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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Beijing Digestive Diseases Center and Beijing Friendship Hospital, Capital Medical University, Beijing, China (X.L., S.T.Z.); and Departments of Pharmacology (W.K.K.W., L.Y., Z.J.L., C.H.C.) and Medicine and Therapeutics (W.K.K.W., J.J.Y.S.) and Institute of Digestive Diseases (W.K.K.W., J.J.Y.S., C.H.C.), Chinese University of Hong Kong, Hong Kong, China
Received November 20, 2007; accepted March 26, 2008.
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Abstract |
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; Ono and Kuwano, 2006). A large number of preclinical studies also indicate that EGFR-mediated signaling directly contributes to angiogenesis (Pore et al., 2006), cell invasion and metastasis (Thomas et al., 2003), and resistance to apoptosis (Kari et al., 2003) that are related to tumor progression. Clinically, the development of EGFR-directed therapeutics represents one of the most promising directions in targeted cancer therapy (Zhang et al., 2006; Harari et al., 2007). EGFR signaling is initiated by the binding of its ligands, such as epidermal growth factor (EGF), transforming growth factor 
, heparin-binding EGF-like growth factor, and amphiregulin, followed by homodimerization or heterodimerization of the receptor with other EGFR family members. The dimerized receptors (i.e., ErbB1/ErbB1, ErbB1/ErbB2, ErbB1/ErbB3, ErbB1/ErbB4) were then internalized and autophosphorylated on their tyrosine residues, which triggered diverse downstream signaling pathways including the Ras/mitogen-activated protein kinases (Roberts and Der, 2007), the phosphatidylinositol 3-kinase/Akt (Sordella et al., 2004), the phospholipase C
(Kauffmann-Zeh et al., 1995), and the Janus kinase-signal transducers and activators of transcription pathways (Park et al., 1996).
β-Adrenoceptor, a classical G-protein-coupled receptor, originally identified as an important regulator of cardiac contractility and smooth muscle relaxation, is now emerging as a multifunctional cell surface catecholamine sensor that regulates a repertoire of cellular processes such as cell proliferation, migration, and apoptosis (Schuller, 2007). In the classic paradigm of β-adrenergic signaling, receptor activation results in dissociation of the heterotrimeric G-protein, in which the Gs subunit stimulates adenylyl cyclase to produce cAMP and activates the downstream protein kinase A (PKA)-mediated pathway. Intriguingly, β2-adrenoceptor may also couple to Gi, which inhibits adenylyl cyclase, in addition to Gs, to elicit a differential response (Daaka et al., 1997). In relation to carcinogenesis, β-adrenergic stimulation has been shown to promote the growth of human breast (Cakir et al., 2002), pulmonary (Masi et al., 2005; Schuller and Cekanova, 2005), and pancreatic (Weddle et al., 2001; Askari et al., 2005) carcinoma cells, as well as colon cancer cell migration and proliferation (Masur et al., 2001; Wu et al., 2005). Furthermore, we discovered that colon cancer cells can secrete epinephrine in an autocrine manner to self-stimulate cell growth via β-adrenoceptors, and the secretion is augmented by the nicotinic stimulation through the up-regulation of catecholamine-synthesizing enzymes (Wong et al., 2007a). It is also reported that the antiapoptotic effects of nicotine on human lung cancer cells can be reversed by the β-adrenoceptor antagonist (Jin et al., 2004). The findings that β-adrenergic antagonists retard the growth of melanoma and fibrosarcoma in the nude mouse xenograft model further reiterate the importance of β-adrenoceptor in carcinogenesis (Hasegawa and Saiki, 2002), and the effect has been suggested to be mediated through the up-regulation of arachidonic acid cascade and the subsequent release of prostaglandin E2 (Cakir et al., 2002; Wu et al., 2005).
The phenomenon that certain signal components can be shared between different signaling pathways, which may in turn interact with one another to form networks, highlights the intriguing complexity of biological system in the mitogenic process of different cells. Therefore, it comes as no surprise that several studies have sought to delineate the interactions between EGFR and the β-adrenoceptor. In this connection, there is culminating evidence suggesting that activation of the β-adrenoceptor and other G-protein-coupled receptors can transactivate EGFR and the subsequent mitogen-activated protein kinase cascades that mediate cell proliferation in different physiological context (Yeh et al., 2005; Noma et al., 2007). Whether the stimulation of EGFR can, in reverse, transactivate β-adrenoceptors, however, has not been studied. In the present study, we aim to elucidate the possible linkage, if any, from EGFR to β-adrenoceptor and its biological significance in the context of cell proliferation in an esophageal squamous cell carcinoma cell line, HKESC-1, that is responsive to the mitogenic stimulation of epinephrine.
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Materials and Methods |
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; Baker, 2005). Tyrosine hydroxylase, β1-adrenoceptor, β2-adrenoceptor, and PKA antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture and Drug Treatment. HKESC-1 cell line was established from a primary moderately differentiated squamous cell carcinoma of the esophagus from a 47-year-old Hong Kong Chinese man (Hu et al., 2000). The cells were cultured in minimum essential medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin G, and 100 µg/ml streptomycin and maintained at 37°C, 95% humidity, and 5% carbon dioxide. HKESC-1 cells were plated at a density of 4 x 104 cells/well in 24-well plates. After 24 h of incubation for cell attachment, the cells were starved in 0.1% fetal bovine serum-containing medium for another 12 h to synchronize the cell cycle. EGF at different concentrations was incubated with the cells for 24 h to study the mitogenic effect. To examine the effects of various inhibitors, cells were pretreated with or without AG1478 (2.5 nM), atenolol (100 µM), or ICI 118,551 (50 µM) for 45 min before EGF treatment. The concentrations of β-adrenoceptor antagonists used in the present study have been previously employed to study the mitogenic function of β-adrenoceptors in colon cancer cells (Wu et al., 2005; Wong et al., 2007a).
Cell Proliferation Assay. Cell proliferation was measured as the amount of DNA synthesis or inferred by the metabolic activity using [3H]thymidine incorporation assay or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) proliferation assay, respectively. For [3H]thymidine incorporation assay, cells were incubated in the absence or presence of EGF for 24 h or epinephrine for 4 h with or without different inhibitors and then incubated with 0.5 µCi/ml [3H]thymidine (GE Healthcare, Chalfont St. Giles, UK) for another 4 h. The cells were then washed with ice-cold 0.15 M NaCl, followed by 10% trichloroacetic acid, and incubated for 15 min at room temperature. After several washings, 1% SDS was added and incubated for another 15 min at 37°C. Finally, hydrophilic scintillation fluid was added into the vial, and the amount of DNA synthesized was measured using liquid scintillation spectrometry on a beta counter (Beckman Coulter, Inc., Fullerton, CA). For MTT proliferation assay, after treatment, MTT solution dissolved in the culture medium at the final concentration of 0.5 mM was added to each well, and the plates were incubated for another 4 h. Dimethyl sulfoxide was then added to solubilize MTT tetrazolium crystal. Finally, the optical density was determined at 570 nm using a Benchmark Plus microplate reader (Bio-Rad, Hercules, CA).
Conventional and Quantitative Reverse Transcription-Polymerase Chain Reaction. The total cellular RNA was isolated from HKESC-1 cells using TRIzol reagent. The RNA concentration was measured by GeneQuant II (GE Healthcare) at 260 nm. The same amount of total RNA (5 µg) was used to generate the first strand of cDNA by reverse transcription (Invitrogen) in accordance with the manufacturer's instructions. Specific primers (Table 1) as described previously (Wu et al., 2005; Wong et al., 2007a) were used to screen the expression of β1- and β2-adrenoceptors and four catecholamine-synthesizing enzymes, namely, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine β-hydroxylase, and phenylethanolamine-N-methyltransferase. The PCR conditions were as follows: the template cDNA was first denatured at 94°C for 5 min. During 35 cycles of amplification, the denaturation step was at 94°C for 1 min, the annealing step at 56°C for 1 min, and the extension step at 72°C for 1 min. The final extension step was at 72°C for 7 min. The PCR products were electrophoresed on a 1.0% agarose gel containing 0.5 µg/ml ethidium bromide. For the quantitation of tyrosine hydroxylase expression, real-time PCR was performed with specific primers purchased from QIAGEN (Valencia, CA). Conditions for quantitative PCR were 94°C for 5 min, 45 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Quantitative PCR was carried out using iQ SYBR Green Supermix (Bio-Rad) and Multicolor Real-Time PCR Detection System (Bio-Rad) as recommended by the manufacturer. The results were analyzed using the comparative threshold cycle method with β-actin as an internal control. The specificity of the PCR product was confirmed by melting curve analysis and DNA gel electrophoresis.
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Western Blot Analysis for β-Adrenoceptors, Tyrosine Hydroxylase, and PKA. HKESC-1 cells were harvested in radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 0.5% -cholate acid, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, and 10% glycerol) containing proteinase and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM NaVO3, and 1 mM NaF). Protein was quantified using a protein assay kit (Bio-Rad). Equal amounts of protein (40 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Hybond C nitrocellulose membranes (GE Healthcare). The membranes were probed with primary antibodies overnight at 4°C and incubated for 1 h with secondary peroxidase-conjugated antibodies. They were developed with an enhanced chemiluminescence system (GE Healthcare) and exposed to an X-ray film (FUJI Photo Film Co., Ltd., Tokyo, Japan). Quantitation was carried out with a video densitometer (ScanMaker III; Microtek International, Inc., Hsinchu, Taiwan).
cAMP Assay. Intracellular cAMP assay was performed according to the manufacturer's recommendation (GE Healthcare). In brief, 1 x 106 cells were treated in the presence or absence of EGF at 10 ng/ml for 12 h. The cAMP level was then measured by the enzyme immunoassay method. The cAMP level was expressed as picomoles per milligram of protein.
Determination of Epinephrine Release. Extracellular epinephrine assay was performed according to the manufacturer's recommendation (IBL-Hamburg, Germany). In brief, 1 x 106 cells were treated in the presence or absence of EGF at 10 ng/ml for 12 h. The epinephrine level was then measured by the enzyme-linked immunosorbent assay method.
Statistical Analysis. Results were expressed as the mean ± S.E.M. Statistical analysis was performed with an analysis of variance followed by the Turkey's t test. P values less than 0.05 were considered statistically significant.
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HKESC-1 Cell Line-Expressed β-Adrenoceptors and Catecholamine-Synthesizing Enzymes. Because β-adrenoceptors appeared to mediate the mitogenic action of EGF in esophageal cancer cells, the mRNA and protein expression of β1- and β2-adrenoceptors was determined in HKESC-1 cells using reverse transcription-PCR and Western blot analysis, respectively. Results showed that both the mRNA and protein of β1- and β2-adrenoceptors were detected in HKESC-1 cells (Fig. 2, A and B). Because de novo synthesis of epinephrine and autocrine stimulation of β-adrenoceptors might occur in cancer cells, we also determined the expression of catecholamine-synthesizing enzymes tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine β-hydroxylase, and phenylethanolamine-N-methyltransferase in HKESC-1 cells. Reverse transcription-PCR revealed that all of the four catecholamine-synthesizing enzymes were expressed in HKESC-1 cells (Fig. 2A). In addition, the protein expression of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, was further confirmed by Western blot analysis (Fig. 2B). Furthermore, epinephrine at the dose of 10 µM enhanced [3H]thymidine incorporation and MTT tetrazolium crystal formation in HKESC-1 cells, which was reduced by β-adrenoceptor antagonists atenolol or ICI 118,551 (Fig. 2, C and D).
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). However, whether EGF could transactivate β-adrenoceptors via a similar mechanism is unknown. Results showed that treating the cells with EGF for 12 h significantly increased the mRNA (Fig. 4A) and protein expression (Fig. 4B) of tyrosine hydroxylase, accompanied by an increased cellular release of epinephrine (Fig. 4C). In this respect, EGF markedly increased epinephrine levels by approximately 2-fold. To this end, the up-regulation of tyrosine hydroxylase and epinephrine release induced by EGF was abrogated by EGFR tyrosine kinase inhibitor (Fig. 4, A–C).
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EGF Increased the Expression of β1-Adrenoceptor Expression and PKA. Although the results presented so far indicated that EGF transactivated β-adrenergic signaling via the increased de novo synthesis of epinephrine; however, whether EGF altered the expression of other components in the β-adrenoceptor-mediated pathway is unknown. Therefore, we determined β1-, β2-adrenoceptor, and PKA expression in EGF-treated HKESC-1 cells. Results revealed that EGF significantly increased the expression of β1-adrenoceptor and PKA but had no effect on β2-adrenoceptor expression, all of which could be abolished by the EGFR tyrosine kinase inhibitor (Fig. 5.
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; Ono and Kuwano, 2006; Schuller, 2007), but the connection between these two receptor systems is not completely understood. In the present study, we demonstrate that the esophageal cancer cell line HKESC-1 expresses the mRNA and protein of β1- and β2-adrenoceptors and increases cell proliferation in response to epinephrine stimulation. In this connection, EGF increases the intracellular cAMP levels and cell proliferation, which are attenuated by EGFR kinase inhibitor or β-adrenoceptor antagonists. These results indicate that β-adrenoceptors are functionally activated by EGF to mediate the mitogenic effect. To date, β-adrenoceptors have been reported to take part in the promotion and progression of different kinds of neoplasm (Masur et al., 2001; Weddle et al., 2001; Cakir et al., 2002; Hasegawa and Saiki, 2002; Askari et al., 2005; Masi et al., 2005; Schuller and Cekanova, 2005; Wu et al., 2005; Wong et al., 2007b). Here, we demonstrate for the first time that transactivation of β-adrenoceptors may act as an effector pathway to mediate the mitogenic effect of EGF (Fig. 6).
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Our results show that the mitogenic effect of EGF on HKESC-1 cells is attenuated by β-adrenoceptor antagonists. Because the antagonistic action of pharmacological blockers depends on the presence of agonists, we hypothesize that the transactivation of β-adrenoceptors by EGF is ligand-dependent. In this respect, we show that HKESC-1 cells express all four enzymes responsible for the synthesis of epinephrine and norepinephrine. Treating the cells with EGF further increases the mRNA and protein expression of tyrosine hydroxylase, which is the rate-limiting enzyme for catecholamine synthesis. Measurement of epinephrine in the culture medium further reveals that EGF enhances the cellular release of epinephrine. Above all, all of these changes can be reversed by the EGFR kinase inhibitor, suggesting that EGF stimulation results in augmented autocrine secretion of epinephrine. Although the concentration of epinephrine was released by EGF from HKESC-1 cells, which is considerably lower than that of exogenous epinephrine to stimulate cell proliferation, the local concentration of epinephrine in the vicinity of the receptors may be remarkably high due to the active synthesis and release of the catecholamine from the cells. This could get easy access to the adjacent receptors at the cell surface to stimulate cell growth. Moreover, it has been reported that high expression of β-adrenoceptors, in particular the β2-adrenoceptors, was found in cancer cells (Schuller and Cekanova, 2005; Wong et al., 2007a). In addition, the possible but unlikely interfering effects of β-adrenoceptor antagonists on EGF binding to EGFR have not been excluded in this study, but further experiments investigating the receptor-binding property of radioisotope-labeled EGF in the presence or absence of β-adrenoceptor antagonists may help resolve this issue.
The finding that tyrosine hydroxylase is a target of EGFR in HKESC-1 esophageal cancer cells is also in line with previous studies reporting that the expression of this gene is regulated by EGFR signaling in the nervous system (Yamada et al., 1996; Iwakura et al., 2005). For instance, it has been reported that EGF increases the levels of tyrosine hydroxylase in the rat pheochromocytoma PC12 cells (Yamada et al., 1996). Coregulation of EGF and tyrosine hydroxylase has also been observed in the prefrontal cortex and the striatum of patients with Parkinson's disease (Iwakura et al., 2005). This evidence, along with our experimental findings, converge to suggest that tyrosine hydroxylase gene expression is under the control of EGFR signaling. To this end, it has been shown that increased activity of transcription factor activator protein-1 up-regulates tyrosine hydroxylase gene expression at the transcriptional level in striatal neurons. Moreover, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitors eliminate tyrosine hydroxylase expression and the associated activator protein-1 changes (Guo et al., 1998). It is therefore speculated that EGFR signaling may cross-talk with β-adrenoceptors through extracellular signal-regulated kinase-dependent activator protein-1-mediated up-regulation of tyrosine hydroxylase and the subsequent release of catecholamine from cancer cells. However, other mechanisms accounting for the release of catecholamine by EGF need further investigation.
Aside from up-regulating the cellular release of epinephrine, EGF stimulation also alters β-adrenergic signaling by increasing the protein expression of PKA and, to a lesser extent, β1-adrenoceptors. The mechanism and functional consequence of these changes, however, remain unknown, but it is highly possible that, by up-regulating PKA and β1-adrenoceptors, the cells are rendered more sensitive to the autocrine stimulation of epinephrine. Moreover, up-regulation of PKA may also switch the coupling of the β2-adrenergic receptor to different G-proteins (Daaka et al., 1997). On the other hand, PKA can in reverse activate the enzymatic activity of tyrosine hydroxylase enzyme and induce the transcription of the tyrosine hydroxylase gene (Joh et al., 1978; Kim et al., 1993), posing the possibility that the up-regulation of PKA may enable a positive feedback loop of autocrine release of epinephrine in EGF-treated cells. To this end, activating adenylyl cyclase by forskolin or cholera toxin to boost the endogenous cAMP production or the use of cAMP analogs may further clarify the role of the cAMP/PKA pathway in the mitogenic effect of EGF and epinephrine. It is also worthwhile to notice that repeated or prolonged β-adrenergic stimulation can desensitize the classical cAMP/PKA pathway and activate alternative signaling pathway mediated by β-arrestin (Tilley and Rockman, 2006). Moreover, the expression of β-adrenoceptors can be down-regulated by the continuous stimulation of β-adrenergic agonists. The relevance of this desensitization mechanism at the receptor level to the responsiveness of cancer cells to the mitogenic stimulation of EGF, however, remains to be examined.
The EGFR signaling pathway plays a pivotal role in the control of cell proliferation, which is fundamental to carcinogenesis. Molecules involved in the EGFR signaling therefore become attractive targets in pathway-directed cancer therapy (Zhang et al., 2006; Harari et al., 2007). Results from clinical trials indicate that therapies directed against EGFR are promising in the treatment of a variety of cancers, including esophageal cancer (Karamouzis et al., 2007). In this study, we show that transactivation of β-adrenoceptors is required for the mitogenic effect of EGF. This finding implicates the β-adrenoceptor antagonists may be used as therapeutic agents, which are of low cost and whose toxicity profiles are well characterized, to target at the EGFR signaling. Indeed, it has been reported that the use of β-adrenoceptor antagonists is associated with a lower cancer risk in human (Pahor et al., 1996; Algazi et al., 2004; Ronquist et al., 2004), suggesting that β-adrenoceptor antagonists may be used as chemoprophylactic agents for the prevention of cancer. Moreover, blockade of β-adrenoceptors in this study almost completely reverses the mitogenic effect of EGF, suggesting that administration of β-adrenoceptor antagonists may achieve a remarkable antimitogenic effect comparable with that of EGFR kinase inhibitors or monoclonal antibodies, which are currently used in clinical settings. Nevertheless, whether blockade of β-adrenoceptors could limit the promoting effects of EGF on other cellular processes such as cell survival, invasion and metastasis, and angiogenesis requires further elucidation. To conclude, the present study not only demonstrates that EGF up-regulates β-adrenergic signaling at multiple levels to mediate its mitogenic signal but also opens up novel therapeutic opportunity for the use of β-adrenoceptor antagonists as inhibitors of EGFR signaling. The therapeutic efficacy of β-adrenoceptor antagonists in the treatment of cancers, however, awaits further clinical investigation.
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L.X. and W.K.K.W. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PKA, protein kinase A; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; atenolol, (+)-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]benzeneacetamide; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PCR, polymerase chain reaction; Ate, atenolol; ICI, ICI 118,551; TH, tyrosine hydroxylase.
Address correspondence to: Dr. Chi Hin Cho, Department of Pharmacology, 4/F Basic Medical Sciences Building, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China. E-mail: chcho{at}cuhk.edu.hk
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, P < 0.05; 
