A differential proteomics approach to identify proteins involved in lung carcinogenesis induced by a mixture of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) and chemoprevention of indole-3-carbinol (I3C) in A/J mice.

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Lung cancer is the most common cause of cancer death in the USA and worldwide. Although primary prevention through cessation of smoking and prevention of smoking initiation is the best approach to reduce lung cancer mortality, secondary prevention using chemopreventive agents could also prevent or reverse the carcinogenesis process. The first step in identifying chemopreventive agents is thorough understanding of the molecular events that take place during carcinogenesis. To assess carcinogen-induced alteration in protein expression and possible modulation of the effect by the chemopreventive agent indole-3-carbinol (I3C), groups of A/J mice were given eight weekly doses of a mixture of B[a]P plus NNK (2 µmol each) in 0.1ml cottonseed oil, cottonseed oil only or the carcinogens and I3C (112 µmol/g diet beginning 50% of carcinogen treatment until sacrifice). Mice were sacrificed 19 weeks after the last dose of carcinogen and tumor incidence and multiplicity were determined. Compared to the group treated with the carcinogens only, tumor multiplicity in the carcinogens plus I3C group was reduced by 77%; no effect was found on tumor incidence. In order to identify proteins that could be differentially expressed, lung homogenates were prepared from pooled lungs (five lungs/group) of each of the three groups (untreated control, carcinogen-treated and carcinogens plus I3C-treated) and processed using the novel proteomics technique, isobaric technique for relative and absolute quantitation (iTRAQ). In each of the three iTRAQ runs with the same pooled sample, we identified and quantified at least 256 proteins with more than one peptide and > 95% confidence. Treatment with a mixture of NNK plus BaP up-regulated the expression of fatty acid synthase, transketolase, L-plastin, annexin A1, haptoglobin and pulmonary surfactant protein C whereas transferrin, alpha-1-antitrypsin precursor and apolpoprotein A-1 were down-regulated. Interestingly, dietary administration of I3C to carcinogen-treated mice decreased the expression of L-plastin, annexin A1, haptoglobin and pulmonary surfactant protein C to the level of the untreated control. We verified these results using Western immunoblotting. Expression of cytochrome P450 2F and glutathione S-transferase mu 1 was up-regulated in the group treated with I3C. Overall, we showed that (1) I3C chemoprevents lung carcinogenesis induced by a mixture of NNK plus BaP, (2) the expression level of nine proteins was altered upon treatment with the carcinogens and (3) dietary feeding of I3C reduced carcinogen-induced up-regulation of L-plastin, annexin A1, haptoglobin and pulmonary surfactant protein C. The chemopreventive effect of I3C might be explained, at least partly, by its effect on these proteins.