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Keywords:

  • lung cancer;
  • epithelial-mesenchymal transition;
  • benzo[a]pyrene;
  • tobacco;
  • smoking

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND.

It is believed that epithelial-mesenchymal transition (EMT) occurs during the development and progression of cancer; however, the correlation between tobacco smoking and EMT remains to be elucidated.

METHODS.

Cells from the bronchioloalveolar carcinoma cell line A549 were exposed to benzo(a)pyrene (B[a]P) for 24 weeks, and morphology, proliferative activity, and gene expression profiles were analyzed.

RESULTS.

Although no apparent morphologic changes were observed, the B[a]P-exposed A549 cells exhibited enhanced proliferative activity in 1% bovine serum that contained medium, and dramatic changes in expression levels were observed in a large number of genes. Of those, the expression of EMT-related genes, such as migration-stimulating factor, plasminogen activator inhibitor-1, fibronectin, twist, transforming growth factor-β2, basic fibroblast growth factor, and electron transport system, were up-regulated; whereas gene expression of E-cadherin was decreased. Most enhanced expression levels remained 8 weeks after the retrieval of B[a]P in culture.

CONCLUSIONS.

The current results indicated that B[a]P seems to induce EMT in lung cancer cells, and it also may drive disease progression in patients with lung cancer. Cancer 2007. © 2007 American Cancer Society.

Cigarette smoking is not only a risk factor for lung carcinogenesis but also may accelerate the biologic behavior of lung cancers. A number of clinical researchers have reported on the aggressiveness of lung cancer in patients who smoke.1–4 The risk of developing lung cancer gradually decreases in individuals who stop smoking within 10 to 15 years after they start, however, thereafter, the risk of lung carcinogenesis becomes steady.5 This phenomenon may be explained by the existence of both reversible and irreversible effects of smoking on lung carcinogenesis. Certain chemical compounds in tobacco smoke, such as aromatic hydrocarbons and nitrosamines, cause loss of heterozygosity (LOH) and mutation, resulting in abnormal proliferation through the dysfunction of tumor suppressor genes or the acceleration of oncogenes.6, 7 Such genetic alterations are almost irreversible. The reversible effects mostly concern changes in molecular expression. Generally, molecules that are involved in the process of invasion or metastasis are not the same as oncogenes or recessive oncogene products, both of which affect the cell cycle. We previously examined several molecules, such as macrophage migration-inhibitory factor (MIF),8 type II hexokinase (HEKII),9 and ubiquitin-ligase protein S-phase kinase-interacting protein (Skp2),10 in clinical lung cancer tissues and observed an association between their expression levels in tumor tissues and the smoking status of patients as well as the clinical aggressiveness of tumors. We hypothesized that tobacco smoke modulates the biologic features of cancer cells. In the current study, we examined the effect of long-term exposure to B[a]P on the morphology, proliferation, and gene expression levels of lung cancer cells. A549, a cell line that was established from bronchioloalveolar carcinoma, which is considered the least relevant to smoking with regard to the effect on carcinogenesis in patients with lung cancer, was used in this study, because the in vitro effect of B[a]P in A549 cells may be more recognizable.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Cell Culture

A549 is a bronchioloalveolar carcinoma cell line with a cytochrome 450P 1A1*1 genotype and was cultured in RPMI supplemented with 10% fetal bovine serum (FBS) under standard cell culture conditions at 37°C and 5% carbon dioxide in a humid environment.

Development of A549 Cells Exposed to B[a]P for a Long Duration

A549 cells were cultured in 10% serum medium that contained a 1-μM solution of B[a]P (C20H12; purity, >96%; purchased from Sigma-Aldrich Coompany, St. Louis, Mo) for a long duration (B[a]P-A549 cells). B[a]P was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 μM and was added to the culture medium at a concentration of 1:100 (final concentration, 1 μM), which was determined through preliminary experiments with reference to our previous report.11 These cells were compared with cells that were exposed only to DMSO (control) (DMSO-A549 cells). After 24 weeks of B[a]P treatment, the A549 cells were cultured in 10% serum medium that contained only DMSO for 8 weeks (R-A549 cells).

3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxy-phenyl)-2-(4-Sulfonyl)-2H-tetrazolium Assay

We used the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, Wis) to evaluate cell growth rates. To evaluate the proliferative activity in a 1% concentration of media, 200 μL of an exponentially growing cell suspension (2 × 103 cells) were seeded in the wells of a 96-well microtiter plate. Then, the cells were incubated for 48 hours at 37°C. To evaluate the growth rate in 10% FBS and 1% FBS, 200 μL of an exponentially growing cell suspension (5 × 102 cells) were seeded in the wells of a 96-well microtiter plate and incubated for 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, or 144 hours at 37°C. Then, 20 μL of CellTiter 96 AQueous One Solution were added to each well, and the plates were incubated for another 4 hours at 37°C. The optical density was measured at 490 nm using a 96-well plate reader. Each experiment was performed using 4 replicate wells for each drug concentration.

Microarray Analysis

Standard Affymetrix Gene Chip protocols (Affymetrix, Santa Clara, Calif) were used. RNA extracted from peripheral blood leukocytes underwent 1 additional round of RNA amplification, because there were limited RNA yields from the early samples of the study. Amplification was performed starting with 100 ng of total RNA using the Ambion MEGAscript RNA amplificaion kit according to the manufacture's protocols. All labeled samples were hybridized to HG-U95Av2 GeneChip data and were analyzed using Microarray Suite (MAS 5.0; Affymetrix) and DNA Chip Analyzer software using the PM only model. Present and absent calls were determined with MAS 5.0. Statistically significant changes in gene expression were measured by using the Significance Analysis of MicroArrays statistical technique. The changes in expression level by culture condition were interpreted as positive when the gene expression levels in B[a]P-A549 cells or R-A549 cells were >2-fold or <0.5-fold the levels in DMSO-A549 cells. We further defined reversible gene expression changes if the degree of increase or decrease from baseline control levels (in DMSO-A549 cells) changed to <50% of the level of B[a]P in the R-A549 cells.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Effect of Long-term Treatment With B[a]P on A549 Cells

B[a]P-A549 cells had greater proliferative activity compared with A549 cells that were cultured with media containing DMSO (DMSO-A549 cells). With this B[a]P treatment, apparent morphologic changes were not observed from the exposure (Fig. 1A and B); however, the proliferative activity was increased (Fig. 1C), and LOH was recognized at the locus of MSH2, a DNA repair protein, by using a microsatellite marker of D2S123.12 In the 54,675 genes that we examined in a microarray analysis, substantial expression was recognized in 21,349 genes in the DMSO-A549 (control) cells. Among them, increased or decreased expression levels were observed in 5491 genes as a result of the treatment. To examine the reversibility of the changes in gene expression, we also analyzed B[a]P-A549 cells 8 weeks after the removal of B[a]P from the medium (R-A549 cells). In an analysis of the R-A549 cells, 1021 genes returned to DMSO-A549 levels (reversible changes), whereas 4470 genes remained unchanged (irreversible changes). Messenger RNA (mRNA) for glutathione-S-transferase M3, which is known to be induced by B[a]P through aryl hydrocarbon receptor (AhR), was increased by 3.17-fold in the B[a]P-A549 cells and was partially decreased by 2.4-fold in the R-A549 cells. AhR was down-regulated in the B[a]P-A549 cells by 0.53-fold and partially recovered by 0.713-fold in the R-A549 cells. The genes for MIF and Skp2 showed no substantial changes; however, the expression of HEKII was up-regulated in the B[a]P-A549 cells by 2.1-fold and was partially reversed in the R-A549 cells by 1.38-fold. In consideration of the large numbers of chemical compounds in tobacco smoke, the B[a]P experiment probably could not reflect all tobacco-related modulations of molecular expression.

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Figure 1. The effects of long-term exposure to benzo[a]pyrene (B[a]P) on lung cancer cells. A549 cells were exposed to 1 μM of B[a]P or dimethyl sulfoxide (DMSO) only (control) for 24 weeks, as described in the text (see Materials and Methods). After 24 weeks of treatment with B[a]P, A549 cells were cultured in 10% serum medium that contained only DMSO for 8 weeks (R-A549 cells). (A,B) Semiconfluent cultures of DMSO-A549 cells (A) and B[a]P-A549 cells (B) showed no difference in morphology under light microscopy. (C) In a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay for the comparison of proliferative activity, B[a]P-A549 cells (black columns) showed significantly higher proliferative activity than DMSO-A549 cells (white columns) at Day 3 and 5, and R-A549 cells (hatched columns) showed intermediate activity. Results are representative of 4 replicate wells, and values are expressed as the mean ± standard deviation (error bars). An asterisk indicates P values < .01.

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Gene Expression Analysis With Special Reference to EMT

Recently, the epithelial-mesenchymal transition (EMT) has been reported as a process of acquiring malignant potential in cancer cells, such as invasion or metastasis.13, 14 For the current report, we hypothesized that there is an association between the tobacco-induced modulation of multiple genes and EMT, and we investigated multiple genes that are involved in the EMT. We observed that the expression levels of a number of important genes for EMT were increased (Table 1). The expression levels of migration-stimulating factor, plasminogen activator inhibitor-1, and fibronectin precursor mRNA were up-regulated remarkably, by 38.9-fold, 32.6-fold, and 21.3-fold, respectively, compared with the levels in DMSO-A549 cells, and remained up-regulated in R-A549 cells. The degrees of increased expression of the same 3 genes in B[a]P-A549 cells were ranked as first, second, and seventh, respectively, among all of the genes that we analyzed. Twist, which is a potent, pivotal nuclear factor for multiple EMT-related genes,15 also was up-regulated by 3.69-fold, and its expression remained elevated in R-A549 cells. Transforming growth factor (TGF)-β2, connective TGF (CTGF), and basic fibroblast growth factor (FGF) increased by 3.98-fold, 3.97-fold, and 3.58-fold, respectively, but decreased to near their normal state in R-A549 cells. Among the epithelial markers, mRNA expression of E-cadherin and protocadherins decreased in B[a]P-A549 cells, whereas expression of N-cadherin and catenins remained stable. The loss or reduction of E-cadherin expression is a representative characteristic of EMT and is an important part of the metastatic process. In lung cancer, reduced expression of E-cadherin reportedly was observed in 42% of patients.16 E-cadherin expression was not normalized in R-A549 cells, probably because Twist, which is a transcriptional repressor of E-cadherin, maintained high expression levels in R-A549 cells.15 These results clearly indicate that B[a]P modulates the expression of a large number of genes and implicate the linkage of EMT, tobacco smoke, and disease progression in lung cancer.

Table 1. Changes in Messenger RNA Expression Involved in Epithelial-mesenchymal Transition in a Bronchioloalveolar Carcinoma Cell Line Exposed to Benzo[a]Pyrene
GeneExposure to B[a]P*Retrieval of B[a]PReversibilityRank
  • B[a]P indicates benzo[a]pyrene; I, irreversible; R, reversible; TGF, transforming growth factor; CTGF, connective tissue growth factor; FGF, fibroblast growth factor; Ets, electron transport system; FGFR, FGF receptor; MMP, matrix metalloproteinase; EGFR, epidermal growth factor receptor.

  • *

    After 24 weeks in culture with B[a]P, gene expression levels in A549 cells were analyzed, and the folds of expression are displayed compared with the expression levels in A549 cells that were cultured with dimethyl sulfoxide (DMSO) for the same period.

  • Eight weeks after the retrieval of B[a]P in culture, gene expression levels in A549 cells were analyzed and compared with the expression levels in A549 cells that were cultured in DMSO for 24 weeks.

  • Rank: Among the 21,349 genes that were expressed substantially in the control cells (DMSO-A549), the degree of increased change was ranked and is presented.

Mesenchymal markers
 Migration-stimulating factor38.950.1I1
 Plasminogen activator inhibitor 132.625.6I2
 Fibronectin precursor21.323.9I7
 Tropomyosin 1α chain4.863.42I111
 Tubulin β4.443.11I147
 TGF-β23.981.64R192
 CTGF3.972.39I194
 Twist3.693.17I229
 Basic FGF3.581.38R249
 Ets3.121.53R366
 Basic FGFR3.004.06I410
 Thrombospondine 12.181.76R954
 Myosin heavy chain2.091.57R1077
 Integrin β52.062.41R1126
 Vimentin1.932.20R1376
 Desmoyokin1.711.29 2011
 TGF-β11.501.64 2080
 MMP-91.661.24 2230
 Snail0.930.97 11,274
Epithelial markers
 Epiregulin4.801.70R114
 EGFR3.260.60R328
 Amphiregulin1.670.68 2193
 c-met1.401.10 2327
 α-Catenin1.261.11 6704
 β-Catenin1.161.17 7171
 Syndecan-11.161.91 8883
 Zo-11.100.91 9546
 N-cadherin0.970.89 10,335
 E-cadherin0.660.77(I)17,790
 Protocadherin a0.501.35R20,029
 Protocadherin b0.360.52I20,875

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Among the various adverse effects of smoking on the respiratory cells, the formation of DNA adducts by its metabolite, B[a]P diol oxide, and the generation of oxidized guanine, 7,8-dihydro-8-oxoguanine, are the direct triggers of DNA alterations, such as LOH or mutations.11, 17 Most of the alterations are considered irreversible, although some of them may be repaired by specific proteins, such as Rad51/BRCA1 or oxoguanin glycosylase 1. We hypothesized that the effects of smoking on respiratory cells included both reversible and irreversible events and that the former may affect cellular behaviors, such as motility, adhesion, and proliferation. In this report, we present direct evidence that B[a]P induces EMT-related gene expression in lung cancer cells; however, the important issue of reversibility remains to be clarified.

In fact, the cellular phenomenon in this experiment is not likely to differ from classic EMT, because no morphologic changes occurred, and the changes in several EMT-related genes were reversible. Classic EMT is considered to be an irreversible event; however, changes in TGF-β2, basic FGF, and CTGF substantially were reversible in this experiment. The expression of epithelial growth factor receptors, such as epidermal growth factor receptor and c-met (a receptor for hepatocyte growth factor), exhibited changes similar to those observed in growth factors for mesenchymal cells (Table 1). Therefore, such reversible changes are reminiscent of reversible scatter, which is a completely reversible and brief period change of transcription to mesenchymal features from epithelial cells.18, 19 Ets, which is a key transcriptional factor, also reverted to the normal state after the removal of B[a]P from the culture, although the levels of Twist, another key transcriptional factor, remained high. The B[a]P exposure in this study had such a strong impact that multiple transcriptional changes of the genes driving EMT and LOH occurred. Therefore the reversibility of these genes may be associated with the duration of the abnormal condition. B[a]P, although it is a strong carcinogen, is only 1 of >4000 chemical compounds in tobacco smoke, and the exposure for 24 weeks to B[a]P may have been too short to mimic the clinical effects of smoking. Classically, the transdifferentiation of epithelial cells into other types of epithelial cells has been observed in the retinal pigmented cells that become lens epithelia20 and in the pancreatic acinar cells that become hepatocytes.21 It remains to be clarified whether the transition to mesenchymal cells from epithelial cells (EMT) is a middle phase of transdifferentiation.

The solid tumors accompanied by fibrosis are characterized by aggressive invasiveness. Schirrhus cancer of the stomach has vast fibrosis in stroma and shows aggressive expansion both horizontally and perpendicularly.22 In this type of gastric cancer, we previously reported high expression levels of Wisp-1 variant, a family of CTGF.23 In bronchioloalveolar carcinoma type lung cancer, central fibrosis occurs over time as the malignant potential, such as invasion to basement membrane and of lymph node metastasis, increases,24 although no critical molecule that works in this process has been identified. Patients with pulmonary fibrosis have a significantly high risk of lung cancer, and mesenchymal transition from alveolar cells recently was reported as a source of increasing fibroblasts in the disease.25 The mechanisms of fibrosis and of EMT have a common molecular profile; therefore, carcinogenesis frequently occurs in the pathologic state of organ fibrosis. Previously, it was reported that immortalized breast epithelial cells, by introduction of c-Ha-ras, resulted in a progression of changes in the morphology, proliferation, and invasiveness by treatment of B[a]P.26 Our results may support such morphologic and functional effects by B[a]P, although we observed no direct evidence of changes in cellular behavior from the B[a]P treatment in this study.

In conclusion, B[a]P, which is an aromatic hydrocarbon, directly caused EMT-like, dynamic changes in gene expression, indicating that EMT is involved in the carcinogenesis and progression of lung cancer. This simple study may indicate that tobacco is a growth factor for lung cancer. Further analyses and a better understanding of this mechanism may lead to developments in the management and treatment of patients with cancer.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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