Effects of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on human small airway epithelial cells and the protective effects of myo-inositol

Harumi Jyonouchi2, Sining Sun, Koji Iijima, Mingyao Wang1 and Stephen S. Hecht1

Department of Pediatrics and
1 Cancer Center, University of Minnesota, School of Medicine, Box 610, UMHC, 420 Delaware Street SE, Minneapolis, MN 55455, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Benzo[a]pyrene (B[a]P), a tobacco-derived carcinogen, induces lung tumors in rodents through its carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE). Tumorigenesis is inhibited by dietary myo-inositol in the post-initiation phase. However, little is known about how B[a]PDE and myo-inositol affect normal human lung cells. We addressed this question using untransformed human small airway epithelial (SAE) cells. SAE cell viability decreased <50% in parallel to an increase of apoptotic cells (>20%) 2 days after the cells were treated for 1 h with B[a]PDE (>100 nM). In contrast, the cell number and viability were not altered in A549 human lung cancer cells by B[a]PDE treatment up to 10 µM with <5% apoptotic cells and <10 U/l LDH in the medium. SAE cells retain the features of basal cells in serum-free, low Ca2+ (4 nM) medium up to 4–5 passages, but in serum-supplemented or serum-free, high Ca2+(1 mM) cultures, they differentiate into non-ciliated epithelial cells expressing Clara cell secretory protein (CCSP). A non-toxic, physiologically relevant dose of B[a]PDE (1 nM) partially inhibited serum and Ca2+-induced SAE cell differentiation. This effect was abolished by wortmannin, a phosphatidylinositol-3 kinase (PI-3K) inhibitor, and PD98059, a mitogen activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor, but not by SB202190, a p38 MAPK inhibitor, or melittin, a protein kinase C inhibitor. Myo-inositol (10–100 µM) did not alter growth or differentiation of untreated SAE or A549 cells, but reversed the inhibitory effect of B[a]PDE on serum and Ca2+-induced SAE cell differentiation when supplemented to the culture after B[a]PDE treatment. This myo-inositol action was not altered by PD98059, wortmannin or melittin, but was partially suppressed by SB202190. Collectively, these results indicate that B[a]PDE inhibits serum-induced SAE cell differentiation, possibly involving activating signals through a PI-3K/MEK1 mediated MAPK pathway, whereas myo-inositol protects SAE cells against this inhibitory effect of B[a]PDE perhaps through both PI-3K/MEK1 and p38 MAPK pathways.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Numerous epidemiological studies indicate a major role of smoking as a cause of lung cancer (1). Tobacco smoke contains multiple carcinogens including benzo[a]pyrene (B[a]P) (1,2). B[a]P is metabolized into anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), as a major ultimate carcinogen of B[a]P (2). B[a]PDE reacts with DNA, forming guanine adducts identical to those produced by B[a]P in mammalian systems (2). B[a]PDE at a concentration of 4 µM and also other carcinogens form DNA adducts preferentially in mutational hotspots in the P53 tumor suppresser gene in HeLa and bronchial epithelial cells (3). However, this B[a]PDE concentration may not be achievable in vivo even in heavy smokers. Moreover, there is no evidence that B[a]PDE selectively reacts with these spots at lower concentrations, since the experimental technique used in the study (3) requires high adduct levels for detection. Currently, mechanisms of carcinogenesis by B[a]PDE are incompletely understood.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
B[a]PDE toxicity
The first set of experiments was designed to determine the toxic effects of B[a]PDE on untransformed SAE human lung cells (Clonetics, San Diego, CA) and A549 human lung adenocarcinoma cells (ATCC, Rockville, MD). SAE and A549 cells were plated at densities of 3x103 and 2x104 cells/ml, respectively, in 24-well tissue culture plates (Costar, Cambridge, MA) and cultured until they became near-confluent (covered >85% of the well). Near-confluence was generally achieved after 1 or 4–5 days of culture in A549 and SAE cells, respectively (12). Then cells were treated for 1 h with B[a]PDE (0.1–10 000 nM) or solvent only [dimethyl sulfoxide (DMSO); 0.5%], the medium was changed, and the cells were cultured for 2–3 days. B[a]PDE forms non-carcinogenic tetraol metabolites in the presence of H2O within 8 min (13). We evaluated SAE and A549 cell growth and differentiation by measuring changes in cell number, cell viability, LDH release (a marker for necrotic changes), apoptotic changes [acridine orange (AO) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain] and cell cytology (Giemsa stain). We chose these parameters, since hyperoxia induced significant changes in these parameters that also differed remarkably between SAE and A549 cells (12).

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 (1–100 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 (10–100 µM). In other experiments, these inhibitors were added to the serum-supplemented medium after B[a]PDE treatment with or without myo-inositol (10–100 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 (651–658); 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 1–2 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 (0.05–0.1%). 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 10–15 min, stained with AO (6 µg/ml; Sigma) in a 2:1 ratio mixture of distilled water and PBS for 3–4 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 500–600 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 1–2 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 1–2 h in a humidity chamber. After washing with PBS, mounting medium (10% glycerol in PBS, pH 8–8.5, 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 300–400 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Toxic effects of B[a]PDE
B[a]PDE did not alter the total number of adherent SAE or A549 cells up to a concentration of 10 µM (Figure 1AGo). However, SAE cell viability declined sharply after treatment of B[a]PDE at >100 nM, paralleled by a sharp increase in apoptotic cells (Figure 1B and CGo). In contrast to SAE cells, B[a]PDE did not affect A549 cell viability significantly even at a concentration of 10 µM with <5% apoptosis (Figure 1CGo). LDH levels in the medium were <10 U/l in both SAE and A549 cell cultures and were not altered by B[a]PDE treatment. The number of non-adherent cells was low (<104 cells/well in SAE cells and <5% of adherent cells in A549 cells) in this culture condition and most of them were not viable, irrespective of B[a]PDE treatment. Thus untransformed SAE cells appear more sensitive to B[a]PDE toxicity than A549 lung cancer cells.




View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1. Changes in (A) the adherent cell number, (B) cell viability and (C) apoptotic changes detected by AO stain in SAE and A549 cells 2 days after treatment with various doses of B[a]PDE. Each datum point represents the mean ± SD, n = 5. *Significantly higher or lower than controls, P < 0.05 at B[a]PDE 100 nmol/l and P < 0.01 at B[a]PDE >100 nmol/l, Wilcoxon signed ranks test.

 
The effects of non-toxic doses of B[a]PDE on serum- or Ca-induced SAE cell differentiation
SAE cells cultured in serum-supplemented medium for 3–4 days differentiated into epithelial cells with <10% basal cells (Figure 2AGo). This morphological change was paralleled by increase in CCSP expression and decrease in cell numbers (Figure 2AGo). SAE cells cultured in serum-supplemented medium did not survive the first subcultivation, most likely due to terminal differentiation of basal cells into epithelial cells. Non-toxic doses of B[a]PDE (0.1–1 nM) partially inhibited the serum-induced SAE cell differentiation as evidenced by less CCSP expression and more basal cells in B[a]PDE-treated SAE cells (Figure 2BGo). B[a]PDE did not alter the SAE cell number (Figure 2BGo). Similar, but less marked morphological changes and increase in CCSP expression were observed in SAE cells cultured in serum-free, high Ca2+ (1 mM) medium. Namely, the percentages of epithelial cells and CCSP expression were 35.0 ± 10.2 and 15.8 ± 6.8 %, respectively, in this culture condition, whereas they were 83.0 ± 7.2 and 24.2 ± 7.0%, respectively, in serum-supplemented cultures (Figure 3Go). This Ca2+ concentration is approximately equivalent to that of the serum-supplemented medium. B[a]PDE (1 nM) also inhibited Ca2+-induced SAE cell differentiation (Figure 3BGo).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. Effects of (A) serum and (B) serum plus B[a]PDE treatment on SAE cell differentiation. The percentages of basal cells, epithelial cells, and CCSP expression and the number of adherent cells recovered were determined in serum-free and serum-supplemented cultures without treatment, or in serum-supplemented cultures after B[a]PDE or DMSO (solvent; control) treatment. Each datum point represents the mean ± SD, n = 5. *Significantly higher in serum-supplemented cultures, P < 0.05. **Significantly higher in serum-free medium or with B[a]PDE treatment, P < 0.01.

 


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Effects of B[a]PDE and myo-inositol on SAE cell differentiation when SAE cells were cultured in (A) low Ca2+ (4 nmol/l) or (B) high Ca2+ (1 mmol/l) medium. Cells were cultured with medium only, DMSO (0.5%), B[a]PDE (1 nmol/l, 1 h treatment), or supplemental myo-inositol (MI; 10 µmol/l) following B[a]PDE treatment. The percentages of basal cells, epithelial cells, and CCSP expression were determined. Each datum point represents the mean ± SD, n = 5. *Significantly lower than control values (P < 0.05).

 
Preventive effects of myo-inositol on the inhibitory B[a]PDE action on serum-induced SAE cell differentiation
Myo-inositol added to the confluent SAE cell cultures did not alter the number of SAE cells, morphological changes or CCSP expression in either serum-free or serum-supplemented conditions (Figure 4AGo and data not shown). In contrast, myo-inositol (10–100 µM) added to serum-supplemented culture medium after B[a]PDE treatment prevented the inhibitory effects of B[a]PDE (1 nM) on Ca2+- or serum-induced SAE differentiation (Figures 3B and 4BGoGo). Myo-inositol did not alter cell number, viability, cell morphology or DNA synthesis of A549 human lung cancer cells (data not shown), which is consistent with previous reports (24,25). Thus myo-inositol protects untransformed SAE cells against B[a]PDE inhibitory action on serum or Ca2+-induced SAE differentiation in the post-initiation phase, but has no effect on A549 lung cancer cells.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. The effects of myo-inositol (MI) added to the medium on differentiation of SAE cells after treatment with (A) DMSO (0.5% control solvent) or (B) B[a]PDE (1 nmol/l). The percentages of basal cells, epithelial cells and CCSP expression were determined. Each datum point represents the mean ± SD, n = 5. *Significantly higher than control values, P < 0.005. **Significantly higher than control values, P < 0.01.

 
The effects of signal transduction inhibitors on the inhibitory action of B[a]PDE on SAE cell differentiation
When confluent SAE cells were treated with wortmannin (10 nM) or PD98059 (100 nM) for 1 h prior to B[a]PDE treatment, the inhibitory effect of B[a]PDE on serum-induced SAE cell differentiation was abolished (Figure 5AGo). The B[a]PDE action was not altered by either SB202196 or melittin (100 nM) (Figure 5AGo and data not shown). When SAE cells were incubated with these inhibitors prior to the serum-supplemented cultures without B[a]PDE treatment, they did not alter serum-induced SAE cell differentiation. They did not alter the preventive effects of myo-inositol on B[a]PDE action either (Figure 5Go and data not shown).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Effects of PI-3K inhibitor (wortmannin), MEK1 inhibitor (PD98059) and MAPK inhibitor (SB202190) on the inhibitory effects of B[a]PDE on serum-induced SAE cell differentiation (A). Their effects were also tested when myo-inositol (MI) was supplemented to the medium after B[a]PDE treatment (B). The percentages of basal cells, epithelial cells and CCSP expression were determined. Each datum point represents the mean ± SD, n = 6. *Significantly higher than controls, P < 0.05, Wilcoxon signed ranks test. **Significantly lower than controls, P < 0.05, Wilcoxon signed ranks test. These values were significantly different when cells were treated with PB98059 and wortmannin prior to B[a]PDE treatment (P < 0.01 by Friedman test).

 
The effects of signal transduction inhibitors on the myo-inositol preventive actions on B[a]PDE-induced changes in SAE cell serum-supplemented cultures: to test the effects of these inhibitors on SAE cells in the post-initiation phase, we examined morphological changes and CCSP expression of SAE cells when these inhibitors were added to medium after B[a]PDE treatment. In the absence of myo-inositol, PD98059 (100 nM) partially reversed B[a]PDE inhibitory action on serum-induced SAE cell differentiation, whereas wortmannin (10–100 nM), SB202190 (100 nM) or melittin (100 nM) had no effect (Figure 6AGo and data not shown). When these inhibitors were added to culture in addition to myo-inositol (100 µM), SB202190 partially abolished the preventive effects of myo-inositol on B[a]PDE inhibitory action on SAE cell differentiation (Figure 6BGo). Neither wortmannin, PD98059 nor melittin altered the effects of myo-inositol (Figure 6BGo).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6. Effects of inhibitors of MEK (PD98059), PI-3K (wortmannin), p38 MAPK (SB202190) and PKC (melittin) on the inhibitory effects of B[a]PDE on SAE cell differentiation when added to the culture after B[a]PDE treatment without myo-inositol (A) or with myo-inositol (MI; 100 µmol/l) (B). Each datum point represents the mean ± SD, n = 5. *Significantly lower than other values (P < 0.05, Friedman test). **Significantly higher than other values (P < 0.01 Friedman test).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was designed to determine the effects of B[a]PDE and myo-inositol on untransformed human lung cells (SAE cells). Our results indicate that B[a]PDE inhibits serum-induced SAE cell differentiation from basal cells to epithelial cells. B[a]PDE may exert this action partly by activating a PI-3K/MEK1 mediated MAPK signal transduction pathway. This occurs at a B[a]PDE concentration of 1 nmol/l, a level attainable in vivo, and myo-inositol reverses this B[a]PDE inhibitory effect on SAE cell differentiation in the post-initiation phase.

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 4–5 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 (0.1–1 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.


    Notes
 
2 To whom correspondence should be addressed Email: jyono001{at}jyono001.email.umn.edu Back


    Acknowledgments
 
The authors thank Drs D.H.Ingbar and E.Wattenberg for critically reviewing this manuscript. This study was supported in part by grant no. CA-46535 from the National Cancer Institute.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. International Agency for Research on Cancer (1986) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, vol. 38, Tobacco Smoking. IARC Scientific Publishers, Lyon.
  2. Conney,A.H., Chang,R.L., Jerina,D.M. and Wei,S.J.C. (1994) Studies on the metabolism of benzo[a]pyrene and dose-dependent differences in the mutagenic profile of its ultimate carcinogenic metabolite. Drug Metab. Rev., 26, 125–163.[ISI][Medline]
  3. Denissenko,M.F., Pao,A., Tang,M. and Pfeifer,G.P. (1996) Preferential formation on benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science, 274, 430–432.[Abstract/Free Full Text]
  4. Stoner,G.D., Morse,M.A. and Kelloff,G.J. (1977) Perspective in cancer chemoprevention. Environ. Health Perspect., 105, 945S–954S.
  5. Wattenberg,L.W. and Estensen,R.D. (1996) Chemopreventive effects of myo-inositol and dexamethasone on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary carcinogenesis in female A/J mice. Cancer Res., 56, 5132–5135.[Abstract]
  6. Wattenberg,L.W. and Estensen,R.D. (1997) Studies of chemopreventive effects of budenoside on benzo[a]pyrene-induced neoplasia of the lung of female A/J mice. Carcinogenesis, 18, 2015–2017.[Abstract]
  7. Clements,R.S. and Darnell,B. (1980) Myo-inositol content of common foods: development of a high-myo-inositol diet. Am. J. Clin. Nutr., 33, 1954–1967.[Abstract]
  8. Clements,R.S. and Reynertson,R. (1977) Myo-inositol metabolism in diabetes mellitus: effects of insulin treatment. Diabetes, 26, 215–221.[Abstract]
  9. Shamsuddin,A.M. (1995) Inositol phosphates have novel anticancer function. J. Nutr., 125, 725S–732S.[Medline]
  10. Hallman,M. (1984) Effect of extracellular myo-inositol on surfactant phospholipid synthesis in the fetal rabbit lung. Biochim. Biophys. Acta, 795, 67–78.[ISI][Medline]
  11. Hallman,M., Bry,K., Hoppu,K., Lappi,M. and Pohjavuori,M. (1992) Inositol supplementation in premature infants with respiratory distress syndrome. N. Engl. J. Med., 326, 1233–1239.[Abstract]
  12. Jyonouchi,H., Sun,S., Chareancholvanich,S. and Ingbar,D.H. (1998) The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells. Am. J. Resp. Cell. Mol. Biol., 19, 426–436.[Abstract/Free Full Text]
  13. Yagi,H., Thakker,D.R., Hernandez,O., Koreeda,M. and Jerina,D.M. (1977) Synthesis and reactions of highly mutagenic 7,8-diol-9,10-epoxides of the carcinogen benzo[a]pyrene. J. Am. Chem. Soc., 99, 1604–1611.[ISI][Medline]
  14. Hackett,B.P. and Gitlin,J.D. (1992) Cell-specific expression of a Clara cell secretory protein-human growth hormone gene in the bronchiolar epithelium of transgenic mice. Proc. Natl Acad. Sci. USA, 89, 9079–9083.[Abstract/Free Full Text]
  15. Russo,J., Calaf,G. and Russo,I.H. (1993) A critical approach to the malignant transformation of human breast epithelial cells with chemical carcinogens. Crit. Rev. Oncogene, 4, 403–417.[ISI][Medline]
  16. Huang,C., Ma,W.Y., Hecht,S.S. and Dong,Z. (1997) Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-3' kinase. Cancer Res., 57, 2873–2878.[Abstract]
  17. Whitmarsh,A.J. and Davis,R.J. (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med., 74, 589–607.[ISI][Medline]
  18. Ward,N.E. and O'Brian,C.A. (1993) Inhibition of protein kinase C by N-myristoylated peptide substrate analogs. Biochemistry, 32, 11903–11909.[ISI][Medline]
  19. Dudley,D.T., Pang,L., Decker,S.J., Bridges,A.J. and Saltiel,A.R. (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl Acad. Sci. USA, 92, 7686–7689.[Abstract/Free Full Text]
  20. Gallagher,T.F., Seibel,G.L., Kassis,S. et al. (1997) Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSB[a]p kinase. Bioorg. Med. Chem., 5, 49–64.[ISI][Medline]
  21. Wendt,C.H., Polunovsky,V.A., Peterson,M.S., Bitterman,P.B. and Ingbar,D.H. (1994) Alveolar epithelial cells regulate the induction of endothelial cell apoptosis. Am. J. Physiol., 267, C893–C900.[Abstract/Free Full Text]
  22. Gorczyca,W., Gong,J. and Darzynkiewicz,Z. (1993 ) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res., 53, 1945–51.[Abstract]
  23. Norsis,M.J. (1993) SPSS for Windows. SPSS Inc., Chicago, IL.
  24. Shamsuddin,A.M., Ullah,A. and Chakravarthy,A.K. (1989) Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice. Carcinogenesis, 10, 1461–1463.[Abstract]
  25. Shamsuddin,A.M., Vucenik,I. and Cole,K.E. (1997) IP6: a novel anti-cancer agent. Life Sci., 61, 343–354.[ISI][Medline]
  26. Kulik,G., Klippel,A. and Weber,M.J. (1997) Antiapoptotic signaling by the insulin-like growth factor I receptors, phosphatidylinositol 3-kinase and Akt. Mol. Cell. Biol., 17, 1595–1606.[Abstract]
  27. Malviya,A.N. and Rogue,P.J. (1998) `Tell me where is calcium bred': clarifying the roles of nuclear calcium. Cell, 92, 17–23.[ISI][Medline]
  28. Lavoie,J.N., L'Allemain,G., Brunet,A., Müller,R. and Pouysségur,J. (1996) Cyclinc D1 expression is regulated positively by the p42p44 MAPK and negatively by the p38/HOG MAPK pathway. J. Biol. Chem., 271, 20608–20616.[Abstract/Free Full Text]
  29. Malkinson,A.M. (1992) Primary lung tumors in mice: an experimentally manipulatable model of human adenocarcinoma. Cancer Res., 52, 2670S–2676S.[Medline]
  30. Malkinson,A.M. and You,M. (1994) The intronic structure of cancer-related genes regulates susceptibility of cancer. Mol. Carcinogen., 10, 61–65.[ISI][Medline]
  31. Fung,H., Quinlan,T.R., Janssen,Y.M.W. et al. (1997) Inhibition of protein kinase C prevents asbestos-induced c-fos and c-jun protooncogene expression in mesothelial cells. Cancer Res., 57, 3101–3105.[Abstract]
Received June 9, 1998; revised August 27, 1998; accepted September 25, 1998.