Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation
ABSTRACT Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 muM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.
- SourceAvailable from: Marta Irigoyen
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- "Cells were then washed with Tris-buffered saline (TBS), fixed in TBS containing 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 5 min, and then washed three times with TBS. Immunofluorescence against neuronal cell adhesion molecule (NCAM), neurofilament M (Nef-M), and Neu-N was performed as described previously (Martínez-García et al., 2008). "
ABSTRACT: Cigarette smoking is strongly correlated with the onset of lung cancer. Nicotine, a major component in cigarette smoke, has been found to promote tumor growth and angiogenesis, as well as protect cancer cells from apoptosis. Among all lung cancer cases, small cell lung cancer (SCLC) is found almost exclusively in smokers; metastasis and chemoresistance are the main reasons for the high mortality rates associated with SCLC. Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnosis display lower response rates and a shorter median survival compared with those who stop smoking. In the current work, we examined the effects of acute and repetitive exposure to nicotine, in the concentrations found in the lungs of active smokers, on the malignant properties of N417 SCLC cells in vitro. We observed that repetitive nicotine exposure induced a neuronal-like appearance in N417 cells along with increased adhesion to the extracellular matrix and chemoresistance. These changes were accompanied by enhanced migration through collagen matrices and adhesion to and transmigration across lymphatic endothelial cell monolayers. SCLC differentiation reverted after cessation of nicotine exposure. Here, we provide evidence for the leading role of the CXCR4/CXCL12 axis in these phenomena. Finally, we show how nicotine-differentiated N417 cells produced bigger and more vascularized tumors in mice, with lower apoptotic rates, than their nondifferentiated counterparts. In short, these findings identify the mechanisms through which nicotine increases SCLC malignancy and provide further evidence that CXCR4 is a potential anticancer target for nicotine-associated SCLC. Lung cancer is a major health issue worldwide (Ian Bray and Weiderpass, 2010; Parkin et al., 2005); it causes approximately 1.2 million deaths per year. Small cell lung cancer (SCLC) accounts for approximately 20% of total lung cancer– associated deaths. This type of tumor is characterized by rapidToxicological Sciences 08/2010; 116:467-476. · 3.85 Impact Factor
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ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are the central regulators of stimulatory and inhibitory neurotransmitters that control the synthesis and release of growth, angiogenic and neurotrophic factors in cancer cells, the cancer microenvironment and distant organs. Data discussed in this Review suggests that smoking and possibly other environmental and lifestyle factors increase the function of nAChRs that stimulate cancer cells and reduce the function of nAChRs that inhibit cancer cells. This novel paradigm necessitates the development of marker-guided cancer intervention strategies that aim to restore the balance between nAChR-mediated stimulatory and inhibitory neurotransmitters and their downstream effectors.Nature Reviews Cancer 03/2009; 9(3):195-205. DOI:10.1038/nrc2590 · 37.40 Impact Factor
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ABSTRACT: To determine whether antiproliferative factor (APF) or epidermal growth factor (EGF) can induce changes in purinergic signaling in normal bladder urothelial cells (BUCs) and/or whether antagonizing EGF activity or blocking adenosine triphosphate (ATP)-purinergic receptors can induce changes in purinergic signaling in interstitial cystitis (IC) cells. IC and normal BUCs were obtained from patients' bladder biopsy specimens. IC BUCs were treated with genistein, which antagonizes EGF's activity, and normal BUCs were treated with EGF, mock APF, or APF. Suramin, which antagonizes ATP activity, was used to treat the APF-treated normal BUCs. ATP release was determined by stimulating the BUCs with 30 microM ATP and then collecting the supernatant for a 3-hour period. ATP quantification was measured by luciferin-luciferase assay. Purinergic receptor P2X, ligand-gated ion channel, 3 (P2X3) expression on BUCs was determined by fluorescence-activated cell sorting. Genistein treatment of IC BUCs resulted in significantly decreased ATP release, thus reverting IC cells to a normal purinergic signaling phenotype. Conversely, normal BUCs treated with EGF or APF resulted in significantly increased ATP release and P2X3 expression, converting normal BUCs to an IC phenotype. Also, suramin treatment of APF-treated normal BUCs significantly reduced ATP release. Genistein and suramin reversed the augmented ATP release in IC BUCs and APF-treated normal BUCs, respectively, suggesting the possibility of intravesical use of these agents in IC treatment. EGF and APF induced augmented purinergic signaling in normal BUCs, as determined by increased ATP release and increased P2X3 expression. These data suggest an association between cytokines and purinergic signaling in human BUCs that should be explored further.Urology 08/2009; 74(5):1163-8. DOI:10.1016/j.urology.2009.02.066 · 2.19 Impact Factor