louis, MO). Melittin [Myristoylated EFG-R fragment (651658); a PKC inhibitor] (18), PD98059 (a MEK-1 inhibitor) (19), and SB202190 (a p38 MAPK inhibitor) (20) were obtained from Calbiochem (Cambridge, MA). These inhibitors were dissolved in DMSO at a concentration of 12 mM, aliquoted, and stored at 70°C. Further dilution was carried out with DMSO on the day of the experiment. Control cultures contained the same final concentration of DMSO (0.050.1%). The DMSO used for dissolving B[a]PDE and the inhibitors did not affect SAE cell differentiation, death and proliferation at concentrations used in our culture system.
Analytical methods
SAE and A549 cell culture. SAE cells were maintained in serum-free SAE basal medium (CCMD 160; Clonetics) supplemented with growth factors as reported before (12). The media was changed every 2 days, subcultivated when confluent (once per week), and all the experiments used cells at less than five passages. Adherent SAE cells were passaged by detaching cells with trypsin (0.25 g/l) and ethylenediamine tetra-acetic acid (EDTA; 0.1 mg/ml) in HBSS (Clonetics) as reported before (11). Serum-supplemented medium used for SAE cultures was RPMI 1640 supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT), penicillin (105 U/l), streptomycin (100 mg/l) and glutamine (2 mM). A549 cells were cultured in F12K medium (Life Technologies, Gaithersburg, MD) supplemented with the same additives used in the SAE cell serum-supplemented medium. A549 cells were passaged twice per week by detaching cells with trypsin (2.5 g/l; Gibco BRL, Gaithersburg, MD) (12). Representative plates were examined with a phase contrast microscope (Nikon, Melville, NY) after the trypsin treatment and contained virtually no residual adherent SAE or A549 cells after trypsin treatment.
Cell number. The numbers of dead and living cells were determined by trypan blue dye exclusion using cells resuspended in phosphate-buffered saline (PBS), pH 7.4. The numbers of non-adherent and adherent cells were determined separately. Cells were counted in a hemacytometer.
Assays for necrosis and apoptotic cell death
Necrotic and apoptotic cell death was assessed by measuring (i) LDH release into the culture media and (ii) morphological changes detected by AO staining (21). The results of AO staining were confirmed by TUNEL stain in selected samples (22).
LDH levels. Culture supernatants were harvested and frozen at 20°C until the time of measurement. LDH levels in the culture supernatants were measured using a commercial LDH kit (EC1.1.1.27 UV-test; Sigma) as reported before (12).
Nuclear and cytoplasmic morphology by acridine orange staining. Cells were cultured in 24-well tissue-culture plates, washed once with PBS and fixed in 70% ethanol for 10 min on ice. Then the plate was air dried for 1015 min, stained with AO (6 µg/ml; Sigma) in a 2:1 ratio mixture of distilled water and PBS for 34 h at room temperature (RT). The staining solution was decanted and the plate was rinsed twice in a 2:1 mixture of distilled water and PBS and examined using a phase-contrast fluorescence microscope (Nikon) (12). Apoptotic cells were identified by their yellow fragmented and condensed nuclei as well as by their condensed red cytoplasm (19). The percentage of cells with apoptotic nuclei was calculated based upon counting 500600 cells per well in duplicate wells in each experiments.
TUNEL stain. To confirm the presence of DNA nicking, TUNEL staining was performed using a commercially available kit (In Situ Cell Death Detection kit, Fluorescein and TUNEL AP; Boehringer Mannheim, Indianapolis, IN) (22). Adherent cells in a 24-well tissue-culture plate were washed twice with PBS with 1% bovine serum albumin (BSA, Sigma), and fixed in paraformaldehyde 40 g/l in PBS, pH 7.4, for 30 min at RT. The plate was washed twice with PBS, placed in a permeabilizing solution of Na citrate (1 g/l) with Triton X-100 (0.1%) for 2 min on ice, washed with PBS again, and stained with the TUNEL reaction mixture for 60 min at 37°C. The plate then was washed with PBS, treated with anti-Fluorescein antibody (Ab) (Fab fragment) conjugated with alkaline phosphatase (AP) for 30 min at 37°C, washed with PBS, and treated with substrate for AP for 10 min at RT. The plates were examined under a light microscope.
Assessment of cell differentiation
Morphological changes. These were assessed directly by Giemsa staining of cells adherent to the culture plate (Diff Quick; Baxter, MacGaw Park, IL). The frequency of basal cells, epithelial cells and mitotic cells were determined under a light microscope by one person in a blind manner.
Expression of CCSP. CCSP expression by SAE cells was assessed by staining them with rabbit anti-human CCSP Ab (Dako, Carpinteria, CA). Fluorescein-conjugated anti-rabbit IgG Ab was used as a second Ab (Harlan, Indianapolis, IN). Confluent SAE cells were washed once with RT PBS and fixed with ice-cold methanol for 15 min. After being air-dried, polyclonal rabbit anti-human CCSP Ab adjusted to 1 µg/well in PBS with 1% BSA was added to each well and incubated for 12 h at 37°C in a humidity chamber. Pre-immune rabbit serum was used as a negative control. Then each well was washed with PBS. A FITC conjugated anti-rabbit IgG (1 µg/well in PBS with 1% BSA) was added and the mixture was incubated at 37°C for 12 h in a humidity chamber. After washing with PBS, mounting medium (10% glycerol in PBS, pH 88.5, 1 ml/well) was added to each well. Cells positively stained were counted under a fluorescent microscope and expressed as a percentage of the total number of cells counted. An average of 300400 cells were counted in each well.
Statistics
For comparison of test values with control values, the paired t-test or Wilcoxon signed ranks test was used. For comparison of effects of myo-inositol, the Friedman test was employed (23). A value of P < 0.05 was considered to be significant. At least five replicate experiments were performed with duplicate or triplicate samples for each parameter in each experiment.
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Results |
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Discussion |
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B[a]PDE, a carcinogenic metabolite of B[a]P, forms guanine adducts in DNA (2,3). However, little is known about the effects of B[a]PDE on normal human lung cells. This partly was due to the difficulty of culturing primary epithelial cells. B[a]PDE preferentially forms DNA adducts in mutation hotspots of the P53 tumor suppresser gene in HeLa and untransformed human bronchial epithelial cells (3). This may be one potential mechanism of B[a]PDE-induced tumorigenesis. However, the concentration of B[a]PDE used (4 µM) may not be achieved in vivo and it is unclear whether B[a]PDE at lower concentrations selectively reacts with these spots: the methodology used requires high DNA adduct levels for detection. The clinical relevance of this finding remains to be seen.
SAE cells maintain the features of basal cells in serum-free medium for up to 45 passages (12). Cellular homeostasis of untransformed SAE cells was tightly regulated by a balance of low-level cell proliferation and apoptotic cell death (12). In contrast, A549 human lung adenocarcinoma cells proliferate rapidly with many cells undergoing necrotic, but not apoptotic, cell death when cultured for >3 days (12). SAE cells were more sensitive to injurious effects of hyperoxia than A549 cells (12). These results indicate the importance of employing primary or untransformed cells in addition to cell lines for studying the effects of carcinogens in certain circumstances.
This study first addressed the toxic effects of B[a]PDE on SAE cells in comparison with A549 lung cancer cells. The SAE cells were far more sensitive to the toxic effects of B[a]PDE and underwent apoptotic cell death at B[a]PDE concentrations >10 nM, whereas A549 cells were resistant to B[a]PDE toxic effects even at a B[a]PDE concentration of 10 µM. A549 cells underwent little apoptosis with B[a]PDE, as was also true in hyperoxia (12). Since lung epithelium likely is exposed to various carcinogens and high oxygen pressure, our current and previous results (12) may reflect the survival advantage of transformed lung cancer cells. However, since <1% of B[a]P is metabolized to B[a]PDE in vivo (2), B[a]PDE concentrations are unlikely to reach >10 nmol/l in the lung, even in heavy smokers. The toxic effects of B[a]PDE observed here are unlikely to occur in vivo except in acute toxic exposures.
The next step of our study, thus, was focused on determining B[a]PDE effects on SAE cells at non-toxic, physiologically attainable concentrations in vivo (0.11 nM). At these concentrations, B[a]PDE had no effect on cell proliferation, death and differentiation of SAE cells cultured in the serum-free, low Ca2+ (4 nM) medium. SAE cells differentiated into Clara cell-like non-ciliated epithelial cells in serum-supplemented or serum-free, high Ca2+ (1 mM) conditions. Since an initial step of transformation is immortalization or dedifferentiation of cells, we hypothesized that B[a]PDE could promote tumorigenesis by altering or diminishing serum or Ca2+-induced SAE cell differentiation. Indeed, B[a]PDE (1 nM) partially inhibited serum or Ca2+-induced SAE cell differentiation, as indicated by an increase in basal cells and decreases in epithelial cells and CCSP expression, but B[a]PDE did not augment SAE cell proliferation. It is possible that B[a]PDE may initiate lung tumorigenesis by inhibiting differentiation of airway epithelial cells.
MAPKs are a group of serine/threonine-specific, proline-directed protein kinases that are activated by a wide spectrum of stimuli (17). Three distinct MAPK transduction pathways have been identified in mammalian cells that activate extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-jun NH2-terminal kinase (JNK)/stress activated protein kinase (SAPK) and p38 MAPK (17,26). ERK1/ERK2 are often activated by growth factors in sequential activation of PI-3K or Ca2+ calmodulin, Ras, Raf-1, MEK1/2 and ERK1/ERK2, in this order (17,27). p38 MAPK is activated by cytokines and the cellular stress that may be associated with stress-induced apoptosis (26,28).
In A/J mice, susceptibility to B[a]P-induced lung tumorigenesis is associated with polymorphism of the K-ras proto-oncogene (29,30). Thus we speculated that activation of a Ras-mediated signal transduction pathway may be associated with the B[a]PDE effect on SAE cells. This study examined the effects of inhibitors of PI-3K, MEK-1, p38 MAPK and PKC on the inhibitory effect of B[a]PDE on SAE cell differentiation; PI-3K activates Ras, which subsequently activates downstream kinases, MEK1 and ERK1/ERK2. The effects of a PKC inhibitor also were tested, since inhibition of PKC prevented asbestos-induced c-fos and c-jun proto-oncogene expression in lung mesothelial cells (31). Our results revealed that inhibitors of PI-3K and MEK-1, but not those of PKC or p38 MAPK, abolished the inhibitory action of B[a]PDE on SAE cell differentiation. Thus, B[a]PDE may inhibit serum or Ca2+-induced SAE cell differentiation partly through activating a IP-3K/MEK-1 mediated MAPK pathway.
Myo-inositol, a common dietary component, prevented B[a]P-induced lung tumorigenesis in A/J mice when given in the post-initiation phase (5,6). Given its safety and excellent intestinal absorption (7,8,10,11), myo-inositol can be an attractive agent for lung cancer chemoprevention. Phosphorylated products of myo-inositol have prevented development of carcinogen-induced colon cancers in rodents (9,24,25). Proposed mechanisms of IP chemoprevention include the inhibition of PI-3K pathway with resultant reduced AP-1 expression and cell proliferation; intracellular Ca2+ mobilization; induction of cell differentiation and apoptosis; and increase in natural killer cell activity (9,16,24,25). IP chelates several key minerals including Fe3+, Mg2+, Zn2+ and Ca2+, which are essential for cell proliferation (9). Thus part of the anti-tumor activity of IP observed in vitro using cancer cell lines might be associated with deprivation of essential minerals from tumor cells. In contrast, myo-inositol was effective only in vivo in preventing carcinogen-induced tumorigenesis (5,6,9,24). Myo-inositol may thus exert its actions in the early post-initiation phase, which can be detected in normal untransformed SAE cells but not in transformed A549 lung cancer cells.
Myo-inositol did not alter cellular homeostasis of SAE cells in serum-free or serum-supplemented cultures or affect growth of A549 cells. However, myo-inositol prevented the inhibitory effects of B[a]PDE on SAE cell differentiation in a dose-dependent manner when added to the culture after B[a]PDE treatment. This is the first report that myo-inositol altered the potentially transforming effects of B[a]PDE on normal human lung cells in vitro. B[a]PDE forms non-carcinogenic tetraol metabolites in culture within 8 min (13), so that it is unlikely that myo-inositol directly prevented the genotoxic effects of B[a]PDE. This myo-inositol effect could be involved in its in vivo chemopreventive activity in the post-initiation phase. It will be interesting to study comparative parameters in vivo, employing rodent models of B[a]P-induced lung tumors or human lung tissues.
PD98059, an inhibitor of MEK-1, but not a wortmannin (a PI-3K inhibitor) exerted a similar effect as myo-inositol, whereas a p38 MAPK inhibitor partially prevented the actions of myo-inositol. We thus suspect that myo-inositol and/or its phosphorylated products may prevent the effects of B[a]PDE partly through inhibiting the signal transduction pathway mediated by PI-3K and/or MEK-1, which is consistent with a previous report (16), and our results that B[a]PDE inhibit SAE cell differentiation by activating PI-3K/MEK-1 mediated MAPK signal transduction pathway. p38 MAPK was implicated with stress-induced apoptotic signals induced by UV light or other inducers (17,26,28). Thus the blockage of this MAPK pathway may increase survival of B[a]PDE-induced pre-cancerous cells, hence the apparent reversal of myo-inositol protective action of SAE cells against B[a]PDE. Taken together, our results generate a testable hypothesis regarding myo-inositol action that should be further investigated in this model, as well as in vivo rodent models.
In summary, we report for the first time the in vitro effects of B[a]PDE on differentiation of untransformed normal human lung cells at concentrations attainable in vivo; this action may be exerted through a PI-3K/MEK-1 mediated MAPK pathway. Moreover, we also show for the first time that myo-inositol can prevent B[a]PDE's effects on normal human lung cells in vitro.
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Notes |
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Acknowledgments |
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References |
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