Introduction

Lung cancer is the most common malignancy in both men and women in the world. In 2003 there will be about 172 000 new cases of lung cancer in the United States and about 157 000 people will die of this disease (American Cancer Society, 2003). Lung cancer, like other types of cancer, develops as a multistage process involving the accumulation of genetic alterations that affect key proto-oncogenes and tumor suppressor genes (You et al., 1989; Herzog et al., 1997). There are a wide variety of genes whose expressions are altered in lung tumors (McDoniels-Silvers et al., 2002; Yao et al., 2002; 2003). Many of these genes are probably altered at the transcription level, and may play important roles in lung tumor development. The A/J mouse has proved to be an extremely valuable animal model for studying lung adenoma and adenocarcinoma formation as well as lung cancer chemoprevention. In addition to histologic similarity between adenomas/adenocarcinomas commonly seen in mice and human lung tumors, genetic changes found in mouse lung tumors also resemble those in humans (Malkinson, 1992; Herzog et al., 1997).

Glucocorticoids and their synthetic analogs have been shown to have cancer chemopreventive efficacy in animal models of lung cancer (Wattenberg and Estensen, 1996; 1997; Wang et al., 2003). In fact, it was initially observed that steroid hormones inhibited croton oil-promoted mouse skin tumorigenesis almost 35 years ago by Belman (Belman and Troll, 1972). Recent studies by Wattenberg et al. have demonstrated that the synthetic glucocorticoid budesonide, has strong preventive activity in the B[a]P-induced pulmonary adenoma formation in female A/J mouse model. This inhibitory effect was achieved with budesonide administrated either by diet or aerosol (Wattenberg and Estensen, 1996; Wattenberg et al., 2000). An inhibitory effect of budesonide was observed even at a very low dose level when administrated by aerosol (Wattenberg et al., 2000). Budesonide is one of a few compounds with lung tumor inhibitory effects even when given after carcinogen initiation (Wattenberg and Estensen, 1997). Recently, we have shown that budesonide has both chemopreventive and chemotherapy effects on lung tumors in mice with different germline mutations and that the degree of efficacy depends on genotypes of mice (Wang et al., 2003).

The mechanisms for chemopreventive effect of glucocorticoid have not been elucidated. Evidence indicates that the possible mechanisms include: (1) directly altering gene transcription by regulating the glucocorticoid response element (GRE) in the target gene DNA sequence; (2) indirectly altering gene transcription by interacting with other transcript factors, such as AP-1, NF-B, and CREB; (3) altering post-transcriptional events and translation of proteins (Greenstein et al., 2002). Through these possible mechanisms, GCs induce apoptosis via activation of death-inducing genes or inhibition of growth/survival genes.

The microarray techniques allow rapid and simultaneous detection of the expression of thousands of genes. In this study, we employed the Affymetrix oligonucleotide arrays representing over 12 000 genes and ESTs for the identification of differentially expressed genes in mouse lung tumors and for analysis of possible modes of action for budesonide in cancer chemoprevention and/or chemotherapy.

Results

Bioassay of the chemopreventive efficacy of budesonide on B[a]P-induced mouse lung tumorigenesis showed that in B[a]P-treated group, mice developed lung tumors in all animals with an average of 12 tumors per mouse and a total tumor volume of 24 mm³ per mouse. In contrast, lung tumor multiplicity in the budesonide group was 3–4 per mouse with a total tumor volume of 1.5 mm³ per mouse. Thus, dietary budesonide significantly inhibited lung tumor formation with a 70% inhibition on multiplicity and a 94% inhibition on total tumor volume. Differences in multiplicity and tumor volume for budesonide-treated mice were highly significant (P< 0.01) (Table 1).

Table 1 - Chemopreventive effects of budesonide on B[a]P-induced lung tumorigenesis in A/J mice.

Full table

The experimental design of microarray includes the use of normal lung tissues and tumors from B[a]P control mice and tumors from mice treated with both B[a]P and budesonide. When comparing the tumors of mice treated with B[a]P only with the tumors of mice treated with both B[a]P and budesonide, 363 genes were found regulated by budesonide of which 243 genes were overexpressed and 120 genes were underexpressed in mouse lung tumors after budesonide treatment (Figure 1). When comparing these genes with the paired normal tissues, 108 genes that changed during lung tumorigenesis induced by B[a]P were modulated back to normal levels by budesonide treatment. Clustering reveals two major gene expression patterns: in Pattern I, 50 genes were found upregulated in B[a]P-induced tumors; however, expression of these genes were modulated back to normal levels with budesonide treatment. In Pattern II, 58 genes were found downregulated in B[a]P-induced tumors compared with normal lungs and were modulated back to the level of normal lung tissues after budesonide treatment (Figure 2). A broad range of functions is associated with these genes, including signal transduction, cell cycle, apoptosis, and transcription (Table 2). These genes may play important roles in mediating the inhibitory effect of budesonide in mouse lung tumorigenesis. From the genes found differentially expressed, nine of 11 genes tested were confirmed by RT–PCR; the confirmation rate is about 80% at the cutoff of two-fold change and P<0.05 (Figure 3).

Figure 1.

Hierarchical clustering of 363 genes and ESTs found to be differentially expressed between B[a]P tumors and budesonide treated tumors (P<0.05, fold change >2). Expression levels for each gene were transformed across samples using a normal (0,1) transformation. Green indicates an expression below the mean value for the gene, black near the mean, and red above the mean. More than 240 genes were overexpressed and 120 genes were underexpressed in mouse lung tumors after budesonide treatment

Full figure and legend (138K)

Figure 2.

Hierarchical clustering of 108 genes and ESTs found differentially expressed between B[a]P induced tumors and budesonide treated tumors (P<0.05, fold change >2) and these 108 genes and ESTs were modulated back to/near normal levels by budesonide treatment. Pattern 1 contains 50 genes upregulated in tumors and modulated back to normal levels after budesonide treatment. Pattern 2 contains 58 genes downregulated in tumors and modulated back to normal levels by budesonide

Full figure and legend (167K)

Figure 3.

Semiquantitative RT-PCR confirmation for selected genes. (a) RT–PCR confirmation. Lanes 1–4 are mouse normal lung tissues, lanes 5–8 are mouse lung tumors induced by B[a]P, and lanes 9–12 are B[a]P-tumors treated with budesonide. (b) Comparison of fold change produced by DNA microarray with relative expression ratio obtained from RT–PCR

Full figure and legend (101K)

Table 2 - Genes changed during mouse lung tumorigenesis and modulated by budesonide.

Full table

GenMAPP is a novel tool for visualizing expression data in the context of biological pathways (Dahlquist et al., 2002). We imported our data set into the program and used it to convert the expression data into illustrations demonstrating the implications of significant expression changes. We found that genes in at least three major regulatory pathways were significantly altered by budesonide during lung tumorigenesis. Figures 4 and 5 represent the G protein signaling pathway and MAPK cascade, respectively.

Figure 4.

GenMAPP – G protein pathways integrating our expression data. Yellow indicates a higher level of expression in budesonide-treated samples. Blue indicates a lower gene expression by budesonide. Grey indicates that the selection criteria were not met but the gene is represented on the array. White boxes indicate that the gene was not present on the array

Full figure and legend (149K)

Figure 5.

GenMAPP – MAPK cascade integrating our expression data. Yellow indicates a higher level of expression in budesonide-treated tumors. Blue indicates a lower level of expression in the budesonide-treated samples. Grey indicates that the selection criteria were not met but the gene is represented on the array. White boxes indicate that the gene was not present on the array

Full figure and legend (98K)

Discussion

In the present study, we have successfully examined chemoprevention effects of budesonide against lung tumor induction in A/J mice and its effects on gene expression. Continual budesonide administration in the diet exhibited a potent protective effect against B[a]P-induced lung tumorigenesis in mice at 40 weeks. We found a strong decrease in tumor multiplicity (70%) and a profound decrease in total tumor volume (94%) in A/J mice. Although glucocorticoids have been used for cancer prevention and treatment in animals for a long time, the precise mechanism of action has not been elucidated. Proposed mechanisms include that glucocorticoids: (1) initiation of the apoptotic cascade by inducing transcription of death-specific genes; (2) induce apoptosis through cell cycle arrest; and (3) initiate apoptosis by inhibiting transcription of cytokines (Greenstein et al., 2002; Schmidt et al., 2004). Using Affymetrix GeneChips that contain about 12 000 genes and ESTs, we identified differentially expressed genes between untreated tumors and tumors treated with budesonide which may lead to the identification of possible mechanisms and pathways through which budesonide inhibits lung tumorigenesis.

Our results support at least two of the three models of budesonide action. As described in more detail below, budesonide induced expression of a number of genes considered to regulate apoptosis and budesonide induced expression of genes involved in cell cycle arrest. Table 2 lists the most prominent genes. Results suggest that budesonide-treated tumors follow a different apoptotic mechanism than do the untreated tumors. In budesonide-treated tumors, Bim, Blk and ALG-2 are all expressed at higher levels than in untreated tumors, suggesting that the Bcl-2-regulated mechanism of apoptosis is activated. Regulation of this pathway has been recently reviewed and showed it to be conserved across many distant species (Puthalakath and Strasser, 2002). In untreated tumors, Raidd/Cradd is expressed at higher levels than in budesonide tumors, suggesting that the Caspase-2 apoptotic pathway is activated in untreated tumors. Differences in apoptotic mechanism could explain, atleast in part, the effectiveness of budesonide.

Results also show budesonide-treated tumors having higher expression of Mad3 and Mad2, which play roles in mitotic checkpoint function. Mad2 and Mad3 are part of a larger complex that interacts with the anaphase-promoting complex (APC) and blocks its ligase activity (Yu, 2002). Cdc28 is the main CDK driving the cell cycle in budding yeast. Genetic interactions with checkpoint and APC mutants suggest Cdc28 may regulate checkpoint arrest downstream of the MAD2 and BUB2 pathways (Kitazono et al., 2003). Yin et al. (2001) recently observed increased expression of Mad2 and Mad3 in response to glucocorticoid stimulation in vitro. Increased expression of these genes therefore suggests a growth arrest pathway for budesonide-treated tumors.

Budesonide-treated tumors also show decreased expression of the proto-oncogene Fyn, which is associated with tumor progression in cancer models including oral squamous cell carcinoma and metastatic murine melanoma (Huang et al., 2003; Li et al., 2003). This suggests a progressive phenotype for the untreated tumors that was ameliorated by budesonide treatment.

It was recently reported that a combination treatment of dexamethasone and cisplatin on solid tumors of the lung and cervix resulted in less apoptosis compared with cisplatin alone (Herr et al., 2003). The mechanism for inhibition of apoptosis was shown to involve downregulation of CD95-L and TRAIL resulting in a reduction of caspase-8/9-mediated apoptosis. In another study, Dorscheid et al. (2001) showed that a number of corticosteroids including both dexamethasone and budesonide elicited apoptosis via a caspase-9 mediated mechanism using epithelial cells. In this study, budesonide treatment appeared to recruit the caspase-8/9 and to downregulate the caspase-2. It would be interesting to carefully optimize combination treatment of these tumors since there are so many advantages to using the glucocorticoid adjuvant.

Next, we imported our data set into the program and used GenMAPP to illustrate the pathways modulated by budesonide treatment. As expected, the G protein pathway coupled with ras MAPK pathway is intimately involved in murine lung tumorigenesis since virtually all of these tumors have mutations in the Kras2 gene. Figures 4 and 5 depict the modulation of G protein pathway and MAPK pathway by budesonide, respectively. Gq/11 pathway is activated by calcium-mobilizing hormones and stimulates PLC- to produce IP3 and DAG. PLC- can also activate the MAPK pathway. Gi can regulate signals from c-Src to the Rap pathway, activated Rap1GAP can effectively block the small GTPase Ras and inhibit the MAPK pathway (Rozengurt, 1998). Modulation of these genes by budesonide may indicate that budesonide exerts its chemopreventive effects on mouse tumorigenesis by influencing the G protein and MAPK pathways in addition to its effects on apoptosis.

The mechanisms of glucocorticoids' chemopreventive effects on tumorigenesis are not known. A number of theories have been proposed, which suggest that there are multiple signal pathways and mechanisms that account for the chemoprevention effects mediated by the glucocorticoids. However, to date, the major molecular changes associated with their chemopreventive effects have not been identified. In this study, we have presented the unique molecular profile of mouse lung tumors in mice treated with budesonide in contrast to untreated tumors, and identified a broad range of budesonide associated genes. Our results suggest that budesonide exerts its effects of chemoprevention through growth arrest via Mad2/3 and through apoptosis via Bim/Blk and by inference caspase-8/9. Continued study is needed to further define the targets of glucocorticoids and to further elucidate the mechanisms by which glucocorticoids prevent tumor development in the clinical setting, and perhaps to novel modes of chemopreventive and/or chemotherapeutic intervention.

Materials and methods

Experimental design and collection of lung tumors and lung tissues

B[a]P (99% pure), budesonide (>99% pure), and tricaprylin were obtained from Sigma Chemical Co. (St Louis, MO, USA). A/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in plastic cages with hardwood bedding and dust covers, in a HEPA-filtered, environmentally controlled room (241°C, 12/12 h light/dark cycle). B[a]P was prepared immediately before use in animal bioassays by dissolving in tricaprylin. All mice were fed AIN-76A purified diet during the experiment. In the vehicle group, 16 6-week-old A/J mice were given a single i.p. injection of tricaprylin (0.1 ml). In the carcinogen control group, 12 6-week-old A/J mice were given a single i.p. injection of B[a]P (100 mg/kg body weight) in 0.1 ml of tricaprylin. In the budesonide group, 19 mice were given a single i.p. injection of B[a]P (100 mg/kg body weight) in 0.1 ml of tricaprylin and were fed AIN-76A purified diet containing budesonide (1.5 mg/kg of diet) beginning 2 weeks prior to B[a]P treatment and continuing for an additional 40 weeks. Food and water were available ad libitum. At termination, the lung tumors were harvested and frozen in liquid nitrogen until RNA analysis. All frozen tumor tissues were microdissected to determine the tumor vs normal cell ratio for each specimen. Tumor tissue sections corresponding to the microscopic sections containing 80% tumor cells were isolated and stored at -80°C for subsequent RNA isolation. Matching normal tissues were also microdissected to ensure that specimens consisted of purely normal lung tissue. Lung tumor development was then evaluated by counting tumor number (N), calculating tumor volume (V) and the total tumor load (total tumor V per mouse).

RNA isolation and amplification

Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA, USA) and purified using the RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocols. In vitro transcription-based RNA amplification was then performed on each sample. cDNA for each sample was synthesized using a Superscript cDNA Synthesis Kit (Invitrogen) and a T7-(dT)24 primer: 5'-GGCCAGTGAATTGTAATACGACT-CACTATAGGGAGGCGG-(dT)24-3'. The cDNA was cleaned using phase-lock gel (Fisher Cat ID E0032005101) phenol/chloroform extraction. Then, the biotin-labeled cRNA was transcribed in vitro from cDNA using a BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY, USA) and purified, again using the RNeasy Mini Kit.

Affymetrix genechip probe array and RT–PCR confirmation

The labeled cRNA was applied to the Affymetrix MG_74Av2 GeneChips (Affymetrix, Santa Clara, CA, USA), which contains >12 000 genes and ESTs on one array according to the manufacturer's recommendations. To validate the microarray results, 11 genes were randomly selected from the genes detected in the microarray assay for further confirmation by semiquantitative RT–PCR as previously described (Yao et al., 2002). After normalization to the level of -actin mRNA, the student's t-test was used to determine the differences in the signal intensity of phosphor imaging among the normal lungs, lung tumors and budesonide treated lung tumors.

Cluster and GenMapp

Four independent samples were collected for each group. Array normalization and gene expression estimates were obtained using Affymetrix Microarray Suite 5.0 software (MAS5). The array mean intensities were scaled to 1500. Genes with negative expression values were removed. Differential expression was determined on the combined basis of statistical testing using t-test and expression ratio with cutoff of P<0.05 and expression ratio (fold change) >2 or <0.5 being called positive for differential expression. Genes passing this filter were allocated to four mutually exclusive groups according to their expression pattern including upregulated or downregulated to near normal level. To perform hierarchical clustering, expression for each gene was transformed across samples to a standard normal deviate. These values were supplied to the Gene Cluster program of Eisen and used without further transformation (Eisen et al., 1998). Signal transduction pathways, metabolic pathways and other functional groupings of genes were evaluated for differential regulation using the visualization tool GenMAPP (UCSF, www.genmapp.org; (Dahlquist et al., 2002). We imported our data set into the program and used GenMAPP to illustrate pathways containing differentially expressed genes. Differential gene expression was based on budesonide treatment vs nontreatment expression change.

References

1. American Cancer Society (2003). Cancer Facts Fig, 4–6.
2. Belman S and Troll W. (1972). Cancer Res., 32, 450–454. | PubMed | ISI | ChemPort |
3. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC and Conklin BR. (2002). Nat. Genet., 31, 19–20. | Article | PubMed | ISI | ChemPort |
4. Dorscheid DR, Wojcik KR, Sun S, Marroquin B and White SR. (2001). Am. J. Respir. Crit. Care Med., 164, 1939–1947. | PubMed |
5. Eisen MB, Spellman PT, Brown PO and Botstein D. (1998). Proc. Natl. Acad. Sci. USA, 95, 14863–14868. | Article | PubMed | ChemPort |
6. Greenstein S, Ghias K, Krett NL and Rosen ST. (2002). Clin. Cancer Res., 8, 1681–1694. | PubMed | ISI | ChemPort |
7. Herr I, Ucur E, Herzer K, Okouoyo S, Ridder R, Krammer PH, von Knebel Doeberitz M and Debatin KM. (2003). Cancer Res., 63, 3112–3120. | PubMed | ISI | ChemPort |
8. Herzog CR, Lubet RA and You M. (1997). J. Cell Biochem. Suppl., 29, 49–63. | Article |
9. Huang J, Asawa T, Takato T and Sakai R. (2003). J. Biol. Chem., 16, 16.
10. Kitazono AA, Garza DA and Kron SJ. (2003). Mol. Genet. Genomics, 24, 24.
11. Li X, Yang Y, Hu Y, Dang D, Regezi J, Schmidt BL, Atakilit A, Chen B, Ellis D and Ramos DM. (2003). J. Biol. Chem., 12, 12.
12. Malkinson AM. (1992). Cancer Res., 52, 2670s–2676s. | PubMed | ChemPort |
13. McDoniels-Silvers AL, Stoner GD, Lubet RA and You M. (2002). Neoplasia, 4, 141–150. | Article | PubMed |
14. Puthalakath H and Strasser A. (2002). Cell Death Differ., 9, 505–512. | Article | PubMed | ISI | ChemPort |
15. Rozengurt E. (1998). J. Cell Physiol., 177, 507–517. | Article | PubMed | ChemPort |
16. Schmidt S, Rainer J, Ploner C, Presul E, Riml S and Kofler R. (2004). Cell Death Differ, 11, S45–S55. | Article | PubMed | ISI | ChemPort |
17. Wang Y, Zhang Z, Kastens E, Lubet RA and You M. (2003). Cancer Res., 63, 4389–4395. | PubMed | ISI | ChemPort |
18. Wattenberg LW and Estensen RD. (1996). Cancer Res., 56, 5132–5135. | PubMed |
19. Wattenberg LW and Estensen RD. (1997). Carcinogenesis, 18, 2015–2017. | Article | PubMed |
20. Wattenberg LW, Wiedmann TS, Estensen RD, Zimmerman CL, Galbraith AR, Steele VE and Kelloff GJ. (2000). Carcinogenesis, 21, 179–182. | Article | PubMed |
21. Yao R, Wang Y, Lubet RA and You M. (2002). Oncogene, 21, 5814–5821. | Article | PubMed |
22. Yao R, Wang Y, Lubet RA and You M. (2003). Neoplasia, 5, 41–52. | PubMed |
23. Yin XY, Grove LE and Prochownik EV. (2001). Oncogene, 20, 2908–2917. | Article | PubMed |
24. You M, Candrian U, Maronpot RR, Stoner GD and Anderson MW. (1989). Proc. Natl. Acad. Sci. USA, 86, 3070–3074. | Article | PubMed | ChemPort |
25. Yu H. (2002). Curr. Opin. Cell Biol., 14, 706–714. | Article | PubMed | ISI | ChemPort |