Introduction
Lung cancer is considered as the leading cause of cancer-related mortality among men worldwide and the second among women (1). Almost as many patients die of lung cancer every year than die of prostate, breast and colon cancer combined in the USA (2). According to the differences in clinical behavior and the aims of treatment, lung cancers can be divided into two broad categories; small cell lung cancer (SCLC) which accounts for 15% of all lung cancer cases and non-small cell lung cancer (NSCLC) which accounts for 85% of cases (3).
A number of studies have documented smoking as the leading risk factor for lung cancers. Since the first Surgeon General's Report on the association of smoking with lung cancer in 1964, increased public awareness of the health risks associated with smoking has gradually decreased the number of smokers over the past five decades in developed countries (4). However, contrary to expectations, the significant decrease in smokers has not yielded a correlating decrease in the incidence and mortality of lung cancer (5); this finding strongly suggests that factors other than smoking play significant roles in the development, progression and responsiveness to therapeutics of lung cancer. Interestingly, increasing evidence suggests that chronic psychological stress has been recognized as an important risk factor in promoting the genesis and development of cancers including lung cancer in recent years (6). In response to stressors, activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to the release of glucocorticoids, catecholamines, including norepinephrine and epinephrine, and other stress hormones from the adrenal gland as well as from the brain and sympathetic nerve terminals (7). The effects of catecholamines are mainly mediated by β-adrenergic receptors (β-ARs) which are expressed in almost all mammalian cell types. Recent findings have indicated that β-ARs can regulate multiple cellular processes that contribute to the initiation and progression of cancer, including inflammation, angiogenesis, apoptosis, cell motility and trafficking, and the activation of tumor-associated viruses (8).
β-ARs, consisting of three subtypes (β1-AR, β2-AR and β3-AR), are members of the superfamily of G protein-coupled receptors (GPCRs). β1-ARs and β2-ARs are expressed in the majority of mammalian cells, while β3-ARs are almost exclusively found in adipocytes (9). β2-ARs can associate with heterotrimeric guanine nucleotide-binding proteins (G proteins), and multiple cytosolic scaffold proteins including β-arrestin and Src, to initiate various signaling pathways and modulate the activity of intracellular effectors such as adenylyl cyclase and mitogen-activated protein kinases (MAPKs) (10). Studies have shown that nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) binds to the β1-AR-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and cyclic adenosine monophosphate response element-binding protein (CREB)/ATF-1 phosphorylation via transactivation of the epithelial growth factor receptor (EGFR) pathway in both human lung adenocarcinoma cell line NCI-H322 and human peripheral airway cell line HPLD1 (11). Moreover, NNK also induced ERK1/2 and CREB phosphorylation through β2-AR in pulmonary adenocarcinoma in the hamster (12). However, the function of β2-AR in human lung epithelial-derived A549 cells and the underlyng mechanisms are not well understood.
To this end, the present study was designed to investigate the function and mechanism of β2-AR-mediated cell proliferation in A549 cells. Our results demonstrated that activation of the β2-AR can significantly enhance the proliferation of lung cancer cells via activation of the ERK1/2/CREB pathway. In addition, matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF) may be involved in isoproterenol (ISO)-induced A549 cell proliferation.
Materials and methods
Antibodies and reagents
Antibodies for western blotting, including phospho-ERK1/2 (Thr202/Tyr204) (cat. 9101, polyclonal, raised in rabbit, 1:2,000), ERK1/2 (cat. 9102, polyclonal, raised in rabbit, 1:2,000), phospho-CREB (Ser133) (cat. 9198, monoclonal, rabbit anti-human, 1:2,000), CREB (cat. 9197, monoclonal, rabbit anti-human, 1:2,000), β-actin (cat. 4970, monoclonal, rabbit anti-human, 1:4,000), anti-rabbit IgG HRP-linked antibody (cat. 7074, goat anti-rabbit, 1:20,000) as well as U0126 (specific ERK1/2 inhibitor) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). ISO (a broad-spectrum β-adrenergic agonist) and ICI-118,551 (ICI) (a selective β2-AR antagonist) were purchased from Tocris Cookson, Inc. (Bristol, UK). Fetal bovine serum (FBS), culture medium and other solutions used for cell culture were from Invitrogen (Shanghai, China). MTT was purchased from Carl Roth GmbH & Co., KG (Karlsruhe, Germany). SYBR® Premix Ex Taq™ II was obtained from Takara Bio, Inc. (Otsu, Japan). Lipofectamine 2000 was procured from Thermo Fisher Scientific (Shanghai, China). The siRNAs against CREB and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and transfection
Human lung adenocarcinoma A549 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured according to the relevant specifications. The cells were grown in Dulbecco's minimal essential medium (DMEM) containing 4.5 g/l glucose supplemented with 10% FBS and 1% glutamine. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. A549 cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions, using siRNAs against CREB for 48 h.
Reverse transcription PCR
Total RNA was isolated from the A549 cells using TRIzol reagent, and reverse transcription was carried out according to the manufacturer's instructions from Invitrogen. PCR amplification was performed on 5 µl cDNA, 12.5 µl 2X Taq PCR Master Mix, 0.5 µl of 10 µM forward primer, 0.5 µl of 10 µM reverse primer and 6.5 µl ddH2O under the following conditions: 94°C for 5 min, 30 cycles of denaturation for 1 min at 95°C, 1 min of annealing at 55°C, elongation at 72°C for 1 min, and extension at 72°C for 1 min. PCR analysis was performed using the following sense and antisense primers: β1-AR forward, 5′-GGGAGAAGCATTAGGAGGG-3′ and reverse, 5′-CAAGGAAAGCAAGGTGGG-3′ which amplify a 270-bp fragment; β2-AR forward, 5′-CAGCAAAGGGACGAGGTG-3′ and reverse, 5′-AAGTAATGGCAAAGTAGCG-3′ which amplify a 334-bp fragment; β-actin forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTT C-3′ which amplify a 101-bp fragment; MMP-2 forward, 5′-CCGTCGCCCATCATCAAGTTC-3′ and reverse, 5′-GCAGCCATAGAAGGTGTTCAGG-3′ which amplify a 90-bp fragment; MMP-9 forward, 5′-TGGTCCTGGTGCTCCTGGTG-3′ and reverse, 5′-GCTGCCTGTCGGTGAGATTGG-3′ which amplify a 111-bp fragment; VEGF forward, 5′-CTGGGCTGTTCTCGCTTCG-3′ and reverse, 5′-CTCTCCTCTTCCTTCTCTTCTTCC-3′ which amplify a 140-bp fragment.
Quantitative real-time polymerase chain reaction (RT-qPCR)
The cDNAs were obtained and primers were used as previously described (13). The analysis using SYBR® Premix Ex Taq™ II from Takara Bio, Inc., the 20 µl PCR system was composed of 10 µl SYBR® Premix Ex Taq™ II, 0.4 µl ROX Reference Dye II, 6 µl ddH2O, 0.8 µl of 10 µM forward primer, 0.8 µl of 10 µM reverse primer and 2 µl cDNA. RT-qPCR amplification was performed under the following conditions: 95°C for 30 sec, 40 cycles of denaturation for 5 sec at 95°C, 34 sec of annealing at 60°C, elongation at 95°C for 15 sec, and extension at 60°C for 1 min. At least three independent experiments were conducted and samples were assessed in triplicate in each experiment.
Western blotting
Lysates from the cultured cells were sonicated and protein concentrations were determined using the Bradford reagent from Bio-Rad Laboratories. Equal amounts of protein (20 µg) were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), which were incubated in blocking buffer (5% non-fat dry milk in Tris-buffered saline and 0.1% Tween-20) for 1 h, followed by incubation with the primary antibodies overnight at 4°C and a 2-h incubation with HRP-conjugated secondary antibodies (1:20,000). Signals were visualised by enhanced chemiluminescence (Pierce, Rockford, IL, USA) associated fluorography, and their quantification was conducted by volume densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and total protein normalization.
MTT assay
Cell proliferation was investigated by MTT assay. Briefly, the cells (5×104/well) were plated into flat-bottom 24-well plates (Costar, Corning, NY, USA). After 24 h, the cells were serum-starved overnight and incubated with different concentrations of ISO and U0126 with or without ICI for different times. Following incubation, 20 µl MTT (5 mg/ml) was added to each well, and the cells were grown in complete media at 37°C for 3 h. The supernatant was removed, and then 500 µl DMSO was added to each well of the 24-well plate and oscillated for 10 min. Subsequently, the absorbance was read at 570 nm using an enzyme-linked immunosorbent assay reader.
Statistical analysis
Data are presented as means ± SEM from at least three independent experiments. Statistical analysis of the data was performed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
β<sub>1</sub>-AR and β<sub>2</sub>-AR expression in lung cancer cells
We first detected whether lung cancer cells express β-ARs. The results of reverse transcription-PCR analysis indicated that both β1-AR and β2-AR were expressed in the A549 cells (Fig. 1A). This was confirmed by RT-qPCR analysis (Fig. 1B). Interestingly, the levels of β2-AR mRNA were significantly higher as compared to β1-AR. This result suggests that β2-AR may be the predominant β-AR in A549 cells and this finding encouraged us to elucidate the role of β2-AR in these cells.
β<sub>2</sub>-AR activation promotes the proliferation of human A549 cells
We then investigated the effect of β-AR agonist ISO on the A549 cell proliferation. We first analyzed the dose-dependent effect of ISO on A549 cell viability. The A549 cells were incubated with 0.1, 1, 5, 10, 20 and 30 µM ISO for 24 h and then cell viability was detected by MTT assay. As shown in Fig. 2A, ISO significantly enhanced the cell viability of the A549 cells maximally by a single treatment with 10 µM ISO while cell viability decreased dramatically with higher doses of ISO. To demonstrate the effects of ISO at different treatment times on A549 cell viability, the cells were incubated with 10 µM ISO for 24, 48 or 72 h. Cell viability assays revealed that ISO significantly increased A549 cell growth at these three time points with the strongest effect at 48 h (Fig. 2B). Based on these results, we chose 10 µM ISO as the reference concentration and a 48-h treatment time for the subsequent studies unless specifically indicated. Importantly, pre-treatment of the A549 cells with the β2-AR-specific antagonist ICI to block endogenous β2-ARs blocked ISO-induced cell growth in a dose-dependent manner and was completely blocked following a 10 µM dose (Fig. 2C). These results suggest that ISO was able to promote A549 cell proliferation through activation of β2-AR. This is consistent with our previous data showing that β2-AR is highly expressed in A549 cells (Fig. 1).
β<sub>2</sub>-AR activation induces ERK<sub>1/2</sub> and CREB phosphorylation in A549 cells
We demonstrated that ISO significantly increased A549 cell growth. MAPK pathways constitute a large modular network that regulates a variety of physiological processes, such as cell growth, differentiation, and apoptotic cell death. To this end, we firstly monitored ERK1/2 phosphorylation levels following ISO treatment. ISO caused a rapid and transient increase in ERK1/2 phosphorylation with no changes in ERK1/2 expression levels (Fig. 3A and B). ERK1/2 phosphorylation peaked at 10 min and then decreased. Similarly CREB, an important transcription factor that plays important roles in cell proliferation, was also phosphorylated in a transient manner and also peaked at 10 min (Fig. 3A and C).
To further confirm the ERK1/2 and CREB phosphorylation mediated by β2-AR, the A549 cells were pre-treated with ICI and then stimulated by ISO. ERK1/2 and CREB phosphorylation was blocked by ICI (Fig. 4A and B). As CREB is an important downstream target of ERK1/2, we therefore tested whether ISO-induced CREB phosphorylation is mediated through ERK1/2. We treated A549 cells with the selective MEK1/2 inhibitor U0126. Consistent with our hypothesis, inhibition of the MAPK pathway led to a strong inhibition of ISO-induced ERK1/2 and CREB phosphorylation (Fig. 4C and D), thus suggesting that the activation of β2-AR by ISO induces ERK1/2 phosphorylation which in turn activates CREB.
MEK<sub>1/2</sub> and CREB inhibition suppresses ISO-mediated proliferation in A549 cells
Given the ability of ISO to significantly promote the proliferation of A549 cells and induce ERK1/2 and CREB phosphorylation, in order to further illustrate whether ISO-mediated ERK1/2 and CREB activation are involved in cell proliferation, A549 cells were pre-treated with U0126, and then treated with 10 µM ISO. The proliferative effects of ISO were suppressed by U0126 (Fig. 5A). Furthermore, knockdown of CREB expression by specific siRNA significantly suppressed the A549 cell proliferation in comparison with control siRNA (Fig. 5B and C). All the above results indicate that ERK1/2 is involved in ISO-mediated proliferation through CREB phosphorylation in A549 cells.
Knockdown of CREB inhibits ISO-mediated increases in MMP-2, MMP-9 and VEGF levels in A549 cells
MMPs, the gelatinases MMP-2 and MMP-9 in particular, and VEGF have been documented as contributing to the aggressiveness of highly metastatic tumors (14). We then examined the effect of ISO on the expression of MMP-2, MMP-9 and VEGF in the A549 cells. The RT-qPCR results showed that expression levels of the MMP-2, MMP-9 and VEGF genes were significantly upregulated after treatment with 10 µM ISO for 48 h (Fig. 6A). Interestingly, these effects were blocked by knockdown of CREB before ISO treatment (Fig. 6B–D), indicating that the action of ISO on A549 cell invasiveness is through β2-AR-mediated CREB phosphorylation.
Discussion
Neurotransmitters are signaling substances that traditionally play important roles in both the central and peripheral nervous systems. However, accumulating studies have demonstrated that the stress neurotransmitters adrenaline and noradrenaline have a direct influence on the migration and invasiveness of multiple tumor cells, including cancers of the lung, prostate, colon, stomach, breast and ovary (15–19). Although several studies have preliminarily investigated the role of β-ARs in lung cancer cell lines such as human lung adenocarcinoma cell line NCI-H322 and human peripheral airway cell line HPLD1 (11), the function of β-ARs especially β2-AR on human lung epithelial-derived A549 cells and the underlying mechanisms have not been well studied. In the present study, we investigated the molecular mechanisms of β2-AR involved in lung cancer cell proliferation. Our results showed that activation of the β2-AR by ISO significantly enhanced the proliferation of A549 cells. These effects were found to be mediated by the MAPK/CREB pathway as blocking the MAPK pathway or knockdown of CREB expression was able to suppress ISO-induced A549 cell proliferation.
β-ARs are constitutively expressed in most mammalian cells. However, the distribution and the expression level of these subtypes may vary from tissue to tissue or even from species to species in a given tissue (20). A much higher level of β2-AR mRNA was detected in hepatocellular carcinoma (HCC) tumor cells, while β1-AR mRNA was almost undetectable (21,22). Moreover, the β2-AR density in HCC cellular membranes was much higher than the β2-AR density in non-adjacent non-tumor liver cell membranes (23). β1-ARs predominated over β2-ARs in NCI-H322 and NCI-H441 cell lines derived from a human pulmonary adenocarcinoma with Clara cell phenotype (11,24). In the present study, both reverse transcription PCR and RT-qPCR results revealed that the expression level of β2-ARs was much higher as compared to β1-ARs in the A549 lung cancer cells. The presence of mRNA does not ensure that the protein is present in the cells in some cases. Nevertheless, none of the commercial antibodies targeting β-ARs work (25) and even much of what is known by using these antibodies has been thrown into doubt (26). However, this has been confirmed by the following functional assay showing that ISO treatment can promote A549 cell proliferation. Moreover, this effect can be completely blocked by ICI, a selective antagonist of β2-AR. Thus, this confirmed that β2-ARs are the predominant β-ARs in A549 cells.
As demonstrated in several studies, agonists binding to β-ARs activate the heterotrimeric Gαs proteins to stimulate adenylyl cyclase synthesis of cAMP. The transient cAMP flux can initiate multiple cellular processes via two major downstream effector systems including cAMP-dependent protein kinase (PKA) and guanine nucleotide exchange protein activated by adenylyl cyclase (EPAC) (8), both of which play important roles in cell morphology and motility. Previous studies suggest that β2-AR exerts an effect on carcinogenesis mainly through activating signaling via adenylyl cyclase and its downstream effectors cAMP, PKA, CREB and STAT3 as well as transactivation of the EGFR pathway. In NCI-H322 and HPLD1 lung cancer cell lines, NNK, an agonist for β-ARs, has been shown to upregulate ERK1/2 and CREB/ATF-1 phosphorylation (11). However, it is not known whether they are also the effectors downstream of β2-AR in A549 cells. In our study, we found that ISO treatment significantly induced ERK1/2 and CREB phosphorylation. The MEK1/2 inhibitor U0126 used to block ERK1/2 activity inhibited CREB activation. However, whether the EGFR transactivation pathway was involved in this process was not investigated. Moreover, in addition to heterotrimeric G proteins, more and more biochemical and cellular studies indicate that β2-ARs may induce ERK1/2 activation through a wide variety of intracellular proteins that are independent of heterotrimeric G protein activation (27). Among these intracellular proteins, β-arrestins and Src have been shown to initiate a broad variety of signaling events such as the activatation of ERK1/2 signaling upon β2-AR activation (28). These are also the possible candidate proteins that may be involved in β2-AR-induced ERK1/2 activaton in A549 cells.
The ability of tumor cells to invade the extracellular matrix plays an important role in invasion and metastasis. MMPs are key factors in the degradation of the components of the extracellular matrix and therefore have been regarded as major critical molecules assisting tumor cells during metastasis (29–31). Studies have shown that the catecholamine hormones may influence cancer progression by modulating the expression of MMPs and VEGF in ovarian cancer cells (19,32). In the present study, we found that ISO had a role in modulating the expression of MMP-2, MMP-9 and VEGF in the A549 cells, which may contribute to the aggressiveness of the highly metastatic types of lung cancer cells. Notably, the ISO-mediated upregulation of MMP-2, MMP-9 and VEGF was inhibited by the knockdown of CREB expression.