Amlodipine, a Ca2+ channel blocker, suppresses phosphorylation of epidermal growth factor receptor in human epidermoid carcinoma A431 cells

Life Sciences 86 (2010) 124–132

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Amlodipine, a Ca2+ channel blocker, suppresses phosphorylation of epidermal growth factor receptor in human epidermoid carcinoma A431 cells Junko Yoshida a,⁎, Takaharu Ishibashi a,b, Mei Yang a, Matomo Nishio a a b

Department of Pharmacology, School of Medicine, Kanazawa Medical University, Daigaku 1-1, Uchinada, Ishikawa 920-0293, Japan Department of Pharmacology, School of Nursing, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan

a r t i c l e

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Article history: Received 1 October 2009 Accepted 18 November 2009 Keywords: Calcium channel blocker Cell proliferation Epidermal growth factor receptor Tyrosine phosphorylation Caveolin-1 Cholesterol A431 cells

a b s t r a c t Aims: Amlodipine, a dihydropyridine Ca2+ channel blocker, inhibits the proliferation of human epidermoid carcinoma A431 cells in vitro and in vivo. This study examined the underlying mechanism of this antiproliferative effect in relation to epidermal growth factor receptor (EGFR) signaling. Main methods: The tyrosine phosphorylated active state of EGFR in A431 cells incubated with the test agents was evaluated by western blot with anti-phosphotyrosine antibody. EGFR phosphorylation levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates. Key findings: In vitro treatment of exponentially growing A431 cells with amlodipine decreased the tyrosine phosphorylation states of EGFR. Amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR and a membrane scaffolding protein, caveolin-1, in serum-starved A431 cells. Amlodipine attenuated the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 without affecting the EGFR/ caveolin-1 interaction. Crosslinking experiments showed that amlodipine also suppressed the EGFstimulated phosphorylation of EGFR predimers. Addition of cholesterol abolished these inhibitory effects of amlodipine plus its inhibition of cell growth. Furthermore, treatment of mice with amlodipine (10 mg/kg/ day × 7 days, i.p.) decreased the levels of phosphorylated EGFR in A431 xenograft tumors. Significance: The results indicated that amlodipine inhibits tyrosine phosphorylation of EGFR in vitro and in vivo, possibly via modulating cholesterol-rich, caveolin-1-containing membrane microdomains. © 2009 Elsevier Inc. All rights reserved.

Introduction Ca2+ channel blockers are used for treatment of hypertension, angina pectoris, and ventricular tachyarrhythmia because they can bind to and block voltage-dependent L-type Ca2+ channels (L-type VDCCs). Published data indicate that Ca2+ channel blockers have multiple actions; antioxidant, eliciting nitric oxide (NO) production, and inhibiting atherosclerosis via mechanisms distinct from L-type VDCCs blockade (Mason et al. 2003). The anti-atherosclerotic action of Ca2+ channel blockers might be due in part to inhibition of vascular smooth muscle cell growth (Pitt et al. 2000; Mancini 2002; Mason et al. 2003). This antiproliferative effect of Ca2+ channel blockers was also demonstrated in tumor cell lines, such as HT-39 human breast cancer cells (Taylor and Simpson 1992), human meningioma cells (Jensen and Wurster 2001), and LNCaP human prostate cancer cells (Rybalchenko et al. 2001). We recently demonstrated that amlodipine, a dihydropyridine derivative, inhibited the growth of human epidermoid carcinoma A431 cells in vitro and in vivo (Yoshida et al.

⁎ Corresponding author. Tel.: + 81 76 218 8107; fax: + 81 76 286 8191. E-mail address: [email protected] (J. Yoshida). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.11.014

2003, 2004), although this cell line lacks L-type VDCCs (Moolenaar et al. 1986). Fluorometric analysis of intracellular free Ca2+ concentrations revealed that Ca2+ channel blockers with antiproliferative activity including amlodipine specifically blunt capacitative Ca2+ entry elicited by intracellular Ca2+ store depletion (Yoshida et al. 2003, 2004). However, diltiazem, verapamil, and dihydropyridine derivative, nifedipine, which do not affect A431 cell growth, did not attenuate the capacitative Ca2+ entry. Therefore, we speculated that the inhibition of capacitative Ca2+ entry by amlodipine might be involved in the antiproliferative effect of Ca2+ channel blockers. In further studies, we reported by reverse transcription-polymerase chain reaction (RT-PCR) that instead of L-type VDCC subunits, A431 cells express canonical transient receptor potential 1 (TRPC1) and TRPC5, which are molecular candidates for store-operated Ca2+ channels mediating capacitative Ca2+ entry (Yoshida et al. 2005). We went on to examine the effect of amlodipine on cell cycle progression because intracellular Ca2+ must increase at several points during cell cycle progression (Clapham 2003; Kahl and Means 2003). We found that amlodipine arrests cell cycle progression at the G1 phase by inducing p21Waf1/Cip1, a cyclin-dependent kinase inhibitor, to reduce the phosphorylation of retinoblastoma protein (pRB), a regulator of G1 to S phase transition (Yoshida et al. 2007). We thus speculated that

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inhibiting capacitative Ca2+ entry probably through TRPC channels was partly responsible for the amlodipine-induced G1 cell cycle arrest and subsequent cell growth inhibition in A431 cells. To further explore the mechanism by which amlodipine inhibits cell growth, this study investigated the effects of amlodipine on EGFRrelated signaling in A431 cells. These cells overexpress EGFRs and the relevant signaling pathways involved in proliferation are well characterized. Amlodipine suppressed tyrosine phosphorylation of EGFR in A431 cells studied here both in vitro and in vivo. The results further suggested that amlodipine exerts its inhibitory effects by modulating plasma membrane microdomains that are abundant in cholesterol and caveolin-1.

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phosphate buffer, then dehydrated and embedded in Quetol 651 epoxy resin. Ultrathin sections (80 nm thickness) were stained with uranyl acetate and Sato's lead solution and examined using a JEOL JEM-1200 EX electron microscope (JEOL, Tokyo, Japan) at 80 keV. Cell viability assay The cells were plated at a density of 5 × 104/ml in 1 ml of DMEM containing 10% FBS in a 24-well plate and incubated for 24 h, followed by administration of agents as indicated. After incubation for another 48 h, viable cells were counted by trypan blue exclusion. Cell growth (% of control) was expressed as the percentage of cells in the test wells compared to the vehicle-control wells.

Materials and methods Preparation of total cell lysate Reagents and antibodies Amlodipine vesilate, donated by Dainippon Sumitomo Pharma (Osaka, Japan), was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. Bis(sulfosuccinimidyl) suberate (BS3) was obtained from Pierce Biotechnology (Rockford, IL). Water-soluble cholesterol and methyl-β-cyclodextrin (MβCD) were obtained from Sigma Aldrich (St Louis, MO). MβCD was dissolved in distilled water at 100 mM (based on a formula weight estimated as 1315). Rabbit polyclonal anti-EGFR, rabbit polyclonal anti-phosphoEGFR (Tyr992), rabbit monoclonal anti-Akt, rabbit monoclonal anti-phospho Akt (Ser473), rabbit polyclonal anti-p44/42 mitogen-activated protein kinase (MAPK), and mouse monoclonal anti-phosphop44/42 MAPK (Thr202/Tyr204) E10 antibodies (Abs) were purchased from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti-phosphocaveolin (pY14) Ab (clone 56) was obtained from BD Biosciences Pharmingen (Franklin Lakes, NJ). Mouse monoclonal anti-phosphotyrosine (clone 4G10) and mouse monoclonal anti-EGFR Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-β-actin Ab (clone AC-74) was purchased from Sigma Aldrich. Rabbit polyclonal anti-caveolin-1 (N-20), horseradish peroxidase-conjugated goat anti-mouse IgG, and horseradish peroxidaseconjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For reprobing western blotting membranes with other primary antibodies, the membranes were stripped for 30 min at 60 °C with WB Stripping Solution Strong (Nacalai Tesque, Kyoto, Japan). Tumor cell lines Human epidermoid carcinoma A431 cells were cultured as described previously (Yoshida et al. 2001, 2004, 2007). Briefly, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM), containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 12.7 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 0.12% sodium bicarbonate, 100 U/ml penicillin G, and 100 µg/ml streptomycin, at 37 °C in humidified air containing 5% CO2. Cells were seeded at a density of 5 × 105/plate in 10-cm-diameter plastic culture dishes and passaged every 3–4 days. Morphological studies with phase-contrast microscopy and electron microscopy A431 cells were grown in DMEM containing 10% fetal bovine serum (FBS) for 24 h and treated with vehicle (0.03% DMSO) or amlodipine for 24 h. Morphological changes in the cell monolayer were observed under an inverted microscope (Olympus IX70-22FL/ PH, Olympus, Tokyo, Japan) equipped with a digital camera system. For electron microscopy, A431 cells (N106 cells) were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4 °C. The cells were postfixed for 2 h at 4 °C with 1% osmium tetroxide in

A431 cells (1.0–1.2 × 106 cells) were grown in 10 ml of DMEM containing 10% FBS for 24 h and treated with vehicle or agents for the indicated time. In some experiments, A431 cells incubated in DMEM containing 10% FBS were serum-starved and treated with agents for the indicated time prior to EGF stimulation. The cells were lysed in 0.5 ml of mammalian cell lysis/extraction reagent (CelLytic™-M; Sigma Aldrich) containing protease inhibitors (Protease inhibitor cocktail; Sigma Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na3VO4. The extract was centrifuged at 12,500 ×g for 15 min at 4 °C and the protein concentration of the supernatant was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). A sample of cell lysate was mixed with a quarter volume of 5× sample buffer (625 mM Tris–HCl, 10% SDS, 25% glycerol, 0.015% bromophenol blue, and 5% 2-mercaptoethanol, pH 6.8) and denatured at 95 °C for 5 min for western blot analysis. Immunoprecipitation Total cell lysates containing equal amounts of protein (400–500 μg) were immunoprecipitated with a specific antibody for 1 h at 4 °C. Twenty μl of Protein A or G PLUS-agarose (Santa Cruz Biotechnology) was then added to the immunoprecipitate and rocked on a platform at 4 °C for 1 h or overnight. The immunoprecipitates were washed three times with radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA], 0.2 mM Na3VO4, 0.2 mM PMSF, 0.5% NP40, and 1% Triton X-100, pH 7.4), and then resuspended in 20 μl of 5× sample buffer and heated at 95 °C for 5 min. The supernatants were subjected to western blot analysis. SDS-PAGE and western blotting The samples (5–10 μg protein) were electrophoretically separated on gradient polyacrylamide gels (Daiichi Pure Chemical Co., Tokyo), and then electrotransferred to nitrocellulose membrane (HybondECL, Amersham Biosciences, Piscataway, NJ). Membranes were incubated with primary antibodies followed by the appropriate secondary antibody conjugates. Immunoreactive bands were visualized using an enhanced chemiluminescence system and exposure to Hyperfilm (Amersham Biosciences). Band intensities were analyzed using Image J 1.37v software (National Institutes of Health, Bethesda, MD). Crosslinking of EGFR Crosslinking experiments were performed as described previously (Ringerike et al. 2002) with minor modification. A431 cells (1 × 106 cells) were grown in 10 ml of DMEM containing 10% FBS for 24 h and then serum-starved for 24 h. Cells were treated with agents for 1 h at

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37 °C prior to EGF stimulation (10 ng/ml EGF for 20 min). The stimulated cells were washed twice with phosphate-buffered saline (PBS) and incubated for 30 min on ice with the membraneimpermeable crosslinking reagent BS3 (Pierce Biotechnology, Rockford, IL) at a final concentration of 2 mM in PBS. The reaction was terminated by adding 1 M glycine to a final concentration of 250 mM and further incubating on ice for 5 min. The cells were scraped loosely, transferred to test tubes and centrifuged at 420 × g for 5 min at 4 °C. The cell pellets were washed with ice-cold PBS and lysed in mammalian cell lysis/extraction reagent (CelLytic™-M; Sigma Aldrich) containing protease inhibitors, 1 mM PMSF, and 1 mM Na3VO4, and the lysate was clarified at 12,000 × g for 15 min. Supernatants were subjected to western blotting to detect EGFR and phosphotyrosine.

Subcellular fractionation Triton X-100-soluble and -insoluble membrane constituents were extracted as described previously (Solomon et al. 1998; Zhuang et al. 2002; Adachi et al. 2007) with minor modification. Serum-starved A431 cells were treated with agents for 1 h at 37 °C and stimulated with EGF (10 ng/ml) for 20 min. The cells were washed twice with 5 ml of PBS and lysed in 0.5 ml of ice-cold buffer A [25 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.5), 150 mM NaCl, 1% Triton X-100, protease inhibitors, 1 mM Na3VO4, and 1 mM PMSF] on ice for 30 min. The cell lysate was centrifuged at 14,000 ×g for 20 min at 4 °C to obtain detergent-soluble fractions (S). The insoluble pellet was washed with ice-cold buffer A by centrifugation at 14,000 ×g for 20 min, and the pellet was solubilized in buffer B [1% Triton X-100, 10 mM Tris (pH 7.6), 500 mM NaCl, 60 mM β-octylglucoside, protease inhibitors, 2 mM Na3VO4, and 1 mM PMSF] for 30 min on ice. Debris were pelleted in a microcentrifuge (14,000 ×g for 20 min) at 4 °C, and the supernatant was collected as the detergent-insoluble membrane fractions (I).

Determination of phosphoEGFR levels in A431 xenograft tumors PhosphoEGFR levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates. Male Balb/c, nu/nu mice (6 weeks of age) were used for these assays. Animal care and experiments were conducted according to the Institutional Animal Care and Use Guidelines. For immunostaining of matrigel plug sections, cultured A431 cell suspension in serum-free DMEM (6×106cells/ml) was mixed with two volumes of phenol red-free Matrigel matrix (BD Biosciences, Bedford, MA) and 500 μl of Matrigel matrix containing A431 cells (1 × 106 cells) was subcutaneously (s.c.) injected into mice anesthetized with diethyl ether by inhalation. Injected Matrigel rapidly formed a single, solid gel plug under the skin. Then, amlodipine (10 mg/kg) or vehicle (0.1 ml of 1% DMSO in PBS/10 g of body weight) was injected intraperitoneally (i.p.) into 5 mice of each group once daily for another 6 days. One day after the last injection, mice were sacrificed by cervical dislocation and the gels were removed by retaining the overlying skin and mucous membrane. These gels were fixed in 4% paraformaldehyde phosphate buffer solution, embedded in paraffin, and sectioned at 5-μm thickness. After deparaffinization and epitope retrieval in 10 mM citrate buffer (pH 6.0), the sections were stained with anti-human phosphorylated EGFR antibody (Tyr992, Cell Signaling Technology) at a dilution of 1:50, and the nuclei were counterstained with hematoxylin. For western blotting for phosphoEGFR in tumor homogenates, A431 cells (1 × 106cells) were inoculated s.c. into mice (3 mice of each group) and treated with amlodipine (10 mg/kg/day) or vehicle as a control for 7 days as described above. One day after the last injection, xenograft tumors were excised from mice and shockfrozen in liquid N2. The frozen tissue samples were pounded with a percussion mortar, and the resulting powder was homogenated in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.1% SDS,

Fig. 1. Effects of amlodipine on A431 cell morphology. (A) Phase-contrast micrographs of cells incubated with vehicle (0.03% DMSO) or amlodipine (20, 30 μM) for 24 h at 37 °C. Control A431 cells grew as a flattened, squamous cell layer on plastic culture plates, while the cells exposed to amlodipine tended to aggregate. Original magnification ×400. (B) Scanning electron micrographs of cells incubated in the absence (a) and presence of vehicle (0.03% DMSO) (b) or 30 μM amlodipine (c) for 24 h. Centrally located nucleus, nuclear membrane liner, mitochondria, and vacuoles or lipid droplets were similarly observed in (a), (b), and (c). Original magnification ×2500 (scale bar = 2 μm).

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0.5% deoxycholate, 1 mM EDTA, 50 mM NaF, 1 mM Na4P2O7, 1 mM Na3VO4, protease inhibitor mixture (Sigma), 2 mM PMSF]. Homogenates were clarified by centrifugation at 14,000 × g for 30 min at 4 °C and the supernatants served for western blot analysis for phosphoEGFR.

Statistical analysis Values were expressed as mean ± SEM. Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by Fisher's PLSD or the unpaired Student's t-test. A P value less than 0.05 was considered statistically significant.

Results Effects of amlodipine on morphology of A431 cells We showed previously that treatment of A431 cells with amlodipine for 48 h inhibited cell growth in a concentrationdependent manner with an IC50 value of 25 μM (Yoshida et al. 2003). Here, we examined the morphological changes in A431 cells incubated with amlodipine for 24 h at 37 °C. Under phase-contrast microscopy, A431 cells exposed to amlodipine tended to aggregate, while control A431 cells grew as a typical flattened squamous cell layer (Fig. 1A). This effect was evident by 1 h incubation with 30 μM amlodipine (data not shown). However, transmission electron microscopic analysis showed no ultrastructural differences between vehicle (0.03% DMSO)-treated and 30 μM amlodipine-treated A431 cells (Fig. 1B).

Fig. 2. Amlodipine attenuates tyrosine phosphorylation of EGFR in the exponentially growing human epidermoid carcinoma A431 cells. Cells were cultured in complete medium and treated with either vehicle (0.03% DMSO) or amlodipine (20, 30 μM) for 24 h. Cell lysates were subjected to immunoprecipitation (IP) with anti-EGFR antibody (Ab) (UBI). Immunoprecipitates were assayed by western blotting for phosphotyrosine and EGFR. Representative result (A) and summary of data for the relative levels of phosphorylated EGFR (phosphotyrosine) to total EGFR protein (% of control; B) are shown. Data in B are mean ± SEM of 5–6 independent experiments. *P b 0.05, **P b 0.01 by one-way analysis of variance followed by Fisher's PLSD.

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Amlodipine attenuates tyrosine phosphorylation of EGFR in proliferating human epidermoid carcinoma A431 cells Cell lysates from A431 cells incubated with amlodipine or vehicle (0.03% DMSO) in the presence of 10% FBS for 24 h were immunoprecipitated with anti-EGFR antibody. The immunoprecipitates were western blotted for phosphotyrosine and EGFR (Fig. 2A and B). Amlodipine reduced the tyrosine phosphorylation state of EGFR (180 kDa) in a concentration-dependent manner. Amlodipine attenuates EGF-stimulated EGFR phosphorylation in serum-starved A431 cells The effect of amlodipine on EGF-stimulated EGFR phosphorylation was then investigated. Serum-starved A431 cells were incubated with either vehicle (0.03% DMSO) or amlodipine (30 μM) for 1 h prior to stimulation with EGF (10 ng/ml) for 20 min. Total cell lysates (5 μg protein/lane) were analyzed by western blot using antibodies for the indicated proteins (Fig. 3). Amlodipine treatment attenuated the phosphorylation of both EGFR at Tyr992 and Akt/protein kinase B at Ser473; Akt/PKB is a downstream regulator of EGFR signaling that plays a critical role in cell proliferation. However, amlodipine did not affect the EGF-stimulated phosphorylation of extracellular-regulated kinase (ERK)1/2, despite this process being activated by binding of EGF to EGFR (Fig. 3). These results suggest that amlodipine treatment attenuates downstream Akt/PKB-related signaling induced by EGFR phosphorylation, but not similarly activated downstream ERK1/2related pathways. In A431 cells, EGF also stimulates the phosphorylation of caveolin-1, a membrane scaffolding protein, on Tyr14 (Lee

Fig. 3. Amlodipine attenuates the EGF-stimulated phosphorylation of EGFR and caveolin-1 in serum-starved A431 cells. Serum-starved A431 cells were incubated in the absence or presence of 30 μM amlodipine for 1 h and stimulated with EGF (10 ng/ ml) for 20 min. Total cell lysates (5 μg protein/lane) were separated on 8–16% gradient SDS-polyacrylamide gels, electrotransferred to membranes, and immunoblotted with Abs for the indicated proteins. Data are representative results of three independent experiments.

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et al. 2000; Kim et al. 2002), although not in adipocytes (Kim et al. 2002) or fibroblasts (Kim et al. 2000). Our results showed that an antiphosphocaveolin-1 antibody (clone 56) recognized tyrosine-phosphorylated caveolin-1 migrated as two bands in EGF-stimulated A431 cells, which was consistent with the report of Lee et al. (Lee et al. 2000). The EGF-stimulated phosphorylation of caveolin-1 was significantly attenuated by amlodipine treatment (Fig. 3). Amlodipine attenuates the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 We next examined whether amlodipine might affect the association between EGFR and caveolin-1 at the plasma membrane, since previous studies have coimmunoprecipitated these two proteins in A431 cells (Couet et al. 1997; Kim et al. 2002). To this end, caveolin-1 immunoprecipitates were electrophoresed on SDS-polyacrylamide gels and western blotted for EGFR, caveolin-1, and phosphotyrosine (Fig. 4). A 180-kDa, EGFR-immunoreactive protein was detected in the caveolin-1 immunoprecipitates (Fig. 4A). Reprobing the membrane with an anti-phosphotyrosine antibody (4G10) showed EGFstimulated tyrosine phosphorylation of the 180-kDa EGFR in the

caveolin-1 immunoprecipitates. Amlodipine did not affect the caveolin-1/EGFR association, but decreased the EGF-induced tyrosine phosphorylation of EGFR (Fig. 4A, lanes 1–3). The detection of caveolin-1 in EGFR immunoprecipitates confirmed the caveolin-1/ EGFR association (Fig. 4B). In this assay, we also examined the effect of cholesterol on the amlodipine-induced inhibition of EGFR phosphorylation, because plasma membrane cholesterol has been implicated in various cell functions, including cell survival (Ringerike et al. 2002; Zhuang et al. 2005; Li et al. 2006). Cholesterol–cyclodextrin complexes were added to the cells in culture to increase the concentration of cholesterol in membranes (Christian et al. 1997), i.e., water-soluble cholesterol premixed in methyl-β-cyclodextrin (MβCD) was added to the culture 10 min prior to amlodipine addition. As shown in Fig. 4A (lanes 4 and 5), an increase in membrane-bound cholesterol did not alter the EGF-induced tyrosine phosphorylation of EGFR in caveolin-1 immunoprecipitates; however, it did reverse the inhibitory effect of amlodipine. Meanwhile, the effect of cholesterol on the growth inhibitory effect of amlodipine in A431 cells was examined. Addition of water-soluble cholesterol as described above effectively diminished the antiproliferative effect of 30 μM amlodipine in A431 cells (Fig. 4C).

Fig. 4. Amlodipine attenuates the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1. Serum-starved A431 cells were treated as described in Fig. 3. Water-soluble cholesterol (20 μM) was added 10 min prior to amlodipine addition. (A) Total cell lysates (450 μg protein) were immunoprecipitated with anti-caveolin-1 Ab. The immunoprecipitates were separated on 8–16% gradient SDS-polyacrylamide gels, electrotransferred to membranes, and then immunoblotted for EGFR. The membranes were then stripped and reprobed with anti-phosphotyrosine Ab (4G10). Equal loading of caveolin-1 in the immunoprecipitates was confirmed by immunoblotting with anti-caveolin-1 Ab. (B) Cell lysate was also immunoprecipitated with anti-EGFR Ab (UBI) and immunoblotted for caveolin-1 and phosphotyrosine. Data are representative results of four independent experiments. (C) Effect of cholesterol on the antiproliferative effect of amlodipine in A431 cells. A431 cells were plated and incubated for 24 h, then incubated further in the presence or absence of amlodipine for 2 days. Water-soluble cholesterol was added 10 min prior to amlodipine addition. Cell growth (% of control) was determined by trypan blue exclusion. Data are mean ± SEM of five independent experiments. *P b 0.05 vs. 30 μM amlodipine alone by one-way ANOVA followed by Fisher's PLSD.

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Amlodipine attenuates the EGF-induced phosphorylation of EGFR dimers in A431 cells

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Amlodipine decreased the phosphoEGFR levels in A431 xenograft tumors

We next examined whether amlodipine affects ligand-induced EGFR dimerization and autophosphorylation (Ferguson 2004) by crosslinking experiments using water-soluble crosslinker BS3, which reacts with primary amino groups (-NH2) in cell-surface proteins via a charged group to form stable amide bonds. The result revealed that EGFR dimers were present in both the presence and absence of EGF stimulation, and that once the cells were stimulated with EGF, EGFR dimers were tyrosine phosphorylated (Fig. 5). Amlodipine decreased this EGF-induced phosphorylation of EGFR dimers without affecting the EGFR dimerization (Fig. 5), and this effect of amlodipine was again abolished by adding water-soluble cholesterol (Fig. 5, lanes 4 and 5).

Our previous study demonstrated that 10 mg/kg amlodipine injected i.p. per day for 20 consecutive days significantly retarded tumor growth and prolonged the survival of mice bearing A431 xenografts (Yoshida et al. 2004). To examine the mechanism underlying this in vivo effect, we measured the status of EGFR phosphorylation in the xenograft tumors. Fig. 7A shows representative immunostaining of matrigel plug sections. Phosphorylated EGFR was clearly membrane-localized in all plugs of control (vehicletreated) mice, while amlodipine treatment decreased the intensity of phosphoEGFR immunostaining. Western blot analysis for phosphoEGFR in tumor homogenates showed that amlodipine treatment (10 mg/kg/day for 7 days) decreased significantly the phosphoEGFR levels in A431 xenograft tumors (Fig. 7B).

Subcellular fractionation

Discussion

Experimental evidence suggests that EGFR activation is regulated by cholesterol-containing membrane microdomains known as lipid rafts (Ringerike et al. 2002). Conversely, EGFR might rapidly move out of caveolae, a specialized form of lipid raft, in response to EGF (Mineo et al. 1999). We examined possible effect of amlodipine on the EGFR localization in the plasma membrane by successive detergent extractions. Serum-starved A431 cells were pretreated with amlodipine for 1 h and stimulated with EGF (10 ng/ml) for 20 min. Triton-soluble (S) and Triton-insoluble (I) fractions were isolated as described in Materials and methods. Under this condition, 3.7 ± 0.9% (n = 6) of total protein (S + I) was fractionated in I fractions of control (vehicle-treated) cells. Western blot analysis of the fractions revealed that cytosolic heat shock protein 90 (Hsp90) was detected predominantly in the S fraction, as expected (data not shown). As is typical, caveolin-1 was more abundant in I than S fractions, while EGFR was predominantly Triton-soluble (Fig. 6). EGF caused substantial phosphorylation of EGFR and caveolin-1 in both fractions. Amlodipine treatment diminished this phosphorylation without affecting the localization of either protein in the plasma membrane. Surprisingly, strongly phosphorylated protein bands at around 360 kDa, corresponding to EGFR dimers, were detected in I fractions in response to EGF stimulation, despite hardly any 360-kDa EGFR protein detected in those insoluble extracts (Fig. 6). The result suggests that trace amounts of EGFR predimers in I fractions were efficiently tyrosine phosphorylated by EGF, and that amlodipine treatment also diminishes the EGF-induced phosphorylation of EGFR predimers.

Taylor and Simpson (1992) initially demonstrated that amlodipine inhibits the growth of HT-39 human breast cancer cells in vitro and in vivo, and suggested a correlation between the ability of amlodipine to lower intracellular calcium levels and its antitumor activity. Liao et al. (2005) then reported that amlodipine ameliorates cardiac hypertrophy by inhibiting EGFR phosphorylation. They demonstrated that amlodipine could inhibit epinephrine-induced EGFR phosphorylation in cultured neonatal rat cardiomyocytes and ameliorate myocardial hypertrophy induced by left ventricular pressure overload in mice. The present study demonstrates for the first time that amlodipine suppresses EGFR phosphorylation both in vitro and in vivo in the A431 tumor cell line. The initial experiments reported here confirmed that amlodipine suppresses EGFR tyrosine phosphorylation in both exponentially growing and serum-starved, EGF-stimulated A431 cells. The same agent also inhibited the EGF-induced phosphorylation of Akt/PKB, a downstream regulator of EGFR signaling, and caveolin-1 in serumstarved A431 cells. To examine whether the inhibition of EGFR phosphorylation in A431 cells is associated with modulating effects of amlodipine on the plasma membrane, the remaining experiments focused on EGFR, caveolin-1 and their assembly in the plasma membrane. Caveolin-1, 22-kDa integral membrane protein, is a major component of membrane invaginations known as “caveolae”, and has been reported to interact with several intracellular signaling molecules via the caveolin-1 scaffolding domain peptide, including EGFR, eNOS, G-protein α subunits, PKCα, H-Ras, and Src-family kinase (Razani et al. 2002). In endothelial cells, amlodipine induced

Fig. 5. Amlodipine attenuates the EGF-induced phosphorylation of EGFR dimers in A431 cells. Serum-starved A431 cells were treated as described in Fig. 3. Water-soluble cholesterol (20 μM) was added 10 min prior to amlodipine addition. The EGFR was crosslinked by BS3 (2 mM) before cell lysis. Cell lysates (10 μg protein/lane) were separated on 2–15% gradient SDS-polyacrylamide gels, electrotransferred to membranes, and then immunoblotted for EGFR. The blots were stripped and reprobed with anti-phosphotyrosine Ab (4G10). Bands at 180 kDa represent EGFR monomers, while 360-kDa bands represent EGFR dimers. Data are representative results of three independent experiments.

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Fig. 6. Subcellular fractionation of serum-starved and EGF-stimulated A431 cells. Serum-starved A431 cells were incubated in the absence or presence of amlodipine (30 μM) for 1 h and stimulated with EGF (10 ng/ml) for 20 min. Cells were fractionated to Triton X-100-soluble (S) and -insoluble (I) fractions as described in Materials and methods. Five μg of each fraction (protein) was electrophoresed then subjected to western blot analysis. The top part of the blot was probed with anti-EGFR Ab (Cell Signaling Technology), stripped, and then reprobed with anti-phosphotyrosine Ab (4G10). The bottom part of the blot was probed with anti-caveolin-1 Ab and reprobed with anti-phosphorylated caveolin-1 Ab. Lane m; protein molecular standard marker. Data are representative results of four independent experiments.

endothelial nitric oxide synthase (eNOS) translocation from caveolinrich low-density membranes to high-density membranes, thus dissociating eNOS from caveolin and potentiating NO production (Batova et al. 2006). In contrast, amlodipine did not affect the coimmunoprecipitation of EGFR and caveolin-1 in this study. However, it attenuated the EGF-induced phosphorylation of EGFR associated with caveolin-1 in the A431 cells. The crosslinking studies performed here further revealed that EGFR predimers exist in A431 cells regardless of the state of EGF stimulation, a result that is consistent with some previous studies (Sako et al. 2000; Yu et al. 2002). Yu et al. (2002) indicated that EGFR forms a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separate events. Sako et al. (2000) also showed preformed EGFR dimers before binding of the second EGF molecule using a single-molecule visualization technique. In the present study, once the serum-starved A431 cells were stimulated with EGF, the EGFR predimers were tyrosine phosphorylated, and this activation by EGF was significantly decreased by pretreatment with amlodipine. Subcellular fractionation using Triton X-100 further showed that EGF effectively tyrosine phosphorylates EGFR dimers in detergent-insoluble membranes, and

that this phosphorylation is suppressed by amlodipine treatment. Taken together with the crosslinking studies, the subcellular fractionation findings thus implied that the inhibitory effect of amlodipine on EGF-stimulated EGFR activation occurred mainly in the detergent-insoluble, caveolin-1-containing membrane microdomains of A431 cells. We next found that the addition of water-soluble cholesterol abolished the inhibitory effects of amlodipine. Both the suppression of EGF-stimulated EGFR phosphorylation and cell growth inhibition induced by amlodipine were reversed by cholesterol, whereas cholesterol itself affected neither EGFR phosphorylation nor cell growth. These results suggest that an increase in the cholesterol content of plasma membrane might affect the bioavailability of amlodipine. Amlodipine is an amphipathic molecule with a charged 2-aminoethoxymethyl substitution at the 2-position of the dihydropyridine ring. This charged side group of amlodipine induces strong electrostatic binding with anionic oxygen groups in membrane phospholipid headgroups (Mason et al. 1989, 1992). By X-ray diffraction and equilibrium binding techniques, Mason et al. (1992) demonstrated that increases in the membrane cholesterol content markedly decreased the binding of dihydropyridine Ca2+ channel blockers to

Fig. 7. Amlodipine suppresses EGFR phosphorylation of A431 cells in vivo. A. Matrigel matrix containing A431 cells were s.c. injected into athymic mice. The mice were then i.p. injected with amlodipine (10 mg/kg per day) or vehicle consecutively for 7 days. The Matrigel plugs were excised and the sections were stained with anti-human phosphorylated EGFR Ab, and the nuclei were counterstained with hematoxylin (see Materials and methods). Representative immunostaining sections of plugs from the vehicle-treated and amlodipine-treated mice are shown. Original magnification × 400. B. Athymic mice inoculated s.c. with A431 cells were treated with amlodipine (10 mg/kg per day) or vehicle for 7 days. Tumors (∼116 mm3, calculated by the formula “π/6 × L × W2, where L is the long axis and W is the short axis of the tumor”) were excised, lysed, and the lysates (100 μg protein each) served for western blot analysis as described in Materials and methods. The blotting membranes were probed with anti-phosphotyrosine Ab (4G10) and reprobed with antiEGFR-Ab (Cell Signaling). The bottom part of the blot was probed with anti-β-actin Ab as a loading control. pEGFR/EGFR: the ratio of phosphorylated EGFR to total EGFR (mean ± SEM, n = 3), *P b 0.05 versus vehicle-control group by the unpaired Student's t-test.

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cardiac phospholipid membranes. Although this previous study indicated that the charged amlodipine was least affected by membrane cholesterol content due to its high membrane affinity, it is possible that the relatively higher concentration of cholesterol (20–30 μM) used in our experiments may have interfered with partitioning of the amlodipine in the membrane bilayer. Furthermore, in smooth muscle cell membranes, amlodipine restored the cholesterol-expanded membrane bilayer width without any effect on membrane fluidity (Kahn et al. 2005). Based on these reports and our results, it is feasible that amlodipine locates to the cholesterol-rich, caveolin-1-containing membrane microdomains, where it acts to suppress EGFR signaling. We reported previously that amlodipine diminishes the capacitative Ca2+ entry elicited by phospholipase C-coupled receptor stimulation with UTP (Yoshida et al. 2004). Many recent studies also demonstrated that cholesterol-rich lipid raft domains in the plasma membrane regulate functions of store-operated Ca2+ channels mediating capacitative Ca2+ entry and the functions of its molecular components, the transient receptor potential canonical (TRPC) family of proteins (Ambudkar et al. 2004; Brownlow et al. 2004; Brownlow and Sage 2005; Kannan et al. 2007; Alicia et al. 2008; Pani et al. 2008). An important role of caveolae/caveolin-1 in capacitative Ca2+ entry was also demonstrated in various cell types including pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension (Patel et al. 2007), endothelial cells (Kwiatek et al. 2006), human submandibular gland cells, and Madin–Darby canine kidney cells (Brazer et al. 2003). Thus, amlodipine might partition in the cholesterol-rich, caveolin-1-containing lipid raft domains of A431 cells to attenuate capacitative Ca2+ entry in a similar manner to its suppression of EGFR activation. It is not clear whether the partitioning effect of amlodipine in the membrane microdomains is associated with the morphological changes under phase-contrast microscopy. To examine whether amlodipine decreases EGFR activation in A431 xenograft tumors, we assessed the EGFR tyrosine phosphorylation levels by the immunostaining of Matrigel plug sections and western blot for phosphoEGFR in A431 xenograft tumors. The mice injected s.c. with A431 cells with or without Matrigel Matrix were treated with amlodipine for 7 days. The immunostaining of Matrigel plug sections showed that amlodipine (10 mg/kg/day) suppressed the tyrosine phosphorylation state of EGFR in the A431 tumor xenograft. Furthermore, western blots for phosphoEGFR showed that amlodipine (10 mg/kg/day) decreased significantly the tyrosine phosphorylation levels of EGFR in the tumor homogenates. Although the steady-state serum drug concentration was not determined, the final concentration of amlodipine was calculated to be approximately 1.53 μM based on an apparent distribution volume of 16 ± 4 l/kg for amlodipine (Meredith and Elliott 1992) and its molecular weight of 408.9, suggesting that amlodipine suppressed the tyrosine phosphorylation of EGFR in vivo at lower dosages than those in vitro. The possibility that host responses to tumors were involved in the antitumor action of amlodipine cannot be ruled out. Further experiments are needed to investigate the mechanism(s) underlying the in vivo antitumor effect of amlodipine. In conclusion, the present study demonstrated that amlodipine suppresses phosphorylation of EGFR in vitro and in vivo, and that this effect might involve, at least in vitro, a membrane-modulating effect on cholesterol-rich, caveolin-1-containing membrane microdomains. Amlodipine is thus a potential lead compound in a new class of agents for potentially inhibiting EGFR-related mitogenic signaling. Further studies on such agents could yield new antitumor drugs that act by associating functionally with mitogenic signaling components in the membrane microdomains of target cells. Conflicts of interest statement None declared.

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