The α1-adrenoceptor-mediated human hyperplastic prostate cells proliferation is impaired by EGF receptor inhibition

Journal Pre-proof The α1-adrenoceptor-mediated human hyperplastic prostate cells proliferation is impaired by EGF receptor inhibition Jessica Barbosa Nascimento-Viana, Rocío Alcántara-Hernández, Eliane OliveiraBarros, Luiza A. Castello Branco, Priscilla R. Feijó, Luiz Antonio Soares Romeiro, Luiz Eurico Nasciutti, François Noël, J. Adolfo García-Sáinz, Claudia Lucia Martins Silva PII:

S0024-3205(19)30975-0

DOI:

https://doi.org/10.1016/j.lfs.2019.117048

Reference:

LFS 117048

To appear in:

Life Sciences

Received Date: 17 June 2019 Revised Date:

24 October 2019

Accepted Date: 5 November 2019

Please cite this article as: J.B. Nascimento-Viana, R. Alcántara-Hernández, E. Oliveira-Barros, L.A. Castello Branco, P.R. Feijó, L.A. Soares Romeiro, L.E. Nasciutti, F. Noël, J.A. García-Sáinz, C.L. Martins Silva, The α1-adrenoceptor-mediated human hyperplastic prostate cells proliferation is impaired by EGF receptor inhibition, Life Sciences, https://doi.org/10.1016/j.lfs.2019.117048. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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The α1-adrenoceptor-mediated human hyperplastic prostate cells proliferation is

2

impaired by EGF receptor inhibition

3 4

Jessica Barbosa Nascimento-Viana1, Rocío Alcántara-Hernández2, Eliane

5

Oliveira-Barros3, Luiza A. Castello Branco3, Priscilla R. Feijó1, Luiz Antonio

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Soares Romeiro4, Luiz Eurico Nasciutti3, François Noël1, J. Adolfo García-

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Sáinz2, Claudia Lucia Martins Silva1*

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*Corresponding author

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Claudia Lucia Martins Silva

11

Laboratory of Molecular and Biochemical Pharmacology, Universidade Federal

12

do Rio de Janeiro. Av Carlos Chagas Filho, 373. Zip code 21941-902, Rio de

13

Janeiro,

14

[email protected]

Brazil.

e-mail:

[email protected],

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1

17

Federal do Rio de Janeiro;

18

Nacional Autónoma de México;

19

Program, Universidade Federal do Rio de Janeiro; 4Pharmaceutical Sciences

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(LAR), Universidade de Brasília

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Laboratory of Molecular and Biochemical Pharmacology, Universidade 2

Instituto de Fisiología Celular, Universidad 3

Cell Biology and Development Research

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Keywords: adrenoceptor, benign prostatic hyperplasia

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Running title: α1-adrenoceptor-mediated BPH cell proliferation is impaired by

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EGFR inhibition

30 31

Abbreviations

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AG – AG1478

33

BPH - benign prostatic hyperplasia

34

CK - cytokeratin

35

EGF - epidermal growth factor

36

EGFR - epidermal growth factor receptor

37

GM – GM6001

38

GPCR – G protein-coupled receptor

39

HB-EGF - pro-ligand heparin-binding epidermal growth factor

40

LUTS - lower urinary tract symptoms

41

NA - noradrenaline

42

Phe – phenylephrine

43

Vim - vimentin

44

3

45

Abstract

46

Benign prostatic hyperplasia (BPH) is an aging related and progressive

47

disease linked to an up-regulation of α1-adrenoceptors. The participation of

48

EGF receptors (EGFR) in the GPCRs´ signalosome has been described but so

49

far data about the contribution of these receptors to prostatic stromal

50

hyperplasia are scanty. We isolated and cultured vimentin-positive prostate

51

stromal cells obtained from BPH patients. According to intracellular Ca2+

52

measurements, cell proliferation and Western blotting assays, these cultured

53

hyperplastic stromal cells express functional α1-adrenoceptors and EGFR, and

54

proliferate in response to the α1-adrenoceptor agonist phenylephrine.

55

Interestingly, in these cells the inhibition of EGFR signaling with GM6001,

56

CRM197, AG1478 or PD98059 was associated with full blockage of α1-

57

adrenoceptor-mediated cell proliferation, while cell treatment with each inhibitor

58

alone did not alter basal cell growth. Moreover, the co-incubation of AG1478

59

(EGFR inhibitor) with α1A/α1D-adrenoceptor antagonists showed no additive

60

inhibitory effect. These findings highlight a putative role of EGFR signaling to

61

α1-adrenoceptor-mediated human prostate hyperplasia, suggesting that the

62

inhibition of this transactivation cascade could be useful to reduce BPH

63

progression.

64 65

4

66 67

1. Introduction Benign prostatic hyperplasia (BPH) is a progressive condition that

68

affects aging men.

The epithelial and stromal hyperplasia occurs in the

69

periurethral prostatic transition zone favoring the occurrence of the lower

70

urinary tract symptoms suggestive of BPH (LUTS/BPH). Prostate enlargement

71

and the enhanced prostatic smooth muscle contraction are responsible for the

72

static and dynamic components of BPH, respectively, and both components

73

reduce bladder outflow favoring urinary retention [1-3].

74

The α1-adrenoceptor belongs to the G protein-coupled receptors

75

(GPCRs) family being composed by three receptor subtypes known as α1A-,

76

α1B- and α1D-adrenoceptors [4]. The adult human prostate expresses mainly

77

α1A-adrenoceptors particularly in the stroma, and its expression is increased

78

during BPH [5-9]. Therefore, it is well accepted that the mechanism underlying

79

the BPH-related raise of smooth muscle contraction involves mainly the

80

increased prostate expression of α1A-adrenoceptors [5,6]. On the other hand,

81

α1D-adrenoceptors have been implicated in prostatic cell proliferation [6,10,11]

82

and their mRNA expression is also up regulated in BPH patients [5,6]. α1A-

83

adrenoceptor blockers such as tamsulosin and silodosin are the first line drugs

84

to treat low to moderate LUTS/BPH favoring bladder emptying, but they do not

85

modify prostatic enlargement progression or the risk of BPH complications [12].

86

The etiology of prostate enlargement is complex with some evidence

87

pointing to the involvement of metabolic and endocrine disorders, including

88

roles of peptide growth factors, such as the epidermal growth factor (EGF)

89

[2,13,14].

5

90

Two large families of membrane receptors, namely GPCRs and receptor

91

tyrosine kinases, regulate cell proliferation among other cell functions. G

92

protein-coupled receptors’ signaling involves canonical and non-canonical

93

pathways. In the case of α1-adrenoceptor, the canonical pathway mediated by

94

G protein involves the activation of phospholipase Cβ and the increase of

95

intracellular Ca2+ [4]. On the other hand, the non-canonical receptor signaling

96

activates other intracellular pathways including growth factor receptors

97

signaling in a process known as transactivation [15-17].

98

The participation of EGF receptors in the GPCRs´ signalosome is

99

essential for physiological and disease-related conditions such as mitogenesis,

100

inflammation, and muscle contraction [17-23]. Moreover, the pioneer work of

101

Oganesian and colleagues [23] showed that the constitutive activity of naturally

102

occurring variant of α1A-adrenoceptor transfected to Rat-1 fibroblasts lead to

103

cell proliferation. EGF receptors belong to the ErbB/HER protein family and are

104

expressed in normal human prostate mainly in the epithelial cells [24,25].

105

However, the expression of these receptors in prostate from BPH patients

106

expands to stromal cells [25], where α1-adrenoceptors are mainly expressed,

107

with EGF inducing stromal cell proliferation [26,27]. Moreover, the availability

108

of EGF receptor ligand depends on matrix metalloproteinases activity, which in

109

turn may be activated by GPCR [15,24].

110

Previously we characterized two N-phenylpiperazine derivatives named

111

LDT3 and LDT5 as new α1A/α1D-adrenoceptor antagonists, and we showed that

112

the α1D-adrenoceptor antagonist BMY7378 as well as LDT3 and LDT5 inhibit

113

human prostatic cell proliferation in vitro [28,29], while another α1D-

114

adrenoceptor antagonist, naftopidil, inhibits cell proliferation in vivo [30].

6

115

Therefore, we explored the possible role of α1-adrenoceptor-mediated

116

transactivation of EGF receptor leading to mitogenesis of stromal cells from

117

BPH patients.

118

involved in the α1-adrenoceptor-mediated proliferation of human hyperplastic

119

prostatic cells providing insight into the understanding of BPH physiopathology,

120

and suggesting that this transactivation cascade could be a target to reduce

121

BPH progression.

Our results indicate that EGF receptor transactivation is

122 123

2. Material and Methods

124

2.1.

Cell culture maintenance:

125

Prostate tissue samples were collected from three patients with

126

LUTS/BPH during transurethral resection and immediately placed in ice-cold

127

Dulbecco's modified Eagle's medium (DMEM) and transported to the

128

Laboratory. The inclusion criteria of the patients were IPSS greater than 8

129

points, 50-70 years of age and PSA less than 2.5 ng/mL. Exclusion criteria

130

were: previous prostatic surgery, prostate cancer, use of drugs that interfere

131

with the function of adrenoceptors such as antagonists, anti-androgens and

132

antidepressants, herbal extract, bladder catheter or neurogenic bladder. This

133

study was approved by the ethics committee of the Federal University of Rio de

134

Janeiro (UFRJ; CAAE-0029.0.197.000-05; 2009) and all experiments were

135

performed in accordance with relevant guidelines and regulations. Informed

136

written consent was signed by all donors. Briefly, prostate tissue was washed

137

in phosphate buffer solution (PBS) before minced. The prostate fragments (1

138

mm3) were added to DMEM supplemented with 10% heat-inactivated fetal

139

bovine serum containing 1 mg/mL type I collagenase. Tissue specimens were

7

140

dissociated by magnetic bar constant stirring for 2 - 4h at 37 oC. The recovered

141

cells were seeded in 25 mm3 flasks cultured in DMEM supplemented with 10%

142

heat-inactivated

143

penicillin/streptomycin (100 IU/mL and 0.1 mg/mL, respectively), and fungizone

144

(25 µg/mL) at 37 °C and 5% CO 2. Cells were observed at phase contrast

145

microscopy (x250) to analyze morphology and confluence. At sub-confluence

146

(approximately

147

trypsin/EDTA, and plated. A homogeneous stromal cell culture was obtained

148

after six passages. The subsequent steps were the sub-culture of the cells and

149

storage at -70 oC until use. Cells were used at 9th - 11th passage since the initial

150

cell harvesting [31].

fetal

90%

bovine

serum,

confluence),

cells

1%

sodium

were

pyruvate

harvested

and

using

1%

0.05%

151

Rat-1 fibroblasts stably expressing human α1A- or α1D-adrenoceptors

152

were cultured in DMEM supplemented with 10% fetal bovine serum, 300 µg/mL

153

neomycin analogue G418 sulfate, 1% penicillin/streptomycin (100 IU/mL and

154

0.1 mg/mL, respectively; 37 °C, 5% CO 2) until confluence. The receptor density

155

of α1-adrenoceptors estimated with [3H]-prazosin is in the range of 1.0 – 1.5

156

pmol/mg of protein [28].

157 158

2.2.

Immunofluorescence assay:

159

To analyze BPH cell morphology we used an anti-human cytokeratin

160

(CK) monoclonal antibody (Dako) and anti-vimentin (Vim) monoclonal antibody

161

(Sigma) as primary antibodies. For this procedure, 2x104 cells (11th passage)

162

were plated and incubated for four days. Following, the culture medium was

163

removed and cells were fixed with absolute cold ethanol for 20 min at room

164

temperature, and then washed three times with PBS. The nonspecific binding

8

165

was blocked with PBS/BSA 5% and then primary antibodies were added to the

166

cultures and incubated for 2h at room temperature. After that, cells were

167

washed with PBS and incubated for an additional 2h with goat anti-mouse

168

Alexa 546 secondary antibody (Invitrogen). Cell nuclei were counterstained

169

with DAPI (Santa Cruz Biotechnology). Finally, cells were washed in distilled

170

water and mounted on histological slides with N-propylgallate (Sigma).

171

Negative control conditions were performed by omitting the primary antibodies.

172

No reactivity was observed when the primary antibody was absent. Images

173

were captured using an inverted microscopy (Olympus IX81) and a

174

Hamamatsu ORCA-R2 digital CCD camera using a 40x objective.

175 176

2.3.

Intracellular [Ca2+] measurement:

177

In order to verify if primary stromal cell cultures from BPH patients

178

express functional α1-adrenoceptors we used a fluorimetric assay to measure

179

the intracellular Ca2+ concentration ([Ca2+]i), a robust marker of the canonical

180

Gq signaling. Cells were serum-starved overnight. In the next day, cells were

181

washed with PBS and loaded with 2.5 µM fura-2/AM in the dark for 60 min at

182

37 °C in Krebs-Ringer-HEPES solution containing (mM ) NaCl 120, KH2PO4

183

1.2, MgSO4 1.2, KCl 4.75, glucose 10, CaCl2 1.2, HEPES 20, and 0.05%

184

bovine serum albumin (pH 7.4). Thereafter, the cells were washed to remove

185

the unincorporated dye, detached by gentle trypsinization, centrifuged (200 x g,

186

7 min, 4 oC), and incubated (106 cells/condition) for 100 sec (baseline) and

187

then stimulated with 10 µM noradrenaline (NA) or 100 µM phenylephrine (Phe).

188

The antagonists (BMY7378, 50 nM; WB4101, 50 nM or tamsulosin, 5 nM) were

189

pre-incubated for 100 sec before addition of the agonists, and their

9

190

concentrations were previously defined based on their affinities [28].

191

Fluorescence measurements were performed at 340 and 380 nm excitation

192

wavelengths and 510 nm emission wavelength, with a chopper interval set at

193

0.5 sec, using an Aminco-Bowman Series 2 luminescence spectrometer

194

(Rochester, NY). Peak fluorescence values were used for data analysis, and

195

the intracellular Ca2+ concentration ([Ca2+]i) was calculated, as described by

196

Grynkiewicz et al. [32]

197 198

2.4.

Cell growth assays:

199

Briefly, cells (3 x 103 cells/well) were plated in 96-well plates in serum-

200

free DMEM for 24h. Sub-confluent cells were kept in DMEM (basal) or

201

stimulated with 3 µM phenylephrine (Phe) for 48h in the absence or presence

202

of GM6001 (10 µM; metalloproteinase inhibitor), CRM197 (200 ng/mL; HB-EGF

203

inhibitor), AG1478 (5 µM; selective EGFR tyrosine kinase inhibitor) or the MEK

204

inhibitor PD98059 (1 µM) added 30 min before addition of the agonist. EGF

205

100 ng/mL (48h) was used as positive control. All concentrations were chosen

206

based on their affinities for the targets and previously reported in the literature

207

[19,22]. Alternatively, cells were also treated with 3 µM phenylephrine for 48h

208

in the absence or presence of LDT3 or LDT5 (50 nM) alone or in combination

209

with AG1478 (5 µM) added 30 min before. The medium was changed every

210

24h with fresh dilutions of drugs. Cell growth was evaluated by counting of

211

viable cells using Trypan blue as an exclusion dye or by the 3-(4,5-

212

dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [28].

213

Otherwise indicated, each assay was performed in quadruplicate to calculate

10

214

the mean value of one experiment. Data were expressed as the percentage of

215

the basal condition which was considered as 100%.

216 217

2.5.

Western blot assays:

218

BPH cells (1 x 106) seeded on 6-well plate were incubated with 100 µM

219

phenylephrine (Phe) or 10 µM noradrenaline (NA; in the presence of 1 µM

220

propranolol added 2 min before) for 2, 5, 15, and 30 min in serum free medium.

221

After treatment, cells were lysed with 200 µL of RIPA buffer (150 mM NaCl, 1

222

mM EGTA, 10% glycerol, 1% Triton X-100, 0.1% SDS, 1.5 mM MgCl2, 10 mM

223

sodium pyrophosphate, 1 mM sodium orthovanadate, 100 mM NaF, 10 mg/mL

224

aprotinin, 10 mg/mL leupeptin, and 50 mM Tris-HCl, pH 7.4) under agitation,

225

centrifuged at 13,000 x g for 5 min (4 oC) and the supernatants were used for

226

evaluation of activation (phosphorylation) of extracellular signal-regulated

227

kinases 1 and 2 (ERK 1/2). Protein content was determined according to the

228

method of Lowry et al. Twenty µg of proteins were boiled for 5 min and

229

resolved using 10% SDS/PAGE. After electrophoresis, proteins were

230

transferred to nitrocellulose membranes and incubated for 1h with Tris buffered

231

saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% non-fat milk. Then,

232

membranes were washed 3 times in TBS-T and incubated overnight with the

233

primary

234

Thr202/Tyr204, #9101; 1:1000), or p44/p42 MAPK Total (ERK 1/2, #4695;

235

1:1000) diluted in TBS-T with 5% non-fat milk at 4oC with gentle shaking. The

236

peroxidase-conjugated anti-rabbit IgG secondary antibody (#7074; 1:10000)

237

was diluted in cold TBS-T and incubated for 1h. Membranes were washed in

238

TBS-T and detection of immunoreactivity was performed by enhanced

monoclonal

antibodies

phospho-p44/p42

MAPK

(p-ERK

1/2

11

239

chemiluminescence (ECL, Westar Cyanagen). Protein blot images were

240

scanned and the optical density was obtained using ImageJ software (NIH,

241

USA). The relative expression of p-ERK was normalized in relation to total ERK

242

(also used as loading control).

243 244

2.6.

Human androgen receptor binding assay

245

Filtration based radioligand binding assays for human prostate androgen

246

receptors (LNCap cells; catalogue number 0933) using 1 nM [3H]-

247

methyltrienolone (4oC, 24h) were performed by Eurofins Cerep SA, France

248

(Study 100018032). Mibolerone (1 µM) was used as positive control and

249

showed an IC50 value of 1.9 nM. LDT3 and LDT5 were tested at the final

250

concentration of 1 µM and the results were expressed as the percentage of the

251

specific binding of [3H]-methyltrienolone (considered as 100%).

252 253

2.7.

Reagents and antibodies:

254

LDT3 and LDT5 were synthesized as previously described [28]. The

255

following reagents were acquired from the sources indicated between

256

parenthesis: (R)-(-)-phenylephrine hydrochloride, (±)-propranolol hydrochloride,

257

(-)-noradrenaline, fungizone, EGF, CRM197 and reagents for RIPA buffer

258

(Sigma Aldrich, USA). Fura 2/AM (Molecular Probes, USA).

259

modified Eagle's medium (DMEM), fetal bovine serum (South America),

260

trypsin, penicillin (10000 IU/mL)/streptomycin (10 mg/mL), PD98059 and other

261

reagents for cell culture (Life Technologies, USA). AG1478 (Calbiochem,

262

USA). GM6001 (Merck Millipore, USA). Rabbit monoclonal primary antibodies

263

against extracellular signal-regulated kinases (ERK) 1/2 (#4695), and phospho-

Dulbecco's

12

264

ERK 1/2 (#9101) (Cell Signaling, USA). Monoclonal anti-human cytokeratin

265

antibody (#0821, Dako, USA).

266

(#V2258, Sigma Aldrich, USA). Anti-rabbit IgG secondary antibody (#7074, Cell

267

Signaling, USA) and anti-mouse Alexa 546 secondary antibody (#A11003,

268

Invitrogen, USA).

Monoclonal anti-human vimentin antibody

269 270

2.8.

Statistical analysis:

271

Otherwise indicated, data are expressed as mean and S.E.M. of 3-5

272

independent experiments. Independent experiments were performed using

273

different primary cell cultures. The significance of the differences among two or

274

more conditions was determined by two-tailed Student´s t test or one-way

275

analysis of variance (one way ANOVA), respectively. ANOVA was followed by

276

Dunnett's multiple comparisons test using the software GraphPad Prism 6.0

277

(Graphpad, La Jolla, CA, USA). Differences were considered statistically

278

significant if P < 0.05.

279 280

3. Results

281

3.1.

BPH stromal cells express functional α1-adrenoceptors

282

The cultured hyperplastic cells are positive for vimentin and negative for

283

cytokeratin staining which is a signature of stromal cells (Fig. 1). The canonical

284

α1-adrenoceptor signaling is linked to the increase of intracellular Ca2+

285

concentration [Ca2+]i.

286

expressing native α1-adrenoceptors with either 100 µM phenylephrine or 10 µM

287

noradrenaline induced similar increments of [Ca2+]i in relation to basal, which

As shown in Figure 2, the stimulation of BPH cells

13

288

indicates that the receptors are functional.

We chose the selective agonist of

289

α1-adrenoceptor phenylephrine to perform the subsequent experiments.

290

The agonist specificity for α1-adrenoceptor was confirmed by measuring

291

the intracellular Ca2+ concentration in Rat-1 fibroblasts stably expressing α1A-

292

or α1D-adrenoceptors subtypes, the two adrenoceptor subtypes relevant for

293

BPH. Cell pre-incubation with the antagonists WB 4101 (50 nM; α1A-

294

adrenoceptor

295

antagonist), but not with BMY7378 (50 nM, α1D-adrenoceptor antagonist),

296

blocked the effect of 100 µM phenylephrine in cells expressing α1A-

297

adrenoceptors, while BMY7378 (50 nM) blocked the agonist effect only in cells

298

expressing α1D-adrenoceptors (Fig. 3A and 3B).

antagonist)

and

tamsulosin

(5

nM,

α1A/D-adrenoceptor

299 300 301

3.2.

The EGF receptor modulates the α1-adrenoceptor-mediated BPH cell growth

302

The presence of EGF receptors in the stromal cells was evaluated by

303

cell proliferation. Cells were stimulated with EGF (100 ng/mL) in the absence

304

and presence of an antagonist. As shown in Figure 4A, the EGF-induced BPH

305

cell proliferation was blocked by the EGF receptor tyrosine kinase inhibitor

306

(AG1478, 5 µM). AG1478 per se did not alter basal cell growth discarding a

307

putative cytotoxicity. Interestingly, phenylephrine (3 µM) increased BPH cell

308

proliferation similarly to EGF. Then, in order to evaluate a possible dependence

309

on the EGF receptor for the α1-adrenoceptor-mediated BPH cell proliferation,

310

we investigated if the blockage of downstream EGF receptor signaling could

311

alter phenylephrine-mediated cell growth.

14

312

GM6001 (10 µM) is a pan inhibitor of the matrix metalloproteinases

313

involved in pro-ligand heparin-binding epidermal growth factor (HB-EGF)

314

cleavage, and consequently EGF receptor signaling [23]. GM6001 treatment

315

did not alter the basal cell proliferation but prevented cell proliferation induced

316

by phenylephrine (Fig. 4B), and the specific inhibitor of human HB-EGF,

317

CRM197 (200 ng/mL) [18], had the same effect. The selective EGF receptor

318

tyrosine kinase inhibitor AG1478 (5 µM) also prevented completely the effect of

319

phenylephrine, without altering the basal cell proliferation. Cells were treated

320

with the MEK inhibitor PD98059 (1 µM) which also inhibited cell proliferation in

321

response to phenylephrine (Fig. 4B). Similar qualitative results were obtained

322

using the MTT assay (Fig. 4C). Moreover AG1478, GM6001 and PD98059

323

blunted the EGF-mediated cell growth (Fig. 4D). None of the inhibitors per se

324

inhibited cell growth. Altogether, the inhibition of sequential steps of EGF

325

receptor signaling impaired the proliferative effect of the α1-adrenoceptor

326

agonist phenylephrine.

327 328

3.3.

α1-adrenoceptor agonists induce ERK phosphorylation in BPH cells

329

Since BPH cell proliferation induced by activation of α1-adrenoceptor

330

was prevented by the inhibition of EGF receptor signaling, which usually

331

involves activation of the mitogen-activated protein kinase pathway, we then

332

evaluated the impact of phenylephrine on ERK 1/2 activation by quantifying the

333

ratio of phospho-ERK (p-ERK) to total ERK protein. Due to BPH cell limitation,

334

we used only one concentration of phenylephrine (100 µM) which has been

335

used elsewhere to activate ERK 1/2 pathway [33,34]. Moreover, previous data

15

336

obtained from different cell systems showed that this concentration usually

337

induces the maximal effect of the agonist [33,34].

338

Phenylephrine stimulated ERK 1/2 activity in stromal BPH cells in a time-

339

dependent manner, and its effect peaked after 15 min. A similar stimulatory

340

effect was also observed with the α1-adrenoceptor agonist noradrenaline (10

341

µM, in the presence of propranolol 1 µM). In these experiments, EGF (100

342

ng/mL, 5 min stimulation) was used as a positive control and it stimulated ERK

343

1/2 activation (Fig. 5A-5C). In isometric contraction assays, 100 µM

344

phenylephrine added in the plateau of the contraction induced by 1 µM 5-HT,

345

and in the presence of 1 µM prazosin, failed to relax rat aorta therefore

346

discarding a putative β-adrenergic effect (data not shown). Previously we

347

showed that the selective α1D-adrenoceptor antagonist BMY7378, as well as

348

the α1A-/α1D-adrenoceptor antagonists LDT3 and LDT5, inhibited human

349

hyperplastic prostate stromal cell growth mediated by phenylephrine [28].

350

Current data showed that AG1478 (5 µM) inhibited the proliferative effect of

351

phenylephrine, and the association with the α1A/α1D-adrenoceptor antagonists

352

LDT3 or LDT5 (50 nM) resulted in similar inhibition suggesting that in this

353

model the EGF receptor pathway is necessary for α1-adrenoceptor-mediated

354

BPH cell proliferation (Fig. 6). Of note, this inhibitory effect of LDT3 or LDT5

355

(50 nM) did not involve the inhibition of prostate androgen receptors (AR) since

356

even at a much higher concentration (1 µM) they did not inhibit the specific

357

binding of the AR agonist (LDT3: -6.9 ± 4.9%, n = 2; LDT5: -17.7 ± 8.1%, n =

358

2).

359 360

16

361

4. Discussion

362

BPH is an aging-related disease linked to an imbalance between

363

prostate cell proliferation and apoptosis, favoring hyperplastic stromal cell

364

growth [35]. Mounting evidence points to the importance of GPCRs for cell

365

proliferation in pathological conditions [17,22,23]. Here we showed that the α1-

366

adrenoceptor-mediated proliferation of human hyperplastic prostatic stromal

367

cells is fully inhibited by EGF receptor and MEK inhibitors suggesting the

368

transactivation of the EGF receptors by α1-adrenoceptors as an important

369

event for BPH cell proliferation. To the best of our knowledge this is the first

370

report about the stromal α1-adrenoceptor-EGF receptor signaling in BPH.

371

The prostatic stroma makes up a large percentage of prostate volume

372

during BPH, and the use of human stromal cells is considered valuable for

373

BPH studies [35,36]. However, the isolation of stromal cells from human

374

prostate yields a small quantity of material. The cell immortalization frequently

375

causes the loss of cell characteristics, making the screening of immortalized

376

cell clones for lines that keep the phenotypic characteristics needed. For

377

instance, immortalized human prostate stromal cells show an increased

378

expression of α1B-adrenoceptors which is not observed in human prostate or

379

primary cultured cells [37]. Therefore we used primary cell culture obtained

380

from BPH patients (i.e., non-transformed cells). In our model, cultured cells

381

stained positively for vimentin, and negatively for the epithelial cell marker

382

cytokeratin, indicating the predominance of stromal cells.

383

The proposed ratio of α1A:α1B:α1D-adrenoceptors mRNA in normal

384

human prostate is approximately 63:6:31%, respectively [6]. In support to these

385

data real time RT-PCR assays corroborate the predominance of α1A- and α1D-

17

386

adrenoceptors mRNA [5]. Moreover, an increased expression of α1A- and α1D-

387

adrenoceptors mRNA has been reported in BPH, and the α1D-adrenoceptor

388

mRNA has a more pronounced increased expression (as high as three times)

389

[5,6]. Therefore these upregulated receptors are supposed to participate in the

390

pathophysiology of BPH. However, most, if not all, commercially available

391

antibodies against each subtype of α1-adrenoceptor lacks selectivity, which

392

limits the quantification of the protein at cellular level [38], and the identification

393

of functional α1-adrenoceptor subtypes relies on the use of selective drugs.

394

Our data showed that BPH stromal cells express functional α1-

395

adrenoceptors since phenylephrine, a selective α1-adrenoceptor agonist,

396

activated the canonical signaling pathway involving the increase of [Ca2+]I.

397

Similar data were obtained using the endogenous nonselective adrenoceptor

398

agonist noradrenaline, when tested in the presence of propranolol for blocking

399

its β–adrenoceptor effects. Moreover, functional data suggest that α1D-

400

adrenoceptor is involved in the BPH cell proliferative effect of phenylephrine

401

since BMY7378, a selective α1D-adrenoceptor antagonist, blocked the agonist

402

effect [28].

403

EGF receptor is a membrane-bound glycoprotein expressed in normal

404

and hyperplastic human prostate [39,40] including stromal cells [27,35] (and

405

present data). One of the proposed mechanisms leading to EGF receptor

406

transactivation by GPCR includes the action of metalloproteinases promoting

407

the shedding of members of EGF family such as HB-EGF, and consequently

408

the binding to its cognate receptor [15,18,20]. As a consequence,

409

metalloproteinase inhibition is reported as a strategy to interrupt EGF receptor

410

transactivation. Here we showed that inhibition of metalloproteinase prevented

18

411

BPH cell growth mediated by phenylephrine, which could suggest that the

412

activation of α1-adrenoceptor induces EGF receptor transactivation. In support

413

of these data it was previously shown in rat lacrimal gland epithelial cells that

414

α1D-adrenoceptor mediates the shedding of EGF and EGF receptor activation

415

[41].

416

In our model EGF induced stromal cell proliferation. As shown by Duque

417

and colleagues [27], both HB-EGF and EGF (100 ng/mL) induce a similar

418

mitogenic effect upon human prostate stromal cell suggesting a role of these

419

agonists in BPH. Moreover, stromal cells express HB-EGF mRNA and the HB-

420

EGF inhibitor CRM197 inhibits in a concentration-dependent manner the

421

stromal cell growth [27].

422

There is evidence that a naturally occurring human α1A-adrenoceptor

423

genetic variant is constitutively coupled to EGFR transactivation [23]. In

424

support of our hypothesis that α1-adrenoceptors might induce transactivation of

425

EGF receptor in BPH cells, the HB-EGF inhibitor CRM197, as well as the EGF

426

tyrosine kinase receptor inhibitor AG1478, prevented hyperplastic cell growth

427

induced by phenylephrine. Of note, the concentration of AG1478 used (5 µM)

428

fully inhibited the phenylephrine- and the EGF-induced cell proliferation. As

429

ERK 1/2 activation is important for cell proliferation, and it may be activated by

430

EGF receptor signaling, we investigated the effect of the MEK inhibitor

431

PD98059, which also inhibited the proliferative effect of phenylephrine.

432

Accordingly, in Western blotting assays, besides EGF (positive control),

433

phenylephrine (and noradrenaline) also stimulated ERK 1/2 phosphorylation

434

corroborating functional data. Therefore, our results suggest that α1-

435

adrenoceptors (most probably α1D-subtype) transactivate EGF receptors in

19

436

stromal cells from BPH patients leading to cell proliferation. However, present

437

data do not rule out that the canonical signaling of α1-adrenoceptor (i.e., Gq

438

canonical signaling) could also activate ERK pathway. On the other hand, as

439

the inhibition of EGF receptor signaling did not reduce cell proliferation in the

440

absence of phenylephrine, we could suggest that α1-adrenoceptor-mediated

441

EGF receptor transactivation is mainly agonist-dependent, rather than

442

constitutively active as previously shown in cardiomyoblasts [42].

443

Different

sets

of

evidence

obtained

by

others

indicate

that

444

transactivation of EGF receptors is involved in α1-adrenoceptor signaling. For

445

instance, it has been shown that α1D-adrenoceptor activation induces the

446

shedding

447

transactivation, and an EGF neutralizing antibody reduces phenylephrine-

448

induced ERK activation in rat lacrimal gland [41]. Moreover, α1-adrenoceptor

449

activation induces ERK 1/2 activity in rat aorta myocytes (mainly α1D-

450

adrenoceptor type) [19], and in human epithelial prostatic cells where ERK 1/2

451

activity was related to cell volume regulation [43]. In good accordance with our

452

data, the metalloproteinase inhibitor GM6001 [41] and the EGF receptor

453

inhibitor AG1478 [19] blocked ERK 1/2 activation in response to phenylephrine

454

linking α1-adrenoceptor to EGF receptor transactivation. On the other hand,

455

the β-adrenoceptor-mediated EGF receptor transactivation may involve (COS-

456

7) or not (brown adipocytes) ERK 1/2 pathway [44, 45]. Therefore ERK 1/2

457

activation during EGF receptor transactivation depends both on GPCR and cell

458

type.

of

biologically

active

EGF

and

subsequent

EGF

receptor

459

It is noteworthy that the co-incubation of EGF receptor inhibitor AG1478

460

with the α1A-/α1D-adrenoceptor antagonists LDT3 or LDT5 resulted in full

20

461

inhibition of cell proliferation, which was similar to the inhibition promoted by

462

each drug alone. A putative nonspecific inhibition of androgen receptors by

463

these α1 adrenoceptor antagonists was ruled out by binding assays. Moreover,

464

as previously shown in our group or by others, the α1D-adrenoceptor selective

465

antagonists BMY7378 and naftopidil also fully inhibited the proliferative effect

466

of phenylephrine in BPH cells [28,30]. However, in these models tamsulosin, a

467

benzenesulfonamide derivative, showed no effect [28,30]. Furthermore,

468

tamsulosin alone did not inhibit the increase of volume of the cell line BPH-1 in

469

response to phenylephrine [43]. In common, BMY7378, naftopidil, LDT3 and

470

LDT5 are α1D-adrenoceptors antagonists that share the N-phenylpiperazine

471

moiety which could shape some inhibitory functional selectivity upon BPH cell

472

proliferation, and therefore other experiments would be welcome. Patients´

473

adherence to current pharmacotherapy is low a fact that favors BPH

474

progression [46] and stimulates the search for new drugs. Understanding the

475

mechanisms involved in prostate cell proliferation may help the development of

476

new drugs.

477

In conclusion, our findings demonstrate that α1-adrenoceptor activation

478

in human hyperplastic prostate cells induces canonical and non-canonical

479

signaling. The α1-adrenoceptor non-canonical signaling involved in mitogenesis

480

of BPH cells depends on EGF receptor transactivation. This mechanism could

481

contribute to prostate enlargement and to the development of LUTS/BPH, and

482

therefore our data give new insight into the physiopathology of BPH. We

483

therefore propose that blockage of this transactivation cascade could be a

484

putative non-hormonal pharmacological strategy to reduce concomitantly LUTS

485

and BPH progression.

21

486 487

Acknowledgements

488

Supported by The Brazilian National Council for Scientific and Technological

489

Development

490

#312709/2017-0). JBNV was a PDJ fellow of CNPq (#166215/2015-5). CLMS,

491

FN, LEN, and LASR are senior fellows of CNPq (Brazil). The Study 100018032

492

was sponsored by Biozeus Desenvolvimento de Produtos Biofarmacêuticos

493

S.A. (Brazil). The funding source had no role in the study design, collection,

494

analysis or interpretation of data, writing or submission of the manuscript.

495

Orlando R. Moreira (UFRJ) for technical assistance.

(CNPq,

Brazil;

Grants

#306431/2015-7,

#455436/2014-2,

496 497

Declaration of interest: none.

498 499

Author Contributions

500

Conducted experiments: JBNV, RAH, LACB

501

Contributed with reagents or cell culture: LASR, LEN, EOB, JAGS

502

Participated in research design and coordinated experiments: CLMS, JAGS

503

Performed data analysis, discussion, revised the manuscript: JBNV, RAH,

504

PRF, FN, JAGS, CLMS

505

Wrote the manuscript with important contributions by all authors: CLMS. All

506

authors read and approved the final version of the manuscript.

507 508

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711 712 713 714 715

Figure legends

716

Figure 1. BPH cells are positive for vimentin and negative for cytokeratin

717

staining. Cells were incubated with anti-human primary monoclonal antibodies

718

against cytokeratin (upper panels) or vimentin (lower panels, red). In all

719

immunostaining-negative controls, reactions were performed by omitting the

720

primary antibody. No reactivity was observed when the primary antibody was

721

absent. Blue staining = DAPI. Bars = 150 µm, objective 40X.

722 723

Figure 2. Phenylephrine and noradrenaline increase intracellular Ca2+

724

concentration ([Ca2+]i) in hyperplastic prostatic stromal cells obtained from BPH

725

patients. Cells were loaded with 2.5 µM fura-2 AM for 60 min before stimulation

726

with the agonists (10 µM NA, 100 µM Phe). Data were expressed as mean and

727

S.E.M. (n = 7 – 10 replicates from 3 individual experiments). *** P = 0.007 vs.

728

basal (two-tailed Student´s t test). Phe = phenylephrine; NA = noradrenaline.

729 730

Figure 3. The increase of intracellular Ca2+ concentration ([Ca2+]i) in Rat-1 cells

731

transfected with human α1A- or α1D-adrenoceptors induced by phenylephrine is

31

732

inhibited by selective antagonists added 100 sec before. Cells were loaded

733

with 2.5 µM fura-2 AM for 60 min before stimulation with phenylephrine (100

734

µM Phe). WB 4101 (50 nM): α1A-adrenoceptor antagonist; tamsulosin (5 nM):

735

α1A/D-adrenoceptor

736

antagonist. Data represent the difference (∆) between the basal and agonist-

737

induced increase of fluorescence, and were expressed as mean and S.E.M. (n

738

= 6-8 replicates from 3 individual experiments). *** P < 0.01 vs. Phe (A, one

739

way ANOVA followed by Dunnett's multiple comparisons test; B, two-tailed

740

Student´s t test).

antagonist;

BMY7378

(50

nM):

α1D-adrenoceptor

741 742

Figure 4. The BPH cell proliferative effect of phenylephrine depends on the

743

activation of EGF receptors. A) Hyperplastic stromal cells in culture were

744

treated with 3 µM phenylephrine (Phe) or 100 ng/mL EGF for 48h in the

745

absence (control, white bar) or presence of the EGF receptor tyrosine kinase

746

inhibitor AG1478 5 µM. *** P < 0.001 Phe and EGF vs. control; AG1478 and

747

EGF + AG vs. EGF (one way ANOVA followed by Dunnett's multiple

748

comparisons test).

749

B and C) Hyperplastic stromal cells were treated with 3 µM phenylephrine

750

(black bar) for 48h in the absence (control, white bar) or presence of the

751

following inhibitors of EGF receptor signaling: GM = GM6001 10 µM; CRM =

752

CRM197 200 ng/mL; AG = AG1478 5 µM; PD = PD98059 1 µM. Cell

753

proliferation was accessed by cell counting using Trypan Blue exclusion dye

754

(B) and MTT assays (C). D. The EGF-mediated cell growth (100 ng/mL) is fully

755

inhibited by AG1478, GM6001 and PD98059. The inhibitors alone did not alter

756

basal cell proliferation (P > 0.05). Data were expressed as mean and S.E.M. of

32

757

3 experiments (performed in duplicate (A) or quadruplicate (B)), or 4

758

experiments performed in quadruplicate (C, D). B and C: ** P < 0.01 and *** P

759

< 0.001 vs. Phe; D: * P < 0.05 and *** P < 0.001 vs. control or EGF (one way

760

ANOVA followed by Dunnett's multiple comparisons test).

761 762

Figure 5. Time-dependent activation of ERK 1/2 by phenylephrine and

763

noradrenaline in hyperplastic stromal cells obtained from BPH patients (full-

764

length representative blots). Cells were treated with 100 µM phenylephrine

765

(Phe; A), 10 µM noradrenaline (NA) in the presence of 1 µM propranolol (B),

766

for the indicated times or 100 ng/mL EGF (5 min, A-C). C. Densitometric

767

analysis. Data were expressed as mean of 2 individual experiments.

768 769

Figure 6. The BPH cell proliferative effect of phenylephrine is completely

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inhibited by α1-adrenoceptor antagonists (LDT3 and LDT5) or AG1478, an EGF

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receptor tyrosine kinase inhibitor, alone or in association. The co-incubation

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with AG1478 (5 µM) with the α1-adrenoceptor antagonists did not potentiate the

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inhibitory effect of LDT3 and LDT5. Phe = phenylephrine. Data were expressed

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as mean and S.E.M. of 2 individual experiments performed in triplicate. *** P <

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0.001 all conditions vs. Phe (one way ANOVA followed by Dunnett's multiple

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comparisons test).

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