pulmicort and vinyl-carbamate

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at Week 20. At Week 20, the rank order for prevention of lung tumors was the combined treatment > budesonide > R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate end-point biomarker for prevention.

Topics: Animals; Anti-Inflammatory Agents; Budesonide; Chemoprevention; Disease Models, Animal; DNA Methylation; Drug Therapy, Combination; Female; Lung Neoplasms; Mice; Mice, Inbred A; Quinolones; Urethane

Biomarkers are being developed that can aid in the evaluation of cancer therapeutic and chemopreventive drugs. Two suggested biomarkers found in mouse lung tumors are DNA hypomethylation and alterations in mRNA expression of genes, such as 18S RNA, caspase 3, cyclin B2, cyclin E1, iNOS and survivin. Budesonide is very efficacious in preventing lung tumors in mice, so that its ability to modulate biomarkers in lung tumors was determined. Lung tumors were induced by vinyl carbamate in female strain A/J mice. Budesonide (2.0 mg/kg diet) was administered for 2, 7 and 21 days or for 14 days followed by a 7-days' holding period prior to the killing of the mice at week 27. After 2 days of budesonide treatment, the size of the lung tumors was reduced. Tumor size continued to decrease during the 21 days of treatment. In the tumors, 2 days of treatment resulted in (i) increased methylation of DNA, reversing DNA hypomethylation, (ii) increased expression of 18S RNA and (iii) decreased mRNA expression of caspase 3, cyclin B2, cyclin E1, iNOS and survivin. Termination of budesonide treatment at 7 days prior to killing did not affect the size of the tumors, but did result in increased mRNA expression of the 5 genes, approaching the expression level in tumors from control mice. Hence, budesonide rapidly decreased the size of lung tumors, reversed DNA hypomethylation and modulated mRNA expression of genes; with the molecular alterations requiring continued treatment with the drug for maintenance.

Topics: Animals; Anti-Inflammatory Agents; Biomarkers, Tumor; Body Weight; Budesonide; Caspase 3; Cell Proliferation; Cyclin B; Cyclin B2; Cyclin E; DNA Methylation; Female; Gene Expression; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Mice; Mice, Inbred Strains; Microtubule-Associated Proteins; Nitric Oxide Synthase Type II; Repressor Proteins; RNA, Messenger; RNA, Ribosomal, 18S; Survivin; Urethane

CpG endonuclease activity was identified in nuclear extracts obtained from mouse lung tumors. Enzyme activity was determined using a 333 bp polymerase chain reaction product of the estrogen receptor-alpha gene that contained either radiolabeled cytosine or tritium-labeled methyl groups at CpG sites. Activity was measured as the release of radioactivity from the substrate. The product of the nuclease activity was identified by high pressure liquid chromatography (HPLC) as either 5-methyl-2'-deoxycytidine when the CpG sites in the substrate were methylated or 2'-deoxycytidine when the CpG sites were not methylated. The CpG endonuclease activity was dependent on nuclear protein and temperature, had a proclivity for double-stranded over single-stranded DNA and was inhibited by ethylenediaminetetraacetic acid or 2-mercaptoethanol. Strain A/J mouse lung tumors induced by vinyl carbamate had a greater level of CpG endonuclease activity than non-involved lung tissue. Budesonide, a potent chemopreventive agent in mouse lung, not only prevented an increase in CpG endonuclease activity in lung tumors but, when administered to mice with established tumors, also decreased the level of endonuclease activity in the tumors. The effect of budesonide on CpG endonuclease activity in lung tumors was inversely related to its published effect on DNA methylation in mouse lung tumors, i.e. the drug decreased CpG endonuclease activity and increased the methylation of DNA. The increased CpG endonuclease activity in mouse lung tumors and its inhibition by budesonide would suggest this endonuclease as a potential molecular target for chemoprevention.

Topics: Animals; Anti-Inflammatory Agents; Budesonide; CpG Islands; DNA Methylation; Endonucleases; Enzyme Activation; Female; Lung Neoplasms; Mice; Urethane

Lung and colon tumors were induced in A/J, C3H, and A/J X C3H (AC3) mice by administering 16 mg/kg vinyl carbamate followed by 6 weekly doses of 12 mg/kg azoxymethane (AOM). Beginning 1 week after carcinogen treatment, the mice received chemopreventive agents, dexamethasone or piroxicam, at 0.1 and 75 mg/kg in the diet, respectively. Both AOM and vinyl carbamate induces lung tumors, but only AOM induced colon tumors. The strain sensitivity for both colon and lung tumors was A/J > AC3 > C3H mice. Dexamethasone and piroxicam reduced the multiplicity of colon and lung tumors in A/J and AC3 mice, demonstrating the advantage of a combined colon and lung bioassay. The ability of budesonide, a drug that prevents mouse lung tumors, to modulate DNA methylation in vinyl carbamate-induced lung tumors was also determined. Budesonide administered for only 7 days prior to sacrifice caused a dose-dependent (0.6 to 2.4 mg/kg diet) reversal in tumors of DNA hypomethylation and hypomethylation of the insulin-like growth factor (IGF)-II gene in the differentially methylated region (DMR) 2 region of exons 4 to 5. Longer treatment with budesonide reversed hypomethylation when administered up to the time of sacrifice. These results indicate that reversal of the hypomethylation of DNA and of specific genes in lung tumors may be applicable as a surrogate end-point biomarker for chemoprevention.

Topics: Animals; Azoxymethane; Biological Assay; Biomarkers; Budesonide; Carcinogens; Chemoprevention; Colonic Neoplasms; Dexamethasone; DNA Methylation; Dose-Response Relationship, Drug; Glucocorticoids; Insulin-Like Growth Factor II; Lung Neoplasms; Mice; Mice, Inbred Strains; Piroxicam; Urethane

Chemopreventive drugs have the potential to decrease the morbidity and mortality of lung cancer. The development of these drugs could be expedited by the application of surrogate end-point biomarkers that demonstrate chemopreventive efficacy. In this study, the ability of budesonide to prevent lung tumors in mice was characterized further and its effects on biomarkers were determined. Lung tumors were induced in female strain A mice by vinyl carbamate (16 mg/kg) administered once weekly for 2 consecutive weeks. Four weeks later the mice started to receive 0.6, 1.2 or 2.4 mg/kg budesonide continually in the diet until killed at week 20. Budesonide caused a dose-dependent decrease in the multiplicity of lung tumors of 25, 58 and 82%, respectively. Budesonide (2.4 mg/kg diet) administered starting at weeks 4, 10 or 16, decreased tumor multiplicity by 82, 66 and 30% at week 20. Administering 2.4 mg/kg budesonide at weeks 4-20 or 20-35 and killing the mice at week 35 did not significantly decrease the yield of tumors, although both treatment regimens did decrease the size of the tumors and the progression of adenomas to carcinomas. Thus, budesonide delayed the appearance of lung tumors and decreased their growth and progression to carcinomas. To determine the effect of limited exposure to budesonide on biomarkers, it was administered for only 7 days prior to death at week 35. Budesonide decreased the proliferating cell nuclear antigen labeling in lung adenomas, carcinomas, parenchyma and bronchial airways by 87.6, 59.0, 41.1 and 25.4%, respectively. Budesonide treatment also increased the protein level of the p21 and p27 genes and increased the mRNA level of p21. Thus, short-term treatment with budesonide modulated biological and molecular end-points in lung tumors that might be developed further as biomarkers for its clinical chemopreventive efficacy in the lung.

Topics: Adenocarcinoma; Adenoma; Animals; Anticarcinogenic Agents; Biomarkers, Tumor; Budesonide; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Primers; Dose-Response Relationship, Drug; Female; Lung Neoplasms; Mice; Mice, Inbred A; Microfilament Proteins; Muscle Proteins; Polymerase Chain Reaction; RNA, Messenger; Urethane