The nicotinic receptor antagonists abolish pathobiologic effects of tobacco-derived nitrosamines on BEP2D cells

Abstract

Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.

Fig. 1

RT-PCR analysis of nAChR subunit expression in BEP2D cells. The cells were grown to ∼80% confluence and used for total RNA extraction. The RT-PCR was performed as described in Sect. ”Materials and methods” using primer pairs and annealing temperature indicated in Table 1. Left lane is a 100-bp molecular weight ladder. C1 is negative control (no cDNA). C2 is β-actin used as a positive control for RNA integrity. The 838-bp product was visualized using primers purchased from BioChain Institute Inc. (Hayward, CA, USA). Amplification of PDH (C3) yielded only a single band of 101 bp, confirming the absence of genomic DNA. C4 is a 500-bp product obtained with control primers provided within First-Strand cDNA Synthesis System (Invitrogen Corporation, Carlsbad, CA, USA) to verify performance of first-strand synthesis

Fig. 2

Displacement curves of NNK and NNN with [³H]nicotine (a) and [³H]epibatidine (b). The numbers on the ordinate indicate the log molarity of the NNK, and NNN solutions used in the radioligand binding inhibition assays with the monolayers of BEP2D cells, as described in the Sect. ”Materials and methods”. The EC₅₀ values reported in the Sect. ”Results” were determined from the displacement curves

Fig. 3

Quantitative analysis of the effects of NNK and NNN on gene expression in BEP2D cells. BEP2D cells were incubated for 24 h in culture medium without any additions (control) or in the presence of 1 μM of NNK or NNN with or without the antagonists αBtx, 1 μM, or Mec, 50 μM. The alterations in gene expression are presented relative to the rates of expression of corresponding genes in control samples, taken as 1. *P<0.05 compared to the same nitrosamine given alone. a. Real-time PCR analysis. b In-cell western analysis

Fig. 4

Assessment of nitrosamine effects on GATA-3 activity using electrophoretic mobility shift assay. The GATA-3 DNA–protein complex (marked GATA-3 complex) was detected by gel mobility shift assay with nuclear extracts prepared from BEP2D cells that were either untreated, or treated with NNK or NNN in the presence or absence of antagonists as described in the legend to Fig. 3. UnO denotes unbound oligonucleotide