Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185, USA
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Abstract |
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Abbreviations: AB, automation buffer; COX, cyclooxygenase; DAB, diaminobenzidine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NSAIDs, non-steroidal anti-inflammatory drugs; PADB, primary antibody diluting buffer; PAR, pulmonary adenoma resistance; PDTC, pyrrolidine dithiocarbamate; PGE2, prostaglandin E2.
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Introduction |
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A new class of compounds targeting cyclooxygenase-2 (COX-2) has recently shown promise for the chemoprevention of several solid tumors. Cyclooxygenases catalyze the rate-limiting step in the synthesis of prostaglandins from arachidonic acid. The COX-2 isoform is generally expressed at low or undetectable levels and induced in response to environmental insults and internal stimuli like cytokines and growth factors. In contrast, COX-1 is constitutively expressed in most tissues and involved in cellular homeostasis. Epidemiological studies have revealed a reduced incidence of colon cancer in people who regularly ingested aspirin, a non-steroidal anti-inflammatory drug (NSAID) (1), and clinical studies have shown the regression of pre-existing colorectal adenomas in patients with familial adenomatous polyposis treated with NSAIDs (2). NSAIDs also significantly reduce tumor multiplicity in animal models of colorectal cancer (3,4).
The anti-inflammatory effects of NSAIDs are mediated in part by inhibition of COX-1 and -2. Approximately half of human colorectal adenomas and 8090% of adenocarcinomas express high levels of COX-2 relative to normal epithelium (5). Similarly, COX-2 protein and mRNA expression levels are also elevated in most adenocarcinomas and in about one-third of premalignant lesions in human lung (6,7). Squamous cell carcinomas, but not small cell carcinomas of the lung also express COX-2 (6,7). NSAIDs and a specific inhibitor of COX-2 (NS-398) inhibit both in vitro and in vivo growth of murine and human lung-cancer-derived cell lines (8,9), suggesting that this pathway may be a good target for chemoprevention in the lung.
Because the in vivo target for chemopreventive compounds is premalignant lung disease, studies in animal models could be invaluable for evaluating the efficacy of the compounds and the interactions between the COX-2 pathway and tumor progression. One model that holds promise for these studies is the A/J mouse. The induction of lung tumors in the A/J mouse progresses through several morphologically distinct stages. Tumor initiation by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is characterized first by a proliferation of type II cells along the alveolar septae. These premalignant hyperplastic lesions contain an activated Ki-ras gene, an alteration present in some human adenocarcinomas, and progress to adenomas and ultimately carcinomas (10). Recent studies have shown that treatment of A/J mice with NSAIDs or NS-398, beginning 2 weeks prior to NNK exposure, decreased lung tumor multiplicity and volume (8). The purpose of our investigation was to extend these findings by delineating COX-2 expression patterns during murine tumor progression, comparing expression seen in A/J lungs to expression in other mouse strains and human tumors, and by examining the effect of a specific COX-2 inhibitor on gene transcription and enzyme activity in xenografted murine lung tumors.
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Materials and methods |
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Lung tumors were induced in 6-week-old A/J and C3H/HeJ mice by i.p. injection of 50 mg/kg NNK three times per week for 7 weeks. Lung tumors were collected 2068 weeks after initiation of treatment. Lungs were fixed by inflation with 4% paraformaldehyde, embedded in paraffin and cut into 5 µm sections. Dissected tumors were stored at 80°C.
The cell lines CL13, CL25, CL30 and IO33 were derived from A/J lung tumors induced by NNK. The MNNK cell line is an NIH 3T3 transformant generated by transfection of A/J lung tumor DNA containing a mutated Ki-ras gene. The Spon4 cell line was derived from a spontaneously arising A/J lung tumor.
Total RNA was isolated from type II cells, tumors and cell lines by homogenization in TRI Reagent (Molecular Research Center, Cincinnati, OH), phenol/chloroform extraction and isopropanol precipitation. RNA pellets were then resuspended in buffer, incubated with DNase I, extracted with phenol/chloroform, precipitated with isopropanol and stored at 80°C.
Immunohistochemistry
Human and murine lung sections were deparaffinized in xylene and rehydrated in a graded series of ethanol/H2O baths. Sections were incubated for 30 min at room temperature in 1% hydrogen peroxide in methanol, rinsed in H2O, then incubated in boiling antigen retrieval citra solution (Biogenex, San Ramon, CA) for 5 min. Sections were cooled to room temperature, rinsed in H2O, and equilibrated in 1:10 automation buffer (AB; Biomeda Corp., Foster City, CA). Sections were incubated for 510 min in PowerBlock (Biogenex), and then overnight at 4°C in a blocking solution composed of 1.5% normal goat serum in AB. A polyclonal rabbit anti-mouse COX-2 antibody (Cayman Chemical Co., Ann Arbor, MI), diluted 1:600 in primary antibody diluting buffer (PADB; Biomeda), was then applied to the sections. Serial sections were incubated with purified rabbit IgG as immunostaining controls. After 2 h the sections were rinsed with AB and incubated for 1 h with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:500 in PADB. After rinsing with AB the sections were incubated for 30 min at room temperature with Avidin DH: biotinylated horseradish peroxidase H complex in AB as described in the instructions accompanying the VectaStain Elite ABC kit (Vector Laboratories). The sections were rinsed in H2O, and a diaminobenzidine (DAB) solution was prepared and applied as instructed (DAB Substrate Kit, Vector Laboratories). After removal of this solution, the sections were rinsed in H2O, counterstained with hematoxylin, dehydrated and coverslipped with a xylene-based mounting medium. Sections incubated with rabbit IgG rather than primary antibody showed little or no staining. Pre-incubation of primary antibody with increasing concentrations of purified peptide antigen (Cayman Chemical Co.) resulted in decreased and, ultimately, a complete absence of staining.
Staining intensity in hyperplastic lesions and neoplasms was subjectively graded in two parts consisting of intensity and distribution. Intensity was graded on a scale of 04+, with 4+ being the most intense (bronchiolar epithelium as a positive control). Distribution of the staining among the cells in a lesion was subjectively graded on a percentage basis (i.e. 1, <25% of the lesion affected; 2, 2550%; 3, 5075%; 4, 75100%). The product of the intensity and distribution gave a subset of the score (e.g. 2550% of cells in a lesion stained with a 4+ intensity: product = 8; 2550% of cells stained with a 3+ intensity: product = 6). For the score of an entire lesion, the subsets were summed.
Ribonuclease protection assay
A COX-2-specific cDNA probe was prepared by PCR amplification of a 300 bp region of a commercially available 1.2 kb COX-2-specific cDNA probe (Cayman Chemical Co.). The resulting cDNA was cloned into a pCR II vector using a TA Cloning Kit (Invitrogen, Carlsbad, CA). Ligation was confirmed by restriction enzyme digestion, as well as sequencing. The insert was then transferred to a pTRIamp18 vector (Ambion, Austin, TX). Linearized plasmid was gel purified using a QIAquick Gel Extraction Kit (Qiagen, Santa Clarita, CA), then 32P-labeled using a MAXIscript In vitro Transcription Kit (Ambion). Radiolabeled transcript was gel purified and combined with sample RNA (310 µg) as described in the RPA III Kit protocol (Ambion). Following incubation, unhybridized RNA was digested with ribonuclease, then hybridized RNAs were separated on a 5% acrylamide/8 M urea TrisborateEDTA gel. The gel was dried and exposed to film. Densitometry was performed using an Alpha Innotech scanning densitometer and accompanying software. A 103 bp radiolabeled cyclophilin probe prepared from a commercially available template (pTRI-cyclophilin, Ambion) was included in all hybridization reactions as a positive/gel-loading control. Yeast RNA was used as a negative control. Radiolabeled marker transcripts were prepared using a Century Marker Template Set (Ambion). All samples were analyzed at least twice.
Lung tumor xenografts
Nude mice were injected s.c. with ~2.5 million CL30 or Spon4 cells in a volume of 0.1 ml. When tumors were palpable, inhibitors were injected i.p. three times per week for 23 weeks. Tumor area was measured twice per week. For each cell line, four mice were treated with vehicle only (0.5% methylcellulose, 0.025% Tween-20), pyrrolidine dithiocarbamate (PDTC, 200 mg/kg; Sigma, St Louis, MO), SC-236 (2 mg/kg; generously provided by Searle-Monsanto, Skokie, IL), or a combination of both PDTC and SC-236. Following treatment, the mice were killed and the xenografts were excised. Portions of each xenograft were quick-frozen and stored at 80°C, and the remainder fixed in formalin.
DNA fragmentation analysis
Apoptotic cells were identified in paraffin-embedded tissue sections using the Klenow-FragEL DNA Fragmentation Detection Kit (Calbiochem, San Diego, CA). 3'-OH groups generated by apoptotic endonucleases were labeled with horseradish peroxidase and visualized with DAB as described in the protocol accompanying the kit. Three sham- and three SC-236/PDTC-treated Spon4 xenografts were analyzed. Apoptotic cells were identified based on positive DAB staining as well as characteristic cellular morphology. Apoptotic cells were counted in 400x microscopic fields. Twenty serial fields were examined per tumor.
Prostaglandin E2 (PGE2) quantitation
Portions of frozen excised xenografts were homogenized on ice in 10 mM TrisHCl (pH 8.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin. The homogenate was then centrifuged at 10 000 g for 20 min at 4°C. The supernatant was re-centrifuged at 10 000 g for 10 min. The resulting supernatant was aliquotted, quick-frozen and stored at 80°C. Protein concentration was determined using a Bradford-based protein assay (Bio-Rad, Hercules, CA). PGE2 concentration was measured using a competitive enzyme immunoassay kit (Cayman Chemicals). The xenograft supernatant was diluted 15 000x prior to analysis by immunoassay. Samples were assayed twice in duplicate. PGE2 concentration was linear with volume of sample assayed, indicating no interfering substances in the xenograft supernatants at this dilution.
Statistical analysis
Statistical analyses comparing data derived from two groups (treated and untreated) were conducted using the Student's t-test (two-tailed, unpaired, equal variance). Statistical analysis of xenograft growth over time, with and without inhibitor treatment, was conducted by means of a repeated measures analysis of variance. The single, independent variable used was treatment group. Multivariate significance was assessed using the HotellingLawley Trace. A multivariate contrast was used to evaluate trend.
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Results |
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In addition to the COX-2 immunostaining of mouse lung tumors, specimens from several human pulmonary neoplasms were evaluated by immunohistochemistry for COX-2 expression. Like the mouse tumors, human tumors displayed variation in staining patterns and intensity (data not shown), with papillary tumors showing locally extensive regions of rather diffuse staining and more solid tumors (such as squamous cell carcinomas) displaying multifocal individual cell staining. Human type II and bronchiolar epithelial cells were routinely negative for COX-2 immunoreactivity.
COX-2 mRNA in murine type II cells, lung tumors and lung tumor-derived cell lines
COX-2 mRNA levels were measured in A/J mouse alveolar type II cells, lung tumors and lung-tumor-derived cell lines to determine whether the heterogeneity seen for COX-2 protein expression was due to differences in transcript levels. In addition to A/J cells, type II cells from two other lung-cancer-sensitive mouse strains (A/WySnJ and SWR/J) and three lung cancer-resistant mouse strains (C3H/HeJ, C57BL/6J and DBA/2J) were analyzed to determine whether COX-2 levels in normal type II cells correlate with lung cancer susceptibility. Similarly high levels of COX-2 mRNA were found in the type II cells from all three lung-cancer-sensitive mouse strains (Figure 3A and B) [A/J level set at 100%, A/WySnJ = 114 ± 8% (average of two measurements ± range), SWR/J = 116 ± 16%]. COX-2 mRNA levels in the lung-cancer-resistant strains were 19 ± 8% and 57 ± 11% of A/J levels in the C3H/HeJ and C57BL/6J strains, respectively. In contrast, COX-2 mRNA levels in the resistant DBA/2J strain were higher than A/J levels (168 ± 3%).
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COX-2 mRNA levels also varied among the six lung-tumor-derived cell lines examined (Figure 3C). Expression varied by an average of 35-fold, with the highest level seen in the Spon4 cell line that was derived from a spontaneously arising A/J mouse lung tumor.
Effect of COX-2 inhibitors on the growth of lung tumor-derived xenografts
A specific inhibitor of COX-2 activity (SC-236) and an inhibitor of COX-2 transcription (PDTC) were tested alone and in combination for their effect on the growth of lung-tumor-derived xenografts implanted s.c. in nude mice. Treatment was initiated ~1 week after implantation, when palpable tumors were clearly evident. Mice were injected with vehicle and inhibitors three times per week. The doses of SC-236 and PDTC were chosen based on doses shown by Chinery et al. (12) to inhibit the growth of colon tumor-derived xenografts. Chinery et al. used the structurally similar COX-2 inhibitor SC-58125, rather than SC-236 (12). SC-236 has a 10-fold lower IC50 than SC-58125, so a 5-fold smaller dose of SC-236 was used. The most dramatic effect observed was the repression of the growth of Spon4 xenografts as a result of combined SC-236/PDTC treatment (Figure 4). After 2 weeks of treatment, the SC-236/PDTC-treated xenografts had increased in size by ~5-fold, whereas untreated xenografts increased in size by ~7-fold. Statistical analysis (Materials and methods) indicated no significant (P > 0.05) differences between the four treatment groups. The effect of these inhibitors on the growth of CL30 xenografts was also examined, with no growth repression observed following 2 weeks of treatment (data not shown). The rapid growth of both cell lines in sham-treated mice precluded extending the treatment period.
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One mechanism proposed for the reduction in tumor growth seen in other studies is increased apoptosis following exposure to COX-2 inhibitors. The average (±SE) number of apoptotic cells in a 400x microscope field in sham- and inhibitor-treated xenografts was 3.5 ± 0.5 and 4.7 ± 0.4, respectively. This increase in apoptotic cells following inhibitor treatment was not statistically significant (P = 0.16).
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Discussion |
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The Ras/ERK signaling pathway appears to play a role in the regulation of COX-2 expression. Human non-small-cell lung cancer cell lines with mutations in Ki-Ras have high expression levels of COX-2, and inhibition of Ras activity in these cell lines decreases COX-2 expression (16). Rat intestinal epithelial cells and fibroblasts transfected with Ha-Ras overexpress COX-2, whereas inhibitors of ERK ameliorate this response (17). However, differences in COX-2 levels between sensitive and resistant mouse strains may also be due to differences in regulatory factors unrelated to the ras pathway. Hida et al. (6) observed increased COX-2 expression in 70% of adenocarcinomas, whereas, historically, mutation of the Ki-Ras gene has been detected in only 30% of this histologic tumor type. Kim and Fischer (18) have identified several cis- and trans-acting factors required for COX-2 expression in mouse skin carcinoma cells, pointing out the complexity of COX-2 regulation. This multi-factorial nature of murine lung cancer susceptibility and COX-2 regulation most likely underlies the lack of full concordance between lung cancer susceptibility and endogenous COX-2 expression levels among the mouse strains examined in our studies.
COX-2 overexpression in normal alveolar type II cells may be directly involved in increasing the sensitivity of these cells to the effects of carcinogens and enhancing tumor development after initiation. Clear evidence for a role for COX-2 in tumorigenesis has been substantiated by a multitude of studies. For example, overexpression of COX-2 in rat intestinal epithelial cells increases their tumorigenic potential by increasing cellular adhesiveness and resistance to apoptosis (19). Overexpression of COX-2 in tumors, and the consequent increase in prostaglandin secretion, may alter the release of cytokines such as IL-10 and IL-12 from lymphocytes, macrophages and the tumors' own cells, resulting in the repression of the host immune response (20). Recent work also indicates that cells overexpressing COX-2 secrete pro-angiogenic factors that may contribute to blood vessel formation in tumors (21).
In addition to being expressed in normal A/J type II cells, expression of the COX-2 protein was frequently seen in hyperplasias in this animal model. This observation, combined with the significant decrease in the number of NNK-induced lung tumors following pre-/early treatment of the A/J mouse with a specific inhibitor of COX-2 activity (8), supports a role for COX-2 in the earliest stages of tumor progression and makes COX-2 a promising target for chemoprevention studies. COX-2 expression has also been reported in early hyperplastic lesions in the human lung (6,7), linking an elevation of this protein to the earliest stages of human lung adenocarcinoma development. Conflicting data have been published regarding COX-2 expression in normal human alveolar epithelial cells (6,7,20), indicating a need for additional investigations.
In our studies of NNK-induced A/J mouse lung tumors, COX-2 expression was more variable and focal in location within adenocarcinomas than observed within hyperplasias. This variation in COX-2 expression has been observed in other types of cancers, including human pancreatic adenocarcinoma, murine skin carcinoma and human breast carcinoma (2224). Most significantly, such variability has also been observed in human lung adenocarcinoma (6,7), further supporting the use of the A/J mouse model for the study of human lung cancer. In cell culture COX-2 expression has proved to be highly responsive, changing rapidly in the presence of many growth factors, cytokines and other inflammatory mediators. For this reason it is not surprising that in some tumors and tumor-regions COX-2 expression becomes down-regulated. Loss of COX-2 expression in some tumors as a result of tumor progression does not alter the value of COX-2 inhibition for lung cancer chemoprevention since, by definition, the target of chemopreventive compounds are early lesions. In fact, examination of the mechanism(s) of such down-regulation could lead to the design of new chemopreventive strategies.
In preliminary chemopreventive studies, two A/J lung tumor-derived cell lines that expressed moderate to high levels of COX-2 were grown s.c. in nude mice and were then treated with COX-2 inhibitors. The repression in growth may have been due, in part, to an increase in apoptosis. Although PDTC, an inhibitor of transcription, was essential for maximum growth repression by SC-236, decreased transcription of COX-2 was not detected. The repression of xenograft growth by PDTC/SC-236 was not as dramatic as seen for xenografts derived from rat intestinal epithelial cells and human colon cancer-derived cell lines (12,25,26). It is possible that the COX-2 inhibitor used in those studies (SC-58125) was more effective, or the appropriate dose of SC-236 for inhibiting this pathway in lung tumor-derived xenografts was not obtained. Another possibility for the lack of significant growth repression of the two lung adenocarcinoma-derived cell lines is that later-stage lung tumors are not dependent on COX-2, a hypothesis supported by the previously discussed variability of COX-2 expression in A/J lung adenocarcinomas. Thus, future chemopreventive studies targeting the lung will be aimed at inhibiting COX-2 during the earliest stages of tumor development. This approach is supported by one study to date in which pre-/early treatment with NS-398, a specific inhibitor of COX-2, was shown to be effective in reducing tumor number in A/J mice initiated with NNK (8).
In a study of six different colon cancer cell lines, only the two cell lines with the highest levels of COX-2 expression showed any repression of proliferation in response to COX-2 inhibitor treatment (27). Similarly, in our studies the lung-tumor-derived cell line that expresses high levels of COX-2 protein (Spon4) showed a partial response, whereas the CL30 cell line that expresses much lower protein levels was refractory. This may provide insight into predicting the response of premalignant lesions to these inhibitors. If efficacy is directly correlated to protein levels, this would further support preneoplasia as a target, where high levels of COX-2 protein are more often seen. The A/J mouse model and derived tumor cell lines should be invaluable for understanding the biology associated with inhibition of COX-2 and for validating this enzyme as a target for chemoprevention of human adenocarcinomas.
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Notes |
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Acknowledgments |
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References |
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