Suppressive oligodeoxynucleotides reduce lung cancer susceptibility in mice with silicosis

Abstract

Silicosis is an inflammatory lung disease induced by the inhalation of silica-containing dust particles. There is conflicting data on whether patients with silicosis are more susceptible to lung cancer induced by cigarette smoke. To examine this issue experimentally, a model was developed in which one of the most abundant and potent carcinogens present in cigarette smoke [4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] was administered to mice at the peak of silica-induced pulmonary inflammation. Results show that the incidence of lung tumors in silicotic mice treated with NNK was significantly increased compared with mice exposed to silica or NNK alone. Synthetic oligonucleotides (ODN) containing repetitive TTAGGG motifs can block pathologic inflammation. We therefore examined whether treatment with these suppressive (Sup) ODN could block silica-induced pulmonary inflammation and thereby reduce susceptibility to lung cancer. Results show that Sup (but not control) ODN inhibit pulmonary fibrosis and other inflammatory manifestations of chronic silicosis. Of greater import, Sup ODN reduced lung tumor incidence and multiplicity in silicotic mice exposed to NNK. These findings establish an experimental model for examining the role of silicotic inflammation in cancer susceptibility and demonstrate that Sup ODN represent a novel therapy for chronic silicosis.

Fig. 1.

Mice treated with NNK plus silica develop lung tumors. Naive A/J mice (○, N = 15) were treated with 1mg of crystalline silica alone (▿, N = 10), 500 μg of NNK alone (□, N = 15) or silica followed by NNK (◆, N = 15, see Materials and methods for details). Lungs were fixed in formalin and analyzed for tumor formation 15–25 weeks posttreatment. Results represent the mean + standard error of the mean (SEM) of all animals per group. Statistic significance was determined by two-way analysis of variation. *P < 0.05; **P < 0.01.

Fig. 2.

Effect of NNK plus silica on pulmonary inflammation. Naive A/J mice (open bar) were exposed to NNK (cross-hatched bar) or NNK plus silica (solid bar). Bronchoalveolar lavage and lung tissue were collected 16 weeks later. (A) The accumulation of inflammatory leukocytes in bronchoalveolar lavage was determined histologically. (B) The expression of genes encoding proinflammatory cytokines in lung tissue homogenates was quantified by reverse transcription–PCR (B). Relative cytokine mRNA levels were determined by comparison with untreated controls after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results represent the combined mean ± SEM of 2–3 independent experiments involving 5–9 mice per group. *P < 0.05; **P < 0.01.

Fig. 3.

Association between IL-1β gene expression and lung tumor development. Mice were treated with NNK plus silica. At 16 weeks, the incidence of lung tumors was quantified histologically, whereas cytokine mRNA levels were measured by reverse transcription–PCR in lung tissue homogenates. Tumor multiplicity versus IL-1β mRNA levels (normalized to GAPDH) from mice in four independent experiments (N = 23) is shown. The solid trend line and P value were calculated using the maximum likelihood estimate derived from a Poisson linear analysis. Note that IL-1β mRNA levels did not vary as a function of tumor multiplicity in mice treated with NNK alone (a model independent of pulmonary inflammation).

Fig. 4.

Effect of Sup ODN on proinflammatory cytokine production in mice treated with NNK plus silica. Mice were treated with NNK plus silica as described in Figure 1. They were then injected i.p. with PBS (N = 16), 300 μg of Sup ODN (N = 20) or 300 μg of control ODN (N = 16) once per week for 4 weeks, starting on day 0. TNFα and IL-1β mRNA levels were measured by reverse transcription–PCR on tissue homogenates at 4 months. Bars show cytokine mRNA levels relative to naive mice (all values normalized to GAPDH). Results from the PBS and control ODN-treated groups were indistinguishable and thus combined to increase statistical power. Each bar represents the mean ± SEM of data combined from three independent experiments. *P < 0.05, **P < 0.01.

Fig. 5.

Effect of Sup ODN on chronic silicosis. Mice were treated with NNK plus silica and then injected with PBS, Sup or control ODN as described in Figure 4. Lung tissue collected at wk 16 was homogenized and examined for (A) IL-10 mRNA levels by reverse transcription–PCR and (B) collagen levels by the Sircol collagen assay. (C) Changes in body weight were measured over 16 weeks. Data represent the combined mean ± SEM of two independent experiments involving greater than or equal to 10 mice per group. Results from the PBS and control ODN-treated groups were indistinguishable and thus combined to increase statistical power. Differences in body weight between groups were analyzed by two-way analysis of variation. *P < 0.05, **P < 0.01

Fig. 6.

Effect of Sup ODN on lung tumor development in mice treated with NNK + silica. (A) Mice were treated with NNK plus silica and then injected with PBS (N = 16), Sup ODN (N = 20) or control ODN (N = 16) as described in Figure 4. (B) Mice were treated with 2000 μg of NNK alone and then injected with PBS (N = 10), Sup ODN (N = 20) or control ODN (N = 10). At 16 weeks, all lungs were analyzed histologically for the presence of tumors. Results from mice treated with PBS or control ODN were indistinguishable and thus combined to increase statistical power. Statistical analysis of tumor incidence was performed using chi square test. *P < 0.05.