Down-regulation of 14-3-3 isoforms and annexin A5 proteins in lung adenocarcinoma induced by the tobacco-specific nitrosamine NNK in the A/J mouse revealed by proteomic analysis

Abstract

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in the A/J mouse model. Here we identified and validated, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry and immunoblotting, proteins that are differentially expressed in the lungs of mice treated with NNK versus vehicle control treatment. We also determined whether protein levels in the lungs of NNK-treated mice could be further modulated by the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). The proteins identified in this study are SEC14-like 3, dihydropyrimidinase-like 2, proteasome subunit alpha type 5, annexin A5, 14-3-3 protein isoforms (theta, epsilon, sigma, and zeta), Rho GDP dissociation inhibitor alpha, myosin light polypeptide 6, tubulin-alpha-1, vimentin, Atp5b protein, alpha-1-antitrypsin, and Clara cell 10 kDa protein (CC10). Among those proteins, we demonstrated for the first time that 14-3-3 isoforms (theta, epsilon, and sigma) and annexin A5 were significantly down-regulated in mouse lung adenocarcinoma induced by NNK and were recovered by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis. Identification of these proteins in surrogate tissue in future studies would be highly useful in early detection of lung adenocarcinoma and clinical chemoprevention trials.

Figure 1

Proteomic profiling of the A/J mouse lung using the 2D-DIGE approach to identify differentially regulated proteins in NNK-treated and (NNK + p-XSC)-treated mice. Representative images of a 2D-DIGE gel scanned for different CyDye fluorophores illustrating fractionated lung proteins from different groups. (A) The vehicle-treated control sample was labeled with Cy5 (red). (B) The NNK-treated sample was labeled with Cy3 (green). (C) An internal pool standard, consisting of all samples in the study, was labeled with Cy2 (blue). (D) Automatic and manual spot detection led to the identification of 796 protein spots outlined in red.

Figure 2

(A) Principal Components Analysis (PCA) of 191 significant differentially expressed protein spots with either carcinogenesis or chemoprevention-based relationships. The two greatest study variances between the three groups are represented on the x- and y- axes (x-axis, 1^st principal component, 92%; y-axis, 2^nd principal component, 8%). Each spot represents the compressed weighted average of the principal components of the 191 proteins for each sample in each treatment group; the vehicle-treated control group (shaded circle), the NNK-treated group (open circle), and the (NNK + p-XSC)-treated group (closed circle). (B) Protein expression heat map for 191 protein spots that were differentially regulated between the three treatment groups (Vehicle-treated Control, NNK-treated, and (NNK + p-XSC)-treated). Up-regulated proteins are in red and down-regulated proteins are in green. Hierarchial clustering of protein expression was performed in two dimensions. The first dimension (top dendrogram) shows the similarity in protein expression profiles of the groups and the second dimension (left hand side dendrogram) shows individual protein expression profiles.

Figure 3

2D-DIGE reference gel image. A selected group of protein spots (indicated by arrows, total number 16) that were differentially regulated were excised and identified using mass spectrometry methods.

Figure 4

Identification of spot #391 (14-3-3 ε). (A) Representative MALDI-ToF/ToF MS and tandem MS spectra used in the identification of the 14-3-3 ε proteins. The peptide sequence YLAEFATGNDR was unique to the 14-3-3 ε protein. (B) 2D-DIGE gel images of 14-3-3 ε expression for all three experimental groups. The three dimensional and graphical presentations of the normalized spot volume for the 14-3-3 ε protein illustrates the expression differences between the vehicle-treated control, NNK, and (NNK + p-XSC)-treated groups. *p<0.05; **p<0.005 (Student’s t-test)

Figure 5

Histopathological analysis of H&E stained NNK-induced lung tumors in the A/J mouse. (A) Tumor section surrounded by normal tissue; tumors were classified as adenocarcinomas, papillary type (magnification, X100). (B) Magnified region of box in (A) (magnification, X600); arrows, indicating multinucleate tumor cells; arrowheads, highlighting variation in nuclear size (anisokaryosis), a feature of dysplasia.

Figure 6

Validation of 14-3-3 isoforms θ, ε, and σ, CC10, and annexin A5 under-expression by immunoblot analysis. (A) Representative immunoblot analysis, from three experiments, of expression of 14-3-3 θ, ε, and σ, CC10, and annexin A5 in dissected NNK-induced lung adenocarcinomas and vehicle-treated control lung tissue. Forty micrograms of total lung protein were loaded into each lane for each sample. (B) Fold-change in mean protein band expression from immunoblot for 14-3-3 θ, ε, and σ, CC10, and annexin A5 in lung tissue from mice treated with vehicle (solid bars) or tumors from mice treated with NNK (open bars). (C) Representative immunoblot analysis, from three experiments, of expression of 14-3-3 θ, ε, and σ, CC10, and annexin A5 in vehicle-treated lung tissue and (NNK + p-XSC)-treated lung tissue. Forty micrograms of total lung protein were loaded into each lane for each sample. (D) Fold-change in mean protein band expression from immunoblot for 14-3-3 θ, ε, and σ, CC10, and annexin A5 in lungs from mice treated with vehicle (solid bars) or (NNK + p-XSC) (gray bars). For comparison between protein expression levels between groups, the fold change was calculated relative to vehicle-treated control levels and normalized with respect to β-actin protein expression levels. ^* p<0.05 (Student’s t-test)

Figure 7

Network Analysis of differentially expressed proteins using the Ingenuity Pathways Analysis (IPA) software. Proteins that were found to be significantly different between lungs of mice treated with the vehicle control versus NNK were analyzed using IPA. The proteins were classified in a cancer, tumor morphology, and cell morphology associated network, along with corresponding protein-to-protein direct (solid lines) or indirect (dashed lines) interactions/regulations, based on information published in the literature. The proteins shown in green were down-regulated and those in red were up-regulated in the NNK-treated group compared to the vehicle-treated control group. Proteins were given shapes related to functionality and were also grouped with respect to cellular location: extracellular space, plasma membrane, cytoplasm, and nucleus.