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Keywords:

  • adrenoceptor;
  • incidence;
  • naftopidil;
  • prostate cancer;
  • transforming growth factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Objectives

Quinazoline-based α1-adrenoceptor antagonists are known to inhibit prostate tumor growth through induction of apoptosis. We investigated the effect of a naphthalene-based α1-adrenoceptor antagonist, naftopidil, on prostate cancer incidence, apoptosis of prostatic cell and transforming growth factor-β signaling.

Methods

Prescription records were linked to pathological data for men who continued naftopidil (n = 766) or tamsulosin (n = 1015) for 3 months or longer between 2003 and 2010. Prostate cancer incidence was analyzed by log–rank test and the Cox proportional hazards model. Apoptosis and cell cycle arrest in human tissues were assessed by immunohistochemical detection of Bcl2 and p21, respectively. Growth inhibition and apoptosis treatment with naftopidil and tamsulosin were assessed in cancer cell lines. Interference with transforming growth factor-β signaling was examined by western blot analysis.

Results

Prostate cancer incidence was significantly lower in men who received naftopidil for 3 months or longer compared with tamsulosin (P = 0.035). Multivariate analysis confirmed a decreased hazard ratio, 0.46, for naftopidil use (P = 0.013), which was more evident with longer treatment. Immunohistochemical positivity for Bcl2, a marker for resistance to apoptosis, was less frequently detected in prostate cancer cells of men who received naftopidil compared with tamsulosin (P < 0.05). Naftopidil inhibited cancer cell growth, induced apoptosis and blocked Smad2 phosphorylation activated by transforming growth factor-β in cell lines, with a half maximal inhibitory concentration of 1.1 µmol/L.

Conclusions

Naftopidil seems to reduce prostate cancer incidence, possibly by inducing apoptosis, preferentially in cancer cells, and blocking transforming growth factor-β signaling.


Abbreviations & Acronyms
BPH

benign prostatic hyperplasia

BSA

bovine serum albumin

DAPI

diamidino-2-phenylindole

DMSO

dimethylsulfoxide

IC50

half maximal inhibitory concentration

PC

prostate cancer

PSA

prostate-specific antigen

R1

receptor 1

RT

room temperature

TGF-β

transforming growth factor-β

TUNEL

terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling

TUR-P

transurethral resection of the prostate

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Some α1-adrenoceptor antagonists, such as doxazosin and terazosin, can reduce PC incidence, whereas other α1-adrenoceptor antagonists; that is, tamsulosin, cannot.[1-5] This inhibitory effect is reportedly mediated by apoptosis induction, and independent from the property of α1-adrenoceptor antagonist; overexpression of α1 adrenoceptor did not influence apoptosis and cell viability;[1] exposure to phenoxybenzamine, an irreversible inhibitor of α1-adrenoceptors, failed to prevent the apoptotic effect of doxazosin.[3] G1 cell cycle arrest has been implicated in the growth inhibitory property of naftopidil, another α1-adrenoceptor, in human PC cell lines.[6]

In the present study, we investigated the effects of naftopidil on PC incidence in an observational cohort and the possible role of TGF-β signaling in its inhibition mechanism.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

A retrospective observational cohort study was carried out on men who received naftopidil or tamsulosin at the Fraternity Memorial Hospital in Tokyo, Japan, between 2003 and 2010. Medications, including α1-adrenoceptor antagonists and hormonal agents, were listed from prescription records of the pharmacy. Patient data, including pathological results, were linked by the identification number. Men receiving hormonal therapy or diagnosed with PC before treatment with α1-adrenoceptors were excluded. All the men had PSA checked before starting medication. The time zero of the present study was the time treatment with α1-adrenoceptor antagonists was started. The end-points of follow up were stopping of the α1-adrenoceptor antagonist or starting hormonal therapy (censored), or diagnosis of PC (evented). The follow up ended in December 2010 in patients who continued the same α1-adrenoceptor antagonists without diagnosis of PC.

The decision to carry out prostate biopsy or TUR-P was made clinically by the primary physicians. Men with follow up less than 3 months were excluded because of minimal exposure to the agents.

Immunohistochemistry

We used fresh prostatic tissues obtained from men with or without PC who received naftopidil, tamsulosin or neither (n = 10, each). The specimens were fixed in formalin, dehydrated, embedded in paraffin and cut into 3-μm sections using a Yamato large-scale microtome (TU213; Yamatokohki, Saitama, Japan). After deparaffinization, the sections were hydrated and incubated for 10 min in 3% hydrogen peroxide in methanol. Sections were heated in a steel pressure oven in 10 mmol/L citrate buffer, pH 6.0, to 120°C for 20 min. Non-specific binding was blocked using 2% normal goat blocking serum (X0907; Dako Cytomation, Glostrup, Denmark). Primary and secondary antibodies were incubated for 1 h and 0.5 h at RT, respectively. After washing, sections were incubated with EnVision (K4063; Dako Cytomation) at RT for 1 h, visualized with 3,3′-diaminobenzidine and stained with hematoxylin. The primary antibodies used were anti-p21 (#2947; Cell Signaling Technology, Danvers, MA, USA) and anti-Bcl2 (M0887; Dako Cytomation), both diluted 1:50. To confirm specificity of staining, primary antibodies were mixed with p21 (#1055; Cell Signaling Technology) and Bcl2 (B25-30G-20; Signalchem, Richmond, BC, Canada) before staining in control specimens. Expression levels were determined by counting positive epithelial cells in 10 separate microscopic fields at ×100 magnification. Cancer cells and non-cancer cells were collected from men with PC, and normal cells were from men without PC. The results were independently reviewed by two blinded investigators.

Cell viability assay

HeLa cell and LNCaP cell were seeded on 12-well dishes (1 × 104 cells/well) in DMEM/F12 (#11320; Life Tech, Carlsbad, CA, USA) and RPMI (#22400; Life Tech) containing 10% fetal bovine serum. Naftopidil, which was kindly provided by Asahi Kasei (Tokyo, Japan), and tamsulosin (T1330; Sigma-Aldrich, St. Louis, MD, USA) were dissolved in DMSO and then diluted 1000 times for use with cells (final 0.1% DMSO). Cells were treated with 10 μmol/L of naftopidil, tamsulosin or control (vehicle) for 2 days. Then cells were detached by trypsinization (#12605; Life Tech) and counted using Coulter counter (Z1; Beckman Coulter, Brea, CA, USA). Cell viability was assessed by trypan blue exclusion assay (#15250; Life Tech) and expressed as a percentage of the control.

Apoptosis detection

HeLa cell and LNCaP cell were seeded on 96-well dishes (1 × 103 cells/well) and on collagen-coated 96 well dishes (3 × 103 cells/well), respectively. The next day, cells were treated with 10 μmol/L of naftopidil, tamsulosin or control (0.1% DMSO) for 1 day, and apoptotic cells were detected by TUNEL assay using an in situ detection kit (#2156792; Roche, Basel, Switzerland). DAPI (D1306; Life Tech) was used to counter staining. Cells were visualized using a fluorescence microscope (BZ9000; KEYENCE, Chicago, IL, USA).

TGF-β induction and western blotting

HeLa cells were seeded on 6-cm dishes. Recombinant human TGF-β1 (100-21; PeproTech, Rocky Hills, NJ, USA) was dissolved in 10 mmol/L citric acid, pH 3.0, in 0.1% BSA, and then diluted 1000 times for use with the HeLa cells (final 10 μmol/L citric acid). Controls contained the same concentrations of citric acid, BSA and DMSO. TGF-β1 1 ng/mL was added the next day, in the presence or absence of naftopidil. After a 30-min incubation, cells were solubilized with radio-immunoprecipitation assay buffer (1.5% Triton X-100, 20 mmol/L Tris, pH 7.5, 150 mmol/L MgCl2 and 1 mmol/L ethylenediaminetetraacetic acid) containing protease inhibitors (dithiothreitol, leupeptin, aprotinin and phenylmethanesulfonylfluoride) and phosphatase inhibitor cocktail3 (P0044; Sigma-Aldrich). Lysates were subjected to western blot analysis. Membranes were detected by anti-phospho-Smad2 (Ser465/467, #3101; Cell Signaling Technology) and anti-Smad2 (#5339; Cell Signaling Technology) with Can Get Signal (NKB-101T; Toyobo, Osaka, Japan). We drew Smad2 and phospho-Smad2 western blotting figures based on three experiments by densitometry scan (Science lab 2005 Multi Gauge ver. 3.0; FUJIFILM, Tokyo, Japan). The average intensity of phospho-Smad2 was analyzed and IC50 was calculated using Bio Data Fit (Chang Bioscience, Castor Valley, CA, USA).

Statistical analysis

Data were analyzed using jmp 9.0.2 (SAS Institute, Cary, NC, USA). PC incidence was analyzed by log–rank test. PC risk was calculated as the hazard ratio using the Cox proportional hazards model. Variables were baseline PSA level and medicine. Other comparisons between groups were analyzed by Wilcoxon rank–sum test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Prostate cancer incidence

We identified 1121 men treated with naftopidil and 1654 men with tamsulosin by tracking 17 497 and 20 870 prescriptions for naftopidil and tamsulosin, respectively, between 2003 and 2010. A total of 355 men on naftopidil and 639 men on tamsulosin were excluded because of treatment was shorter than 3 months (226 and 382, respectively), combination with hormonal therapy (32 and 49, respectively), interrupted prescriptions (29 and 36, respectively), PC before prescription (50 and 150, respectively) and PC diagnosed during the first 3 months of treatment (18 and 22, respectively). The remaining men received continuous treatment with either medicine for 3 months or longer; naftopidil group (n = 766) and tamsulosin group (n = 1015). No significant differences were detected for baseline age, serum PSA level, and the methods for examination of the prostate histology between the naftopidil and tamsulosin group (Table 1).

Table 1. Backgrounds in men treated with naftopidil or tamsulosin for 3 months or longer
 3 Months or longer12 Months or longer36 Months or longer
Naftopidil (n = 766)Tamsulosin (n = 1015)P-valueNaftopidil (n = 433)Tamsulosin (n = 506)P-valueNaftopidil (n = 203)Tamsulosin (n = 208)P-value
  1. †Median (quartile range). ‡Cases (%).

Baseline age (years)71 (64–76)71 (65–78)0.0971 (64–76)72 (66–78)0.1071 (64–76)72 (66–79)0.12
Baseline PSA level (ng/mL)2.5 (1.1–4.9)2.3 (1.0–4.6)0.142.5 (1.2–4.9)2.3 (1.0–4.4)0.092.3 (1.1–4.5)2.1 (1.1–3.9)0.27
Methods for examination of the prostate         
Biopsy197 (25)250 (24)0.60124 (29)134 (26)0.4664 (32)56 (27)0.30
TUR-P93 (12)120 (11)0.8349 (11)67 (13)0.3712 (6)21 (10)0.11
Either239 (31)313 (30)0.86147 (34)169 (33)0.8568 (33)69 (33)0.94

During the observational period ranging 3–96 months (median 13 months), 46 men (2.5%) were diagnosed with PC. The incidence of PC was significantly higher in men with the baseline PSA levels greater than 4 ng/mL (P < 0.001, 6.2% vs 0.8%, Fig. 1b and Table 2), and significantly lower in men receiving naftopidil (P = 0.035, 1.8% vs 3.1%, Fig. 1c and Table 2). Multivariate analysis showed that the odds ratio of developing PC was 9.00 for men with high PSA (P < 0.0001) and 0.46 for men on naftopidil (P = 0.013, Table 2). Along with the extension of observation, the increased risk of PC by high PSA became less evident; the ratio was 9.00, 7.51 (P < 0.0001) and 3.56 (P = 0.083) for men treated for 3 months or longer, 12 months or longer and 36 months or longer, respectively. By contrast, a reduced odds ratio by naftopidil treatment was more pronounced: 0.46, 0.46 (P = 0.081) and 0.16 (P = 0.039) for men treated for 3 months or longer, 12 months or longer and 36 months or longer, respectively. The Gleason scores in men diagnosed with PC did not differ between the two groups (P = 0.86, median 6 and 6, respectively).

figure

Figure 1. Cumulative PC incidence in men during treatment with α1-adrenoceptor antagonists. (a) There was no significant difference between men aged 71 years or older (image) and men aged less than 71 years (image). (b) The incidence was higher in men with PSA ≥4.0 ng/mL (image) than in men with PSA <4.0 ng/mL (image; P < 0.001). (c) It was lower in men receiving naftopidil (image) than in men receiving tamsulosin (image; P = 0.035).

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Table 2. Risk of PC diagnosis
 Variable3 Months or longer12 Months or longer36 Months or longer
Cancer/total (%)Univariate analysisMultivariate analysisCancer/Total (%)Univariate analysisMultivariate analysisCancer/Total (%)Univariate analysisMultivariate analysis
P-valueHazard ratio95% CIP-valueP-valueHazard ratio95% CIP-valueP-valueHazard ratio95% CIP-value
  1. †Log–rank test. ‡Cox proportional hazards model.

Baseline age (years)Less than 7119/852 (2.2)0.44   9/434 (2.0)0.58   3/188 (1.5)0.58   
71 or higher27/929 (2.9)    13/505 (2.5)    5/223 (2.2)    
Baseline PSA level (ng/mL)Less than 4.010/1206 (0.8)<0.00011.00  6/641 (0.93)<0.00011.00  4/298 (1.3)0.081.00  
4.0 or higher36/575 (6.2) 9.004.63–19.2<0.000116/298 (5.3) 7.513.07–21.0<0.00014/113 (3.5) 3.560.83–15.10.083
MedicineTamsulosin32/1015 (3.1)0.0351.00  15/506 (2.9)0.151.00  7/208 (3.3)0.071.00  
Naftopidil14/766 (1.8) 0.460.23–0.840.0137/433 (1.6) 0.460.17–1.090.0811/203 (0.4) 0.160.008–0.920.039

Immunohistochemical analysis

Typical immunohistochemical staining for p21 and Bcl2 in prostatic epithelial cells are shown in Figure 2. The specificity of staining was ensured by completely negative staining in specimens pre-incubated with the corresponding proteins. Expression of p21 was more enhanced in cancer cells than normal cells, and it was significantly more enhanced in men treated with naftopidil than in men treated with neither α1-adrenoceptor antagonists (P < 0.05, Table 3). By contrast, expression of Bcl2 was more suppressed in cancer cells than normal cells. In men receiving naftopidil, as compared with men treated with tamsulosin or neither, Bcl2 expression was significantly suppressed in cancer cells (P < 0.05) and significantly increased in non-cancer cells (P < 0.05, Table 3).

figure

Figure 2. Immunohistochemical expression of p21 and Bcl2 in prostatic cells. Cancer cells from men exposed to naftopidil showed higher p21 expression and lower Bcl2 expression compared with men exposed to tamsulosin or neither. In contrast, non-cancer cells from men exposed to naftopidil showed higher Bcl2 expression compared with men exposed to tamsulosin or neither. Normal cells showed no significant difference in expression among medication groups. Staining with p21 and Bcl2 (without protein) was specifically diminished of pre-incubation with the corresponding protein (with protein).

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Table 3. Proportion of epithelial cells positive for p21 and Bcl2 (%)
   Neither (n = 10)Tamsulosin (n = 10)Naftopidil (n = 10)
  1. *P < 0.05: versus neither. **P < 0.05: versus neither and versus tamsulosin. Median (quartile range).

Men with prostate cancerCancer cellsp211 (0–5.5)4 (0.7–9.5)7 (3.5–10)*
Bcl211 (4.5–23)11 (5–13)4 (2.5–5)**
Non-cancer cellsp210 (0–1)0 (0–0)0 (0–2.2)
Bcl261 (27–78)60 (44–69)82 (66–94)**
Men without prostate cancerNormal cellsp211 (0–1)0 (0–1.2)0.5 (0–1.2)
Bcl271 (50–82)71 (68–76)74 (67–88)

Cell viability and apoptosis

Cell viability was significantly suppressed by naftopidil in HeLa and LNCaP cell lines (Fig. 3a). TUNEL assay showed apoptosis by naftopidil for both cell lines (Fig. 3b). Naftopidil significantly suppressed cell variability and induced apoptosis in both cell lines, whereas tamsulosin did not.

figure

Figure 3. Cell viability and apoptosis assay. (a) Cell viability (% of control). Cells treated with 10 μmol/L of naftopidil, showed significantly reduced viability compared with tamsulosin or vehicle (control). *P < 0.05, **P < 0.01. (b) TUNEL assay. Double labeling of cells with DAPI (blue) and TUNEL (red) showed significant increments of apoptotic cells treated with 10 μmol/L naftopidil.

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Western blotting

TGF-β induced Smad2 phosphorylation, which was inhibited by naftopidil in a dose-dependent manner (Fig. 4a). The IC50 against Smad2 phosphorylation was 1.1 μmol/L (Fig. 4b). Smad2 expressions were not changed by TGF-β or naftopidil (Fig. 4c).

figure

Figure 4. Western blot analysis and model of inhibition. (a) Intensity of three times western blot analyses and representative data of phospho-Smad2. Phosphorylation of Samd2 induced by TGF-β was suppressed by naftopidil dose-dependently. (b) Model of phosphorylate inhibition of Smad2 by naftopidil. Y = a exp (–bX) + c; a = 1.0 × 105 b = 0.58 c = 7.6 × 104 IC50 was 1.1 μmol/L. (c) Intensity of three times western blot analyses and representative data of Smad2. Expressions of Smad2 were not changed by TGF-β or naftopidil treatment.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

The growth inhibitory and apoptotic effects of adrenoceptor antagonists were initially reported in 1994.[7] Subsequently, PC incidence was found to be significantly lower in men exposed to terazosin or vasopressin than in unexposed men.[2] However, another α1-adrenoceptor antagonist, tamsulosin, failed to induce apoptosis or inhibit tumor cell growth in PC cell lines.[1] In the present study, we found a significant reduction of PC in men receiving naftopidil (1.8%) compared with men receiving tamsulosin (3.1%). The odds ratio of PC was 0.46 for men on naftopidil by multivariate analysis, and the ratio was further lowered along with longevity of naftopidil administration.

The histological analysis showed higher cell cycle arrest and higher apoptosis in human PC cells compared with non-cancer cells, and this reciprocal relationship was more pronounced in men treated with naftopidil. Compounds with inhibitory effects on PC cell growth all contain a piperazine group, which is absent from tamsulosin (Fig. 5). Naftopidil shares structural similarly with terazosin and doxazosin in terms of containing piperazine and naphthalene groups, which might be important for PC inhibition. Some signaling pathways that induce apoptosis through α1-adrenoceptors include the death receptor,[8] vascular endothelial growth factor,[9] Smad4 and TGF-β pathways.[10] Cancer cells are known to be more susceptible to apoptosis because of their cellular deviation and it exerts protective effect against neoplasms.[11-13] Actually, PC with less apoptosis index tends to show biochemical failure after total prostatectomy.[14] In this context, it is notable that TGF-β induces apoptosis in normal cells, but promotes the proliferation of cancer cells.[15-17] There are five types of TGF-β inhibitors: oligonucleotides, antibodies, small-molecule inhibitors, interacting peptides and vaccines.[18] Naftopidil is made up of small molecules and might therefore inhibit TGF-β R1 kinase-like pyrazole inhibitors (Fig. 5), which attach to the adenosine triphosphate-binding site of the TGF-β R1 kinase domain.[19, 20] Smad2, which is rapidly phosphorylated by TGF-β R1 bound to TGF-β, is known to function as a signal transducer for TGF-β signaling. The observed rapid inhibition (less than 30 min) after TGF-β stimulation in the present study supports the idea that naftopidil might interact directly with TGF-β R1 and block TGF-β signaling. Based on the pharmacokinetics of naftopidil,[21, 22] it is estimated that oral administration of 320 mg is required to attain serum concentration of 1.1 μmol/L (IC50 for Smad2 phosphorylation). The normal dose of naftopidil for BPH (25–75 mg in Japan) is unlikely to achieve a therapeutic effect on PC, but might prevent prostate carcinogenesis.

figure

Figure 5. Structural formula of medicines. Naftopidil, KMUP-1, terazosin and doxazosin all contain piperazine, but tamsulosin does not. Naftopidil also contains naphthalene, which is similar to xanthine, quinazoline and naphthyridine.

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The limitations of the present study included its retrospective nature of a cohort study on BPH men and restricted exploration of TGF-β signaling pathways. The relative risk between users and non-users of α1-adrenoceptor antagonists was not evaluated in the present study. Further investigations are warranted to clarify the cancer inhibitory property of naftopidil and its mechanisms involved.

The results of the present study show for the first time that naftopidil use might reduce the PC incidence by possibly inducing apoptosis preferentially in cancer cells and blocking TGF-β signaling.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Dr Shinichi Teshima and Masakazu Takahira for assisting with the immunohistochemical analyses. This study was supported in part by a Grant-in-Aid #21592066 (to HN) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Professor Y Homma received grants from Asahikasei (Tokyo, Japan) and Astellas (Tokyo, Japan).

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
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    Revelos K, Petraki C, Gregorakis A, Scorilas A, Papanastasiou P, Koutsilieris M. Immunohistochemical expression of Bcl2 is an independent predictor of time-to-biochemical failure in patients with clinically localized prostate cancer following radical prostatectomy. Anticancer Res. 2005; 25: 31233133.
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
iju12156-sup-0001-si.tif583K

Fig. S1 Demographic chart of the male population in Sumida-ku and Japan. The ubiety if the Fraternity Memorial Hospital is this Sumida-ku, whereabouts of the Fraternity Memorial Hospital, represent well the average Japanese population.

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