Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-κB activation in normal human bronchial epithelial cells

Abstract

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (−)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (20–80 μ M) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-κB (NF-κB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-κB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IκBα, and activation and nuclear translocation of NF-κB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-κB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-κB-regulated proteins cyclin D1, MMP-9, IL-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-κB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.

Introduction

The negative health consequences of smoking are widely recognized, but smoking still remains the number one cause of preventable death in developed countries. Epidemiological evidence confirms that exposure to cigarette smoke, a complex mixture of more than 4000 particulate and volatile components, increases the incidence of lung carcinogenesis, a leading cause of cancer deaths in the US and other developed countries (Sasco et al., 2004). The risk of lung cancer development is 20–40 times higher in lifelong smokers compared to non-smokers accounting for 90% of male and 79% of female lung cancers (Ozlu and Bulbul, 2005). Airway inflammation is ubiquitous in the lungs of smokers, regardless of the presence or absence of lung disease. Inhaled smoke may induce airway inflammation locally as well as causing systemic side effects. A strong correlation between inflammation and cancer has long been suggested (Coussens and Werb, 2002), and it is now clear that the tumor microenvironment, which is largely orchestrated by inflammatory cells, contributes to the neoplastic process, fostering proliferation, survival and migration. Finally, changes in the structure and function of the bronchial epithelium as a result of exposure to the carcinogens present in cigarette smoke may result in aberrant proliferation and differentiation of these cells, leading to the development of respiratory diseases, including lung cancer (Wistuba et al., 2002).

NF-κB is one of the key transcription factors involved in the inflammatory responses to cigarette smoke in the lungs. NF-κB activity is regulated by cytoplasmic degradation of the IκB inhibitor. NF-κB dimers localize to the nucleus, once IκBα is inactivated and undergo further modification, mostly through phosphorylation of the Rel proteins (Sizemore et al., 2002). In the nucleus activated NF-κB binds to promoters of its target genes and regulates the expression of genes involved in immune responses, inflammation and cell survival (Lin and Karin, 2003). There is evidence that cigarette smoke induces AKT-dependent proliferation and NF-κB-dependent survival of lung cancer cells (Tsurutani et al., 2005). Many of the agents that activate MAPKs can also activate NF-κB suggestive of the involvement of multiple kinases in the activation of NF-κB. It is known that the SAPK and p38 pathways are regulated by stress signals such as the pro-inflammatory cytokines, TNF-α or IL-1 (Kyosseva, 2004). ERKs on the other hand, are activated by growth signals resulting in the formation of well-defined signaling complexes at the membrane which then culminates in either cell proliferation or differentiation dependant on the cell type (Kyosseva, 2004).

Epigallocatechin-3-gallate (EGCG) comprises approximately 60% of the catechins in tea, and has been shown to have a protective effect against a variety of malignant proliferative disorders such as lung cancer, breast cancer and prostate cancer (Doss et al., 2005). Various mechanisms have been elucidated for its cancer protective activity that include inhibition of metabolic activation of carcinogens along with concomitant induction of detoxifying enzymes and/or through inhibition of signaling pathways that control cell proliferation and tumor growth (Yang et al., 2005). Even with the abundance of literature investigating the inhibitory role of EGCG, most studies have been carried out in already established models of lung carcinogenesis. However, it is well known that once tumor progression has occurred it is an irreversible process with irretrievable structural changes in the airways. It is therefore important to study the effect of EGCG on primary lung cells exposed to cigarette smoke in order to assess the relevant changes that may have a role in the development of lung tumors. Here, we provide evidence of the inter-relationships between the signaling networks activated by CSC-induced stress and the anti-proliferative effect of EGCG in normal human bronchial cells (NHBE). In addition, our studies in nonmalignant human bronchial cell culture as a model system can give clear and more specific information about the molecular mechanisms involved in smoking-induced lung disease.

Results

CSC exposure to NHBE results in increased NF-κB DNA-binding activity

Cigarette smoke contains several carcinogens known to initiate and promote tumorigenesis as well as metastasis. NF-κB and its regulators are linked to various signal transduction pathways as well as transcriptional activation events that regulate critical stages of cell proliferation (Philip et al., 2004). It has been reported that nicotine from cigarette smoke can promote survival of many cell types including lung cancer cells through activation of NF-κB (Tsurutani et al., 2005). To investigate the effect of varying doses of CSC on NF-κB DNA-binding activity in NHBE, cells were treated with different concentrations of CSC (5–20 μg/ml) for 30 min, nuclear extracts were prepared and NF-κB DNA-binding activity examined by EMSA. As shown in Figure 1a, treatment of the NHBE cells with CSC for 30 min resulted in a dose-dependent increase in the NF-κB DNA-binding activity. As, it is evident from the data that CSC exposure even at lower doses can result in NF-κB DNA-binding activity, thereafter we selected a CSC dose of 10 μg/ml for our further studies.

Figure 1
figure1

EGCG inhibited CSC-induced activation of NF-κB in NHBE cells. (a) CSC exposure results in increased NF-κB DNA-binding activity in NHBE. NHBE cells were treated with 5–20 μg/ml CSC for 30 min. Nuclear extracts were prepared, and assayed for NF-κB transcriptional activity by EMSA as described in Materials and methods. The data shown here are from representative experiment repeated two times with similar results. (b) EGCG treatment of NHBE inhibits CSC-induced NF-κB DNA-binding activity: Cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h, and then exposed to 10 μg/ml CSC for 30 min, nuclear extracts were prepared and assayed for NF-κB transcriptional activity by EMSA. The data shown here are from representative experiment repeated three times with similar results. (c) EGCG inhibited CSC-induced NF-κB/p65 activity. The effect of EGCG on NF-κB/p65 was evaluated using ELISA as detailed in Materials and methods. Results were expressed as means±s.d. (d) EGCG inhibited CSC-induced phosphorylation of NF-κB/p65, as shown by an increase in the total cytosolic NFκB/p65 levels with a concomitant decrease in nuclear NF-κB/p65 levels in EGCG treated lanes. Cytosolic and nuclear extracts were prepared, as detailed earlier, for immunoblot analysis. Equal protein loading was evaluated by β-actin and lamin. (e) EGCG inhibited CSC-induced nuclear translocation of NF-κB/p65. NHBE cells were seeded in tissue culture glass slides and treated with CSC with/without EGCG (40 μ M). Briefly, cells were fixed in 2% Paraformaldehyde, and incubated with NF-κB/p65 antibody followed by incubation with donkey anti-rabbit Rhodamine Red TM-X-conjugated antibody. Samples were mounted using Prolong antifade kit and observed using a Zeiss Axiophot DM HT microscope. NF-κB/p65 translocation was detected in the nuclei of cells exposed to CSC alone whereas EGCG treatment effectively inhibited this translocation as determined by significant diminution in nuclear p65 immunostaining. (f) EGCG inhibited NF-κB-dependent reporter gene expression induced by CSC. NHBE cells were seeded at a concentration of 1.5 × 105 cells per well in six-well plates and co-transfected with NF-κB-driven luciferase reporter construct and pSV40-β-gal plasmid. After 48 h, cells were treated with CSC with or without EGCG and Luciferase activity was measured and normalized with respect to β-galactosidase activity. Results were expressed as means±s.d.

EGCG treatment of NHBE inhibits CSC-induced NF-κB DNA-binding activity

While low concentrations of EGCG have been shown to inhibit DNA damage induced by reactive oxygen and nitrogen species, higher concentrations of the compound may itself result in damage to cellular DNA (Johnson and Loo, 2000). We first determined the effect of EGCG on cell viability by performing MTT assay. EGCG pretreatment to NHBE cells for 24 h, at doses 20–80 μ M had an insignificant effect on cell viability, as assessed by MTT assay (data not shown). Next, to determine the effect of EGCG on the CSC-mediated transcriptional activation of NF-κB, NHBE cells were treated with different concentrations of EGCG (20–80 μ M) for 4 h and then exposed to CSC (10 μg/ml) for 30 min. Nuclear extracts were prepared and examined for NF-κB DNA-binding activity employing EMSA. EGCG pretreatment to NHBE cells resulted in a significant inhibition of CSC-mediated NF-κB DNA-binding activity in a dose-dependent manner (Figure 1b; lane 6, 7, 8). Moreover, when compared to the untreated group (Figure 1b; lane 1) no significant alteration in the DNA-binding activity was observed in cells treated with EGCG alone (Figure 1b; lane 2, 3, 4) suggesting that the NF-κB DNA-binding activity expression may not be substantially affected by physiological concentrations of EGCG.

EGCG treatment of NHBE inhibits CSC-induced phosphorylation and nuclear translocation of NF-κB/p65

NF-κB comprising of p65/RelA and p50/p105 subunits, is inactive in the cytoplasm by remaining in a complex with its inhibitory unit, IκB. IκB masks the NF-κB nuclear localization domain and inhibit its DNA-binding activity. In response to a large variety of stimuli, IκB is rapidly phosphorylated and degraded by proteasomes, freeing NF-κB/p65 to translocate to the nucleus and bind to DNA, leading to expression of target genes. Immunoblot analysis shows that CSC induces the phosphorylation of NF-κB/p65 at Ser536 resulting in the translocation of NF-κB/p65 to the nucleus. EGCG treatment of NHBE cells suppressed CSC-induced NF-κB/p65 phosphorylation in a dose-dependent manner (Figure 1d). Also, total cytosolic NFκB/p65 levels were seen to increase in EGCG pretreated lanes as opposed to the nuclear levels of the protein signifying that EGCG inhibits CSC-induced nuclear translocation of NF-κB/p65 in NHBE cells (Figure 1d). This was further confirmed by performing ELISA with an epitope for p65 bound to the target DNA. As expected, we observed that nuclear NF-κB/p65 activity was significantly induced by CSC and was effectively inhibited by EGCG (Figure 1c). Immunocytochemical analysis further elucidated this effect. In untreated cells or cells treated with EGCG alone, a slight cytoplasmic immunostaining was seen with anti-p65 antibody whereas cells treated with CSC showed an intense nuclear fluorescence (Figure 1e). Predictably, EGCG pretreated cells exposed to CSC showed a significant diminution in nuclear p65 immunostaining, signifying an inhibitory effect of EGCG on the translocation of the NF-κB/p65 subunits into the nuclei (Figure 1e). Taken together, these observations indicate that CSC exposure to NHBE resulted in phosphorylation of NF-κB/p65 with its consequent translocation from the cytosol to the nucleus and pretreatment of cells with EGCG inhibited CSC-induced phosphorylation of NF-κB/p65 and its migration to the nucleus (Figure 1).

EGCG treatment of NHBE inhibits CSC-induced NF-κB reporter activity

The availability of promoter assays makes it possible to efficiently dissect signaling pathways that mediate the effects of diverse insults to host cells. CSC appears to activate NF-κB (Figure 1), which should result in the transcriptional activation of NF-κB-inducible genes. Whether EGCG can suppress CSC-induced NF-κB promoter activity was investigated. For this, cells were transiently transfected with NF-κB promoter-luciferase reporter plasmid then exposed to CSC in the presence and absence of EGCG. Treatment with 10 μg/ml CSC resulted in significant enhancement of luciferase reporter expression compared to control (Figure 1f) indicating activation of NF-κB. Our data show that EGCG treatment to NHBE cells not only inhibited NF-κB-DNA-binding as seen in Figure 1b but also resulted in a significant decrease in CSC induced NF-κB promoter activity (Figure 1f).

EGCG treatment of NHBE inhibits CSC-mediated phosphorylation of of IKKα and IκBα

A crucial step in the activation of NF-κB is the phosphorylation of IκB by a 700- to 900-kDa multimeric complex, referred to as the IκB kinase (IKK) complex (Valen et al., 2001). The IKK complex consists of two highly homologous kinase subunits, IKKα and IKKβ, and a nonenzymatic regulatory component, IKKγ/NEMO. Several other protein kinases have been reported to be able to phosphorylate IκBα in vitro. However, only phosphorylation of IκBα on serine residues 32 and 36 followed by ubiquitination of lysine residues 21 and 22 marks IκBα for proteasome degradation (Magnani et al., 2000). To determine whether the inhibitory action of EGCG towards NF-κB activation was due to its effect on IκBα degradation, the cytosolic levels of IKKα and IκBα were determined by Western blot analysis after pretreating NHBE cells with EGCG (20–80 μ M) followed by exposure to CSC. Western blot analysis showed that CSC exposure resulted in phosphorylation of IKKα and pretreatment of NHBE with EGCG inhibited this phosphorylation in a dose-dependent manner. Further examining the effect of EGCG on IκBα phosphorylation by Western blot analysis, using an antibody that detects only the serine-phosphorylated form of IκBα, we found that CSC exposure resulted in increased phosphorylation of IκBα. Our data show that EGCG treatment of NHBE suppressed this phosphorylation in a dose-dependent manner (Figure 2). These results indicate that EGCG treatment of NHBE resulted in inhibition of CSC-induced activation of IKKα, phosphorylation and degradation of IκBα, and subsequent activation of NF-κB (Figures 1 and 2).

Figure 2
figure2

EGCG inhibited CSC-induced phosphorylation of IκBα. NHBE cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h, and then exposed to 10 μg/ml CSC for 30 min. To examine the effect of EGCG on the level of expression of IKKα and IκBα proteins, cytosolic lysates were prepared for Western blot analysis as detailed in ‘Materials and methods’. Equal protein loading was evaluated by β-actin (bottom). The data shown here are from representative experiment repeated three times with similar results.

EGCG treatment of NHBE inhibited CSC-induced activation of cyclin D1, MMP-9 and VEGF

NF-κB has been recognized as a major regulator of pathogen and inflammatory cytokine-inducible genes (Baeuerle and Baltimore, 1994) and is linked to various signal transduction pathways as well as transcriptional activation events that mediate critical stages of cell proliferation. As cyclin D1, MMP-9 and VEGF are NF-κB-regulated genes (Ho et al., 2005; Vlahos et al., 2006), we next investigated whether expression of these gene products is abrogated by EGCG. Immunoblot analysis showed that CSC exposure to NHBE cells resulted in an increase in the protein expression of cyclin D1 and MMP-9 and EGCG pretreatment resulted in a significant reduction in the expression of these proteins (Figure 3). VEGF, a dimeric 42-kDa protein, is a multifunctional cytokine that plays a pivotal role in angiogenesis. Expression of VEGF has been localized to perivascular cells in many organs, including the lung and its inhibition prevents pathological angiogenesis in a wide variety of tumor models, a phenomenon that has led to the clinical development of a variety of VEGF inhibitors (Ferrara et al., 2003). Our data show that CSC exposure to NHBE cells resulted in an increased expression of VEGF protein and EGCG treatment abrogated the CSC-induced increase in the expression of VEGF in NHBE cells (Figure 3).

Figure 3
figure3

EGCG inhibited CSC-induced activation of Cyclin D1, MMP-9 and VEGF. NHBE cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h, and then exposed to 10 μg/ml CSC for 30 min after which cells were harvested and total cell lysates were prepared for Western blot analysis as detailed in ‘Materials and methods’. Equal protein loading was confirmed by β-actin (bottom). The data shown here are from representative experiment repeated three times with similar results.

EGCG treatment of NHBE inhibited CSC-induced activation of inflammatory markers

Effect of cigarette smoke involves a multitude of direct and indirect effects on pulmonary epithelial cells but principally centers around an increase in airway inflammation resulting in changes in lung function, morphology and gene expression. Studies show that short-term cigarette smoke exposure results in an enhanced release of IL-8, IL-1β and sICAM-1 in epithelial cell cultures (Rusznak et al., 2001). iNOS, expressed by both macrophages and epithelial cells during inflammation, plays an important role in inflammatory reactions via the production of nitric oxide. In order to determine whether EGCG could downregulate proinflammatory cytokine production, whole cell extracts were analysed by Western blotting. Our data show that CSC exposure to NHBE cells resulted in an increased expression of inflammatory marker proteins ICAM, IL-8 and iNOS. EGCG treatment of cells resulted in a significant inhibition of the CSC-induced increase in the expression of these proteins (Figure 4) signifying the anti-inflammatory potential of EGCG.

Figure 4
figure4

EGCG inhibited CSC-induced activation of inflammatory markers. NHBE cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h, and then exposed to 10 μg/ml CSC for 30 min after which cells were harvested and total cell lysates were prepared for Western blot analysis as detailed in ‘Materials and methods’. Equal protein loading was confirmed by β-actin (bottom). The data shown here are from representative experiment repeated three times with similar results.

EGCG treatment of NHBE inhibited CSC-induced activation of MAPK pathway

Generation of oxidants by cigarette smoke appears to be the primary stimulus for activation of MAPK cascades in lung epithelium (Mossman et al., 2006). Targeting of MAPKs and interrelated signaling cascades may be imperative to prevention of lung cancer. Whether the inhibitory effect of EGCG on CSC-induced NF-κB activation pathway extends to MAPK signaling pathway was investigated. Employing Western blot analysis, we found that CSC exposure to NHBE resulted in a significant increase in the phosphorylation of p38, ERK1/2, and JNK1/2 MAPK proteins (Figure 5). EGCG treatment to NHBE cells resulted in a marked reduction in the CSC-induced phosphorylation of p38, ERK1/2 and JNK1/2 MAPK proteins as shown in Figure 5, suggesting that EGCG is an effective inhibitor of pathways other than NF-κB in NHBE cells.

Figure 5
figure5

EGCG inhibited CSC-induced activation of MAPK pathway. NHBE cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h, and then exposed to 10 μg/ml CSC for 30 min after which cells were harvested and total cell lysates were prepared for Western blot analysis as detailed in ‘Materials and methods’. Equal protein loading was confirmed by β-actin (bottom). The data shown here are from representative experiment repeated three times with similar results.

EGCG treatment of NHBE inhibited CSC-induced PI3K/AKT/mTOR signaling pathway

It has been reported that nicotine in cigarette smoke induces multisite phosphorylation of Bad with consequent suppression of apoptosis through three signal pathways, including ERK, PI3K/AKT and PKA in human lung cancer cells (Jin et al., 2004). Another study shows that exposure to nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, two components of cigarette smoke, result in activation of the serine/threonine kinase AKT in nonimmortalized human airway epithelial cells (West et al., 2003). We therefore tested the effect of EGCG on CSC-induced activation of PI3K/AKT signaling pathway. For this, protein lysates were analysed by Western blot using antibodies that detect the phosphorylated form of these proteins. As shown in Figure 6, CSC exposure to NHBE cells resulted in increased phosphorylation of AKT at Ser473 and Thr308, and EGCG pretreatment suppressed the phosphorylation of the protein at both its phosphorylation sites. In addition, CSC exposure resulted in increased protein expression of PI3K (p85) and EGCG pretreatment to cells resulted in the downregulation of the protein (Figure 6). Several biochemical events identified downstream of AKT enhance cell survival, increase cell proliferation, and alter cell metabolism. In this respect, phosphorylation of tuberin leads to the activation of mammalian target of rapamycin (mTOR), a critical mediator of protein translation (Manning et al., 2002). Whether EGCG modulates CSC induced mTOR phosphorylation was investigated. Immunoblot analysis shows that CSC exposure resulted in significant phosphorylation of mTOR at Ser2448 and EGCG treatment abolished this effect. These data suggest that EGCG may exert an inhibitory effect on PI3K/AKT signaling molecules and their downstream effectors and consequently restore the cell growth to a controlled state.

Figure 6
figure6

EGCG inhibited CSC-induced PI3K/AKT/mTOR pathway. NHBE cells were pretreated with various concentrations of EGCG (20–80 μ M) for 4 h at 37°C, and then exposed to 10 μg/ml CSC for 30 min after which cells were harvested and total cell lysates were prepared for Western blot analysis as detailed in ‘Materials and methods’. Equal protein loading was confirmed by β-actin (bottom). The data shown here are from representative experiment repeated three times with similar results.

Discussion

Most of the studies evaluating the chemopreventive potential of EGCG have been conducted on lung cancer cell lines rather than targeting primary cells. In the present study, we show that EGCG treatment of normal bronchial cells results in downregulation of the signaling networks activated by cigarette smoke induced stress. It is conceivable that one of the mechanisms of cancer chemoprevention exerted by EGCG may be through inhibition of these biomarkers in tumor promoting cells.

There is sufficient evidence to establish a causal association between cigarette smoking and lung cancer as well as several other organs (Sasco et al., 2004). Cigarette smoke contains a complex mixture of chemical compounds that are delivered to the lung in both gas and tar phases, of which more than 40 have been identified as mutagens and carcinogens, and exert their biologic effects through the formation of DNA adducts in target tissues and mutations in transforming genes (Patel, 2005). Inflammation and oxidative stress induced by smoking results in proteolytic imbalance and progressive lung structural derangement, with disease susceptibility being controlled by inherited variations in protective or inflammatory genes (Anderson and Bozinovski, 2003). Nevertheless, cigarette smoke is directly mutagenic. Acquired somatic mutations, rather than inherited polymorphisms, might therefore be major determinants in the development of lung cancer that may alter the nature of signal transduction in the lung. Studies show that environmental tobacco smoke is composed of emissions from cigarette smoke and contains a higher concentration of tobacco smoke carcinogens than mainstream smoke (Miller et al., 2003). This implies that environmental tobacco smoke may be another etiologic factor for lung cancer because lung adenocarcinoma was observed as the major histologic type in women and never smokers. Chemopreventive strategies might then be developed for high-risk smokers or those exposed to secondhand smoke.

Studies suggest that pathways activated by EGCG in normal epithelial versus tumor cells create different oxidative environments, favoring either normal cell survival or tumor cell destruction. It is thought that cells in frequent contact with plant-derived polyphenols, such as those found in the epidermis, oral mucosa and digestive tract, develop mechanisms to mitigate the toxicity from these compounds (Yamamoto et al., 2003). However, green tea polyphenols, when used in high doses, may be cytotoxic to other human cells that lack this tolerance and to cancer cells that have lost these protective mechanisms. An optimum dose is therefore important in enhancing the effectiveness of the agent to selectively promote cancer cell death while protecting normal cells.

Dose-dependent activation of NF-κB, with induction of multiple genes associated with cellular inflammation, innate immunity and repair processes was observed with sub-chronic exposure of cigarette smoke in a murine model (Vlahos et al., 2006). Activation of the NF-κB pathway is closely linked to an increase in the growth-promoting potential and anti-apoptotic responses of cells to a broad spectrum of stimuli that may result in malignant transformation of cells. In the present study, CSC-induced activation of NF-κB, leading to the activation and nuclear translocation of the NF-κB/p65 (Figure 1) was largely abolished by EGCG treatment suggesting that EGCG has the potential to suppress cigarette smoke induced carcinogenesis.

Cigarette smoke causes accumulation of polymorphonuclear leukocytes in alveolar septum and an increased number of neutrophils has been demonstrated in bronchoalveolar lavage fluid of smokers (Hunninghake and Crystal, 1983). A wide array of cytokines including IL-1, IL-6, IL-8, MCP-1 and gro/MGSA, may be secreted during this inflammatory response. Activation of transcriptional factors especially NF-κB have been shown to play an important role in the induction of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin), cytokines, acute phase proteins, growth factors, COX-2 and iNOS expression (Sahnoun et al., 1998). Studies show that the recruitment of neutrophils into the airways is associated with increases in both NF-κB DNA-binding activity and IL-8 mRNA expression (Nishikawa et al., 1999). Under the influence of these inflammatory mediators, other enzymes are also activated. iNOS, the inducible isoform of nitric oxide synthase is considered more important in cancer development than its other isoforms, eNOS and nNOS, involved in the production of sustained levels of nitric oxide in response to inflammation. Nitric oxide has been implicated in processes of tumor initiation, promotion and progression and can directly damage DNA, inhibit DNA repair, enhance oncogene expression, modulate transcriptional factors, block apoptosis and contribute to angiogenesis (Rao, 2004). Moreover, studies have shown that the ablation of iNOS or inhibition of its activity inhibits the development of rodent lung cancer (Kisley et al., 2002). Increased nitric oxide generation in a cell may contribute to tumor angiogenesis by upregulating VEGF (Xu et al., 2002). A recent study strongly suggests that nitric oxide regulates mast cell expression of VEGF and nitric oxide may function as an upstream regulator of expression as well as a downstream effector of the action of VEGF (Konopka et al., 2001). MMP-2 and MMP-9 are thought to be the key enzymes involved in the degradation of type IV collagen, the main component of extracellular matrix. Increased expression of these has been shown to correlate with an invasive phenotype of cancer cells (Vihinen and Kahari, 2002). Inhibition of MMP activity has been shown to suppress lung metastasis with arrest of cancer progression seen on combined use of inhibitors of MMPs and inhibitors of the plasmin/plasminogen activation system, in several in vivo models (Shiraga et al., 2002). It is evident from our data that EGCG inhibits CSC-induced activation of inflammatory markers, implying that EGCG can be useful in preventing the occurrence of an inflammatory environment apparently involved in the pathogenesis of cigarette smoke induced lung disease.

NF-κB activation has been shown to result in the induction of the cyclin D1 gene (Ho et al., 2005). Also, increased cyclin D1 levels are found in different types of lung tumor tissues, as well as other tumors (Petty et al., 2003). Our results show that EGCG treatment to NHBE cells effectively downregulates CSC-mediated increase in the expression of cyclin D1 (Figure 4). Thus, cyclin D1 levels might be a useful marker to predict the occurrence of smoking-associated lung carcinogenesis.

An important role of MAPKs in the development of cigarette smoke-induced pathogenesis has been elucidated. Differential response to cigarette smoke components has been shown to result in MAPKs activation leading to aberrant cell proliferation and squamous cell metaplasia in the lungs of rats (Zhong et al., 2005). MAPKs and PI3K/AKT pathways are downstream signaling molecules activated via the epidermal growth factor receptor (EGFR) that in turn regulate cell survival and proliferation. Kurie et al. (1996) have shown that there is increased EGFR expression in the metaplastic bronchial epithelium of smokers. Furthermore, a recent report suggests that CSC can directly stimulate the activity of the MMPs, leading to EGFR activation (Zhang et al., 2005), in addition to causing ligand independent transactivation of the EGFR (Takeyama et al., 2001). This provides a compelling rationale for targeting MAPKs by a variety of chemopreventive measures. Our data suggest that EGCG has the potential to attenuate CSC-induced MAPK pathways associated with high risk of carcinogenesis.

Active AKT has been detected in airway epithelial cells and lung tumors from NNK-treated A/J mice, and in human lung cancers derived from smokers (West et al., 2003). A more recent report revealed increased p-AKT expression in bronchial hyperplasia, squamous metaplasia and bronchial dysplasia, supporting the observation that AKT activation is an early event in lung cancer progression (Tsao et al., 2003). mTOR, a serine/threonine kinase downstream of AKT, plays a vital role in the control of cell growth and proliferation through integration of mitogenic signals and intracellular nutrient levels. mTOR signaling has been shown to be activated in precursors of lung adenocarcinoma with a role in lung tumor progression (Wislez et al., 2005). Our data show that EGCG may have a potential chemopreventive role by blocking PI3K/AKT/MTOR signaling pathway, a critical target in the proliferation and malignant transformation of normal cells (Figure 6).

In summary, this study shows that EGCG inhibits activation of pro-survival pathways such as NF-κB, PI3K/AKT/mTOR and MAPKs and reduces the production of proinflammatory cytokines such as ICAM-1, IL-8 and iNOS in addition to inhibiting the CSC mediated activation of cyclin D1, MMP-9 and VEGF in normal bronchial cells (Figure 7). On the basis of our data, it may be suggested that long-term exposure to low levels of EGCG attained through diet alongwith modulation of environmental factors could suppress the development of lung cancer in smokers as well as those exposed to second hand smoke. Epidemiological studies however, are needed to better characterize the relationship between tea consumption and human lung cancer caused by smoking.

Figure 7
figure7

Schematic representation of the signaling networks activated by cigarette smoke induced stress and the anti-proliferative effect of EGCG in human bronchial cells.

Materials and methods

Materials

A purified preparation of EGCG (>98% pure) was kindly provided by Dr Yukihiko Hara of Mitsui Norin Co., Ltd (Shizuoka, Japan), and cigarette smoke condensate (CSC) was kindly provided by Dr Harish C Sikka (State University New York College, Buffalo, NY, USA).

Antibodies

p-ERK1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr204), IκBα, p-IκBα (Ser32/36), cyclin D1, p-AKT (Ser473) and PI3Kp85 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). IKKα and VEGF antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). NF-κB/p65, p-NF-κB/p65 (Ser536) and p-Akt (Thr308) antibodies were obtained from Upstate Cell Signaling Solutions; MMP-9 and IL-8 antibodies were purchased from Chemicon International (Temecula, CA, USA); and iNOS antibody was obtained from BD Transduction Laboratories (San Jose, CA, USA).

Cell culture

Normal Human Bronchial Epithelial (NHBE) cells were obtained from Cambrex Bio Science Inc. (Rockland, MD, USA). The cells were cultured, as recommended in BEBM – Bronchial Epithelial Basal Medium from Cambrex Bio Science Inc. (Rockland, MD, USA) and supplemented with BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, gentamicin/amphotericin-B and retinoic acid. The cells were maintained at 37°C and 5% CO2 in a humid environment.

Treatment of cells

EGCG dissolved in 1X phosphate-buffered saline (PBS) was used for the treatment of cells. For dose-dependent studies, the cells (70–80% confluent) were treated with EGCG (20, 40, 80 μ M) for 4 h at 37°C in BEBM, after which the media was removed, cells were washed with PBS, and harvested/treated with CSC for 30 min and harvested. CSC dissolved in dimethyl sulfoxide was used for the treatment of cells.

Preparation of total cell lysate

After treatment of cells with EGCG or CSC (or both), the medium was aspirated and the cells were washed twice in PBS (10 mM, pH 7.4). The cells were incubated in 0.4 ml ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA), 1 mM ethylene diaminetetraacetic acid (EDTA), 20 mM NaF, 100 mM Na3VO4, 0.5% Nonidet P-40 (NP-40), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) (pH 7.4)) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA, USA). The cells were then centrifuged at 13 000 g for 25 min at 4°C, and the supernatant (total cell lysate) was collected, aliquoted and stored at −80°C. The protein concentration was determined by the BCA protein assay kit using the manufacturer's protocol (Pierce, Rockford, IL, USA).

Preparation of cytosolic and nuclear lysate

Following treatment of cells with EGCG or CSC (or both), the medium was aspirated and the cells were washed twice in PBS (10 mM, pH 7.4). The cells were incubated in 0.4 ml ice-cold lysis buffer (HEPES (10 mM, pH 7.9), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 mM), PMSF (1 mM)) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA, USA) for 15 min, after which 12.5 μl of 10% Nonidet P-40 was added and the contents were mixed on a vortex and then centrifuged for 1 min (14 000 g) at 4°C. The supernatant was saved as cytosolic lysate and stored at −80°C. The nuclear pellet was resuspended in 50 μl of ice-cold nuclear extraction buffer (HEPES (20 mM, pH 7.9), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (1 mM)) with freshly added protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA, USA) for 30 min with intermittent mixing. The tubes were centrifuged for 5 min (14 000 g) at 4°C, and the supernatant (nuclear extract) was stored at −80°C. The protein concentration was determined by the BCA protein assay kit using the manufacturer's protocol (Pierce, Rockford, IL, USA).

Western blot analysis

For Western blot analysis, 25–30 μg of protein was resolved over 6–16% PAGE and transferred to a nitrocellulose membrane. The blot containing the transferred protein was blocked in blocking buffer (5% nonfat dry milk, 1% Tween 20; in 20 mM TBS, pH 7.6) for 1 h at room temperature followed by incubation with appropriate monoclonal/polyclonal primary antibody in blocking buffer for 1 h to overnight at 4°C. This was followed by incubation with anti-mouse or anti-rabbit secondary antibody horseradish peroxidase (Amersham Biosciences, UK) for 1 h and several washes and finally detection by chemiluminescence (ECL kit, Amersham Biosciences, UK) and autoradiography using XAR-5 film obtained from Eastman Kodak Co. (Rochester, NY, USA). Densitometric measurements of the bands in Western blot analysis were performed using the digitalized scientific software program UN-SCAN-IT (Silk Scientific Corporation, Orem, UT, USA).

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) for NF-κB was performed using lightshift™ chemiluminiscent EMSA kit (Pierce, Rockford, IL, USA) as per manufacturer's protocol. To start with, DNA was biotin labeled using the Biotin 3′ end labeling kit (Pierce, Rockford, IL, USA). Briefly, in a 50 μl reaction buffer, 5 pmol of double-stranded NF-κB oligonucleotide 5′-IndexTermAGT TGA GGG GAC TTT CCC AGG C-3′; 3′-IndexTermTCA ACT CCC CTG AAA GGG TCC G-5′ was incubated in a microfuge tube with 10 μl of 5 × TdT (terminal deoxynucleotidyl transferase) buffer, 5 μl of 5 μ M biotin-N4-CTP, 10 U of diluted TdT, and 25 μl of ultrapure water at 37°C for 30 min. The reaction was stopped with 2.5 μl of 0.2 M EDTA. To extract labeled DNA, 50 μl of chloroform:isoamyl alcohol (24:1) was added to each tube and centrifuged briefly at 13 000 g. The top aqueous phase containing the labeled DNA was removed and saved for binding reactions. Each binding reaction contained 1X-binding buffer (100 mM Tris, 500 mM KCl, 10 mM dithiothreitol, pH 7.5), and 2.5% glycerol, 5 mM MgCl2, 50 ng/μl poly (dI-dC), 0.05% NP-40, 5 μg of nuclear extract, and 20–50 fmole of biotin end-labeled target DNA. The contents were incubated at room temperature for 20 min. To this reaction mixture was added 5 μl of 5 × loading buffer, subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. When the transfer was complete, DNA was crosslinked to the membrane at 1200 mJ/cm2 using a UV crosslinker equipped with 254 nm bulbs. The biotin end-labeled DNA was detected using streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film (XAR-5 Amersham Life Science Inc., Arlington Heights, IL, USA) and developed using a Kodak film processor.

Enzyme-linked immunosorbent assay

Trans-AM enzyme-linked immunosorbent assay (ELISA) kit from Active Motif (Carlsbad, CA, USA) was employed for NF-κB/p65 assay. A 96-well plate was used, to which oligonucleotide containing an NF-κB consensus site (5′-IndexTermGGGACTTTCC-3′) that has been immobilized and binds to the nuclear extract and can detect NF-κB, and recognize an epitope on p65 activated and bound to its target DNA. In the absence of competitive binding with the wild-type or mutated consensus oligonucleotide, 30 μl of binding buffer was added to each well in duplicate. Alternatively, 30 μl of binding buffer containing 20 pmol (2 μl) of appropriate oligonucleotide, in duplicate, was added to the corresponding well. Nuclear lysate protein (10 μg) of each sample diluted in 20 μl lysis buffer was loaded per well. For positive control, 20 μl of lysis buffer containing 1 μl of control cell extract per well, was used and for blank 20 μl of lysis buffer per well was used. The plate was sealed with the adhesive film and incubated for 1 h at room temperature with mild agitation (100 r.p.m. on a rocking platform), after which the wells were washed three times with 200 μl of 1 × wash buffer, and 100 μl of diluted primary antibody (1:1000 dilution in 1 × antibody-binding buffer) was added to each well and incubated at room temperature for 1 h without agitation. The wells were washed again three times with 1 × wash buffer, and 100 μl of diluted HRP-conjugate antibody (1:1000 dilution in 1 × antibody-binding buffer) was added to each well and incubated for 1 h. The wells were again washed four times with 1 × wash buffer followed by the addition of 100 μl of developing solution after which the contents were incubated for 5 min at RT. This was followed by the addition of 100 μl of stop solution to each well, and the absorbance was read within 5 min at 450 nm.

Luciferase Activity

To determine the effect of EGCG on NF-κB transcription activity, NHBE cells were seeded at a concentration of 1.5 × 105 cells per well in six-well plates and co-transfected with 4 μg NF-κB-driven luciferase reporter construct and 6 μg pSV40-β-gal plasmid (Clontech, Mountain View, CA, USA) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). After 48 h exposure to the transfection mixture, the cells were incubated in medium containing EGCG for 4 h and thereafter exposed to CSC (10 μg/ml) for 30 min as described earlier. Lysates were made after washing each well with PBS and then incubating with Passive Lysis Buffer (Promega, Madison, WI, USA) for 15 min on a shaker. Luciferase activity was measured by using the Reporter luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's protocol and normalized with respect to β-galactosidase activity (Pierce Biotechnology, Rockford, IL, USA). All experiments were repeated at least three times to prove their reproducibility.

Immunocytochemistry

NHBE cells were seeded in two chamber tissue culture glass slides and treated with/without EGCG and CSC as described earlier. After treatment, cells were washed with 1 × PBS and fixed in 2% Paraformaldehyde/1 × PBS for 10 min at RT. Cells were then permeabilized in cold methanol (−20°C), after three washes with 1 × PBS and blocked with 2% donkey serum (1 × PBS) for 1 h and incubated with NF-κB/p65 antibody (1:50 in 5% donkey serum (1 × PBS)) overnight at 4°C. After three washes with 2% donkey serum (1 × PBS) cells were incubated with donkey anti-rabbit Rhodamine Red™-X-conjugated antibody (1:50) for 45 min at room temperature. After rinsing in three changes of 2% donkey serum (1 × PBS), samples were mounted using Prolong antifade kit (Invitrogen, Eugene, OR, USA), and observed using a Zeiss Axiophot DM HT microscope. Images were captured with an attached camera linked to a computer. Images and figures were composed using ADOBE PHOTOSHOP 7.0 (Adobe Systems, Mountain View, CA, USA).

Abbreviations

EGCG:

(−)-epigallocatechin-3-gallate

NHBE:

normal human bronchial epithelial cells

CSC:

cigarette smoke condensate

NF-κB:

nuclear factor-κB

mTOR:

mammalian target of rapamycin

References

  1. Anderson GP, Bozinovski S . (2003). Acquired somatic mutations in the molecular pathogenesis of COPD. Trends Pharmacol Sci 24: 71–76.

    CAS  Article  Google Scholar 

  2. Baeuerle PA, Baltimore D . (1994). Function and activation of NF-κB in the immune system. Annu Rev Immunol 12: 141–179.

    CAS  Article  Google Scholar 

  3. Coussens LM, Werb Z . (2002). Inflammation and cancer. Nature 420: 860–867 (Review).

    CAS  Article  Google Scholar 

  4. Doss MX, Potta SP, Hescheler J, Sachinidis A . (2005). Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases. J Nutr Biochem 16: 259–266.

    CAS  Article  Google Scholar 

  5. Ferrara N, Gerber HP, LeCouter J . (2003). The biology of VEGF and its receptors. Nat Med 9: 669–676.

    CAS  Article  Google Scholar 

  6. Ho YS, Chen CH, Wang YJ, Pestell RG, Albanese C, Chen RJ et al. (2005). Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation. Toxicol Appl Pharmacol 205: 133–148.

    CAS  Article  Google Scholar 

  7. Hunninghake GW, Crystal RG . (1983). Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 128: 833–838.

    CAS  PubMed  Google Scholar 

  8. Jin Z, Gao F, Flagg T, Deng X . (2004). Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis. J Biol Chem 279: 23837–23844.

    CAS  Article  Google Scholar 

  9. Johnson MK, Loo G . (2000). Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Mutat Res 459: 211–218.

    CAS  Article  Google Scholar 

  10. Kisley LR, Barrett BS, Bauer AK, Dwyer-Nield LD, Barthel B, Meyer AM et al. (2002). Genetic ablation of inducible nitric oxide synthase decreases mouse lung tumorigenesis. Cancer Res 62: 6850–6856.

    CAS  PubMed  Google Scholar 

  11. Konopka TE, Barker JE, Bamford TL, Guida E, Anderson RL, Stewart AG . (2001). Nitric oxide synthase II gene disruption implications for tumor growth and vascular endothelial growth factor production. Cancer Res 61: 3182–3187.

    CAS  PubMed  Google Scholar 

  12. Kurie JM, Shin HJ, Lee JS, Morice RC, Ro JY, Lippman SM et al. (1996). Increased epidermal growth factor receptor expression in metaplastic bronchial epithelium. Clin Cancer Res 2: 1787–1793.

    CAS  PubMed  Google Scholar 

  13. Kyosseva SV . (2004). Mitogen-activated protein kinase signaling. Int Rev Neurobiol 59: 201–220.

    CAS  Article  Google Scholar 

  14. Lin A, Karin M . (2003). NF-kappaB in cancer: a marked target. Semin Cancer Biol 13: 107–114.

    CAS  Article  Google Scholar 

  15. Magnani M, Crinelli R, Bianchi M, Antonelli A . (2000). The ubiquitin-dependent proteolytic system and other potential targets for the modulation of nuclear factor-κB (NF-κB). Curr Drug Targets 1: 387–399.

    CAS  Article  Google Scholar 

  16. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC . (2002). Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 10: 151–162.

    CAS  Article  Google Scholar 

  17. Miller DP, De Vivo I, Neuberg D, Wain JC, Lynch TJ, Su L et al. (2003). Association between self-reported environmental tobacco smoke exposure and lung cancer: modification by GSTP1 polymorphism. Int J Cancer 104: 758–763.

    CAS  Article  Google Scholar 

  18. Mossman BT, Lounsbury KM, Reddy SP . (2006). Oxidants and signaling by mitogen-activated protein kinases (MAPK) in lung epithelium. Am J Respir Cell Mol Biol 34: 666–669.

    CAS  Article  Google Scholar 

  19. Nishikawa M, Kakemizu N, Ito T, Kudo M, Kaneko T, Suzuki M et al. (1999). Superoxide mediates cigarette smoke-induced infiltration of neutrophils into the airways through nuclear factor-kappaB activation and IL-8 mRNA expression in guinea pigs in vivo. Am J Respir Cell Mol Biol 20: 189–198.

    CAS  Article  Google Scholar 

  20. Ozlu T, Bulbul Y . (2005). Smoking and lung cancer. Tuberk Toraks 53: 200–209.

    PubMed  Google Scholar 

  21. Patel JD . (2005). Lung cancer in women. J Clin Oncol 23: 3212–3218.

    CAS  Article  Google Scholar 

  22. Petty WJ, Dragnev KH, Dmitrovsky E . (2003). Cyclin D1 as a target for chemoprevention. Lung Cancer 41 (Suppl 1): S155–S161.

    Article  Google Scholar 

  23. Philip M, Rowley DA, Schreiber H . (2004). Inflammation as a tumor promoter in cancer induction. Semin Cancer Biol 14: 433–439.

    CAS  Article  Google Scholar 

  24. Rao CV . (2004). Nitric oxide signaling in colon cancer chemoprevention. Mutat Res 555: 107–119.

    CAS  Article  Google Scholar 

  25. Rusznak C, Sapsford RJ, Devalia JL, Shah SS, Hewitt EL, Lamont AG et al. (2001). Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells. Clin Exp Allergy 31: 226–238.

    CAS  Article  Google Scholar 

  26. Sahnoun Z, Jamoussi K, Zeghal KM . (1998). Free radicals and antioxidants: physiology, human pathology and therapeutic aspects (part II). Therapie 53: 315–339.

    CAS  PubMed  Google Scholar 

  27. Sasco AJ, Secretan MB, Straif K . (2004). Tobacco smoking and cancer: a brief review of recent epidemiological evidence. Lung Cancer 45 (Suppl 2): S3–S9.

    Article  Google Scholar 

  28. Shiraga M, Yano S, Yamamoto A, Ogawa H, Goto H, Miki T et al. (2002). Organ heterogeneity of host-derived matrix metalloproteinase expression and its involvement in multiple-organ metastasis by lung cancer cell lines. Cancer Res 62: 5967–5973.

    CAS  PubMed  Google Scholar 

  29. Sizemore N, Lerner N, Dombrowski N, Sakurai H, Stark GR . (2002). Distinct roles of the Ikappa B kinase alpha and beta subunits in liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and in phosphorylating the p65 subunit of NF-kappa B. J Biol Chem 277: 3863–3869.

    CAS  Article  Google Scholar 

  30. Takeyama K, Jung B, Shim JJ, Burgel PR, Dao-Pick T, Ueki IF et al. (2001). Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 280: L165–L172.

    CAS  Article  Google Scholar 

  31. Tsao AS, McDonnell T, Lam S, Putnam JB, Bekele N, Hong WK et al. (2003). Increased phospho-AKT Ser(473) expression in bronchial dysplasia: implications for lung cancer prevention studies. Cancer Epidemiol Biomarkers Prev 12: 660–664.

    CAS  Google Scholar 

  32. Tsurutani J, Castillo SS, Brognard J, Granville CA, Zhang C, Gills JJ et al. (2005). Tobacco components stimulate Akt-dependent proliferation and NF-κB-dependent survival in lung cancer cells. Carcinogenesis 26: 1182–1195.

    CAS  Article  Google Scholar 

  33. Valen G, Yan ZQ, Hansson GKJ . (2001). Nuclear factor kappa-B and the heart. Am Coll Cardiol 38: 307–314.

    CAS  Article  Google Scholar 

  34. Vihinen P, Kahari VM . (2002). Matrix metalloproteinases in cancer: prognostic markers and therapeutic Targets. Int J Cancer 99: 157–166.

    CAS  Article  Google Scholar 

  35. Vlahos R, Bozinovski S, Jones JE, Powell J, Gras J, Lilja A et al. (2006). Differential protease, innate immunity and NF{kappa}B induction profiles during lung inflammation induced by sub-chronic cigarette smoke exposure in mice. Am J Physiol Lung Cell Mol Physiol 290: L931–L945.

    CAS  Article  Google Scholar 

  36. West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM et al. (2003). Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest 111: 81–90.

    CAS  Article  Google Scholar 

  37. Wislez M, Spencer ML, Izzo JG, Juroske DM, Balhara K, Cody DD et al. (2005). Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras. Cancer Res 65: 3226–3335.

    CAS  Article  Google Scholar 

  38. Wistuba II, Mao L, Gazdar AF . (2002). Smoking molecular damage in bronchial epithelium. Oncogene 2: 7298–7306.

    Article  Google Scholar 

  39. Xu W, Liu LZ, Loizidou M, Ahmed M, Charles IG . (2002). The role of nitric oxide in cancer. Cell Res 12: 311–320.

    Article  Google Scholar 

  40. Yamamoto T, Hsu S, Lewis J, Wataha J, Dickinson D, Singh B et al. (2003). Green tea polyphenol causes differential oxidative environments in tumor versus normal epithelial cells. Pharmacol Exp Ther 307: 230–236.

    CAS  Article  Google Scholar 

  41. Yang CS, Liao J, Yang GY, Lu G . (2005). Inhibition of lung tumorigenesis by tea. Exp Lung Res 31: 135–144.

    Article  Google Scholar 

  42. Zhang Q, Adiseshaiah P, Reddy SP . (2005). Matrix metalloproteinase/epidermal growth factor receptor/mitogen-activated protein kinase signaling regulates fra-1 induction by cigarette smoke in lung epithelial cells. Am J Respir Cell Mol Biol 32: 72–81.

    Article  Google Scholar 

  43. Zhong CY, Zhou YM, Douglas GC, Witschi H, Pinkerton KE . (2005). MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and squamous metaplasia in the lungs of rats. Carcinogenesis 26: 2187–21895.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This study was supported by the developmental funds from US Public Health Service Grant 5P30 CA 14520 and also used resources of USPHS grants R01 CA 78809 and R01 CA 101039.

Author information

Affiliations

Authors

Corresponding author

Correspondence to H Mukhtar.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Syed, D., Afaq, F., Kweon, MH. et al. Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-κB activation in normal human bronchial epithelial cells. Oncogene 26, 673–682 (2007). https://doi.org/10.1038/sj.onc.1209829

Download citation

Keywords

  • (−)-epigallocatechin-3-gallate
  • cigarette smoke condensate
  • normal human bronchial epithelial cells
  • nuclear factor-κB

Further reading

Search

Quick links