Original Paper Pharmacology 2014;94:163–169 DOI: 10.1159/000368050

Received: May 16, 2014 Accepted after revision: August 29, 2014 Published online: October 4, 2014

Naftopidil Is Useful for the Treatment of Malignant Pleural Mesothelioma Koji Mikami a Hisao Nagaya b Akinobu Gotoh b Takeshi Kanno c Ayako Tsuchiya c Takashi Nakano a Tomoyuki Nishizaki c a Division of Respiratory Medicine, Department of Internal Medicine, b Laboratory of Cell and Gene Therapy, Institute for Advanced Medical Sciences, and c Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, Nishinomiya, Japan

Key Words Naftopidil · Malignant pleural mesothelioma · Apoptosis · Tumor necrosis factor-α · FasL · Cancer therapy

Abstract Naftopidil, an α1-adrenoceptor blocker, induced apoptosis of human malignant pleural mesothelioma NCI-H2052 cells. Naftopidil upregulated the expression of tumor necrosis factor-α (TNF-α) mRNA in these cells. Naftopidil, alternatively, increased FasL secretion from NCI-H2052 cells, without affecting the expression of FasL mRNA and protein, and activated caspase-3 and -8 in NCI-H2052 cells. Naftopidil drastically suppressed tumor growth in mice inoculated with these cells. The results of the present study indicate that naftopidil induces apoptosis of NCI-H2052 cells by upregulating the expression of TNF-α and stimulating the secretion of FasL, a ligand for the death receptor Fas, both to activate caspase-8 and the effector caspase-3, leading to the suppression of NCI-H2052 cell proliferation in vivo. This raises the possibility that naftopidil could be developed as an effective drug for the treatment of malignant pleural mesothelioma. © 2014 S. Karger AG, Basel

Introduction

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor originating from the mesothelium. The response of MPM patients after conventional chemotherapy and radiotherapy is limited [1–3], and the median overall survival of the patients ranges from 12 to 24 months [4]. MPM is histologically classified into 3 main types: epitheloid, sarcomatoid, and mixed/biphasic, with a longer survival in the epitheloid and a shorter survival in the sarcomatoid types [4]. Cisplatin and pemetrexed are widely used in MPM patients undergoing chemotherapy, but half of the patients are partially responsive and have a prolonged survival from 9 to 12 months [5]. Vascular endothelial growth factor inhibitors have been examined, but this resulted in limited success [6, 7]. The type 1 receptor tyrosine kinase EphB4 and its cognate ligand Ephrin-B2 have been highlighted as novel targets for the treatment of MPM [8]. Naftopidil, an antagonist of α1A/1D-adrenoceptor, has been developed as a drug for the treatment of benign

© 2014 S. Karger AG, Basel 0031–7012/14/0944–0163$39.50/0 E-Mail [email protected] www.karger.com/pha

Prof. Tomoyuki Nishizaki, MD, PhD Division of Bioinformation, Department of Physiology Hyogo College of Medicine 1-1 Mukogawa-cho, Nishinomiya 663-8501 (Japan) E-Mail tomoyuki @ hyo-med.ac.jp

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K.M. and H.N. contributed equally to this work.

RT-PCR

FasL

sense anti-sense

CACTACCGCTGCCACCCCTGA CATCATCTTCCCCTCCATCATCACC

Fas

sense anti-sense

ATTATCGTCCAAAAGTGTTAATGCCCAA TGCACTTGGTGTTGCTGGTGAGTG

FADD

sense anti-sense

GCCTGGGGAAGAAGACCTGTGTG CTGGCTTCCTGCTGGGTCTTCAC

TNF-α

sense anti-sense

CCTCCCCTGCCCCAATCCC GCTGGGCTCCGTGTCTCAAGG

TNFR1

sense anti-sense

ATGCCGAAAGGAAATGGGTCAGG AAAATGACCAGGGGCAACAGCAC

TNFR2

sense anti-sense

CCTGCCCCTTGGAGTGCCTG GCGGGGCAGGTCACAGAGAGT

TRADD

sense anti-sense

GGGTTCCTTCTGCGGCTATTGCT TGAGTGAAACTGTAAGGGCTGGCTGTA

TRAIL

sense anti-sense

GGACCCCAATGACGAAGAGAGTATGAA TGGACCATTTGTTTGTCGTTCTTTGTG

TWEAK

sense anti-sense

CCCCAGACAGAAGAAAGCCAGGAT CAGCACACCATCCACCAGCAAG

DR3

sense anti-sense

GGAGTGCCAGGTCAGCCAATGTG CCAGCAGTGGCGGTATGTGTAGGTC

DR4

sense anti-sense

GTGACTTTGGTTGTTCCGTTGCTGT GGCGTTCCGTCCAGTTTTGTTGA

DR5

sense anti-sense

GGTCGGTGATTGTACACCCTGGAG CAGGAGTCAAAGGGCACCAAGTCTG

DR6

sense anti-sense

TACCTTGTGCTGCCTTGACTGACC AGGTTTGGGAGGGTCTTGTTCACG

GAPDH

sense anti-sense

GACTTCAACAGCGACACCCACTCC AGGTCCACCACCCTGTTGCTGTAG

prostate hyperplasia and hypertension [9]. A topic that has recently been discussed is that naftopidil exerts its antitumor action on a variety of cancers [10–13]. We have earlier found that naftopidil induces apoptosis of MPM cells by interacting with protein kinase C but regardless of the α1D-adrenoceptor-dependent manner, with the mechanism independent of other α1-adrenoceptorinfluencing substrates such as prazosin [13]. The present study was conducted to assess whether naftopidil is useful for the treatment of MPM. Here, we show that naftopidil induces apoptosis of NCI-H2052 MPM cells by upregulating the expression of tumor necrosis factor-α (TNF-α) and stimulating FasL secretion, both of which are responsible for the activation of caspase-8 followed by the effector caspase-3, leading to the suppression of tumor growth in mice inoculated with NCI-H2052 cells.

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Materials and Methods Cell Culture NCI-H2052 human MPM cells were purchased from the American Type Culture Collection (Manassas, Va., USA). Cells were grown in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum, 0.003% L-glutamine, penicillin (final concentration 100 U/ml), and streptomycin (final concentration 0.1 mg/ml) in a humidified atmosphere of 5% CO2 and 95% air at 37 ° C.  

 

Assay of Cell Viability Cell viability was evaluated by using 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as previously described [14]. TUNEL Staining TUNEL staining was performed to detect in situ DNA fragmentation as a marker of apoptosis using an in situ apoptosis detection kit (TaKaRa Bio, Otsu, Japan). Briefly, fixed and permea-

Mikami /Nagaya /Gotoh /Kanno / Tsuchiya /Nakano /Nishizaki  

 

 

 

 

 

 

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Table 1. Primers used for real-time

 

Enzymatic Assay of Caspase-3, -8, and -9 Activities Caspase activity was measured using a caspase fluorometric assay kit (Ac-Asp-Glu-Val-Asp-MCA for a caspase-3 substrate peptide, Ac-Ile-Glu-Thr-Asp-MCA for a caspase-8 substrate peptide, and Ac-Leu-Glu-His-Asp-MCA for a caspase-9 substrate peptide) by a method previously described [14]. Briefly, cells were harvested before and after treatment with naftopidil or prazosin and then centrifuged at 1,200 rpm for 5 min at 4 ° C. The cell pellet was incubated on ice in cell lysis buffer for 10 min and reacted with the fluorescently labeled tetrapeptide at 37 ° C for 2 h. Fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm with a fluorometer (Fluorescence Spectrometer F-4500; Hitachi High-Tech, Tokyo, Japan).  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Monitoring of FasL Secretion NCI-H2052 cells were treated with naftopidil (100 μmol/l). Then, extracellular culture medium and cells were collected. Proteins were extracted from the extracellular medium using 10% (w/v) trichloroacetic acid/acetone. Cells were lysed in a lysate solution [150 mmol/l NaCl, 20 mmol/l Tris, 0.1% (v/v) Tween 20 and 0.1% (w/v) sodium dodecyl sulfate, pH 7.5] containing 1% (v/v) protease inhibitor cocktail and 1% (v/v) phosphatase inhibitor cocktail (Nacalai Tesque). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Blotting membranes were blocked with TBS-T containing 5% (w/v) bovine serum albumin and subsequently reacted with antibodies against FasL (Cell Signaling Technology, Danvers, Mass., USA) and β-actin (Sigma, St. Louis, Mo., USA). After washing, membranes were reacted with a horseradish peroxidase-conjugated goat antirabbit IgG antibody. Immunoreactivity was detected with an ECL kit (Invitrogen, Carlsbad, Calif., USA) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, N.J., USA).

Naftopidil-Induced Anticancer Effect

80 60 40 20 0

0

1 10 100 Naftopidil (μmol/l)

 

Real-Time Reverse Transcription-Polymerase Chain Reaction Total RNAs from NCI-H2052 cells were purified by an acid/ guanidine/thiocyanate/chloroform extraction method using the Sepasol-RNA I Super kit (Nacalai Tesque, Kyoto, Japan). After purification, total RNAs were treated with RNase-free DNase I (2 units) at 37 ° C for 30 min to remove genomic DNAs, and 10 μg of RNA was resuspended in water. Then, random primers, dNTP, 10 × RT buffer, and MultiScribe Reverse Transcriptase (Life Technologies, Carlsbad, Calif., USA) were added to an RNA solution and incubated at 25 ° C for 10 min followed by 37 ° C for 120 min to synthesize the first-strand cDNA. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) was performed using a SYBR Green Real-Time PCR Master Mix (TaKaRa Bio) and the Applied Biosystems 7900 Real-Time PCR detection system (ABI, Foster City, Calif., USA). The thermal cycling conditions were as follows: 94 ° C for 4 min (first step); 94 ° C for 1 s, 65 ° C for 15 s, and 72 ° C for 30 s (ensuing 40 cycles). The expression level of each mRNA was normalized by that of GAPDH mRNA. The primers used for real-time RT-PCR are shown in table 1.  

100

Fig. 1. The effect of naftopidil on cell viability. An MTT assay was carried out on NCI-H2052 cells untreated and treated with naftopidil at the indicated concentrations for 24 h. Each point represents the mean percentage (± SEM) of the control cell viability (MTT intensities of cells untreated with naftopidil). n = 4 independent experiments.

Inoculation of NCI-H2052 Cells Nude BALB/c-nu/nu mice (male, 6 weeks) were obtained from Japan SLC, Inc. (Shizuoka, Japan). NCI-H2052 cells (1 × 107 cells) suspended in 200 μl of culture medium with 50% (v/v) matrigel (BD Biosciences, San Jose, Calif., USA) were subcutaneously inoculated into the right flank of the mice under pentobarbital general anesthesia. Naftopidil was diluted with a physiological salt solution, and an intraperitoneal injection of that solution or naftopidil twice a week was started 1 week after inoculation. The longer (L) and shorter length (S) of inoculated tumors was measured using calipers, and tumor volume (V) was calculated according to the following equation: V = L × S2 × 1/2. Statistical Analysis Statistical analysis was carried out using the unpaired t test, Dunnett’s test, and repeated-measures analysis of variance followed by Tukey’s test.

Results

Naftopidil Induces Apoptosis of NCI-H2052 MPM Cells Naftopidil reduced NCI-H2052 cell viability in a concentration-dependent manner (1–100 μmol/l), the extent reaching almost 0% of basal levels at 100 μmol/l (fig. 1). In the TUNEL staining, naftopidil significantly increased the number of TUNEL-positive cells as compared with that of the untreated control in NCI-H2052 cells (fig. 2). Collectively, these results indicate that naftopidil induces apoptosis of NCI-H2052 MPM cells.

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Cell viability (% of control)

bilized cells were reacted with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-deoxyuridine triphosphate for 90 min at 37 ° C. Fluorescein isothiocyanate signals were visualized with a confocal laser scanning microscope (LSM 510; Carl Zeiss Co., Ltd., Oberkochen, Germany).

6

Naftopidil Caspase activity

Cont

DIC

Caspase-3 Caspase-8 Caspase-9

4

2

TUNEL 0

p < 0.001

Fig. 3. Naftopidil-induced activation of caspase-3 and -8. NCI-

80

H2052 cells were treated with naftopidil (100 μmol/l) for 3–6 h, and then the activities of caspase-3, -8, and -9 were enzymatically assayed. Each point represents the mean ratio (± SEM) to the basal caspase activities (before treatment with naftopidil). n = 4 independent experiments.

60 40 20 0

Cont

Naft

Fig. 2. TUNEL staining was carried out on NCI-H2052 cells un-

treated (Cont) and treated with naftopidil (Naft; 100 μmol/l) for 12 h. TUNEL-positive cells were counted in a randomly selected area (0.4 × 0.4 mm). In the graph, each column represents the mean percentage (± SEM) of TUNEL-positive cells relative to total cells. n = 4 independent experiments. The p value was calculated with the unpaired t test. DIC = Differential interference contrast. Scale bars = 50 μm.

Naftopidil Activates Caspase-3/-8 in NCI-H2052 MPM Cells Naftopidil apparently activated caspase-3 and -8, whereas caspase-9 was not activated except in NCI-H2052 cells (fig. 3). In the real-time RT-PCR, the expression of TNF-α mRNA in NCI-H2052 cells was markedly upregulated by naftopidil (100 μmol/l) (fig. 4d), and expression of death receptor (DR)5 (fig. 4l) and DR6 mRNAs (fig. 4m) was slightly upregulated. In contrast, naftopidil (100 μmol/l) had no significant effect on the expression of mRNAs for FasL (fig. 4a), Fas (fig. 4b), Fas-associated death domain protein (FADD; fig. 4c), TNFR1 (fig. 4e), TNFR2 (fig. 4f), TNFR1-associated death domain protein (TRADD; fig. 4g), TNF-related apoptosis-inducing ligand (TRAIL; fig. 4h), TNF-related weak inducer of apoptosis (TWEAK; fig. 4i), DR3 (fig. 4j), or DR4 (fig. 4k). These results suggest that TNF-α may participate in naftopidil-induced caspase-8 activation.

166

3 6 Naftopidil treatment (h)

Pharmacology 2014;94:163–169 DOI: 10.1159/000368050

Naftopidil (100 μmol/l) did not affect the expression of FasL protein in NCI-H2052 cells (fig. 5a). Notably, naftopidil (100 μmol/l) significantly increased the concentrations of extracellular FasL protein in a bell-shaped, treatment time-dependent manner (3–6 h; with the peak at 6 h) in NCI-H2052 cells (fig. 5b). This indicates that naftopidil stimulates FasL secretion, possibly to activate Fas, without affecting the expression of FasL mRNA and protein, which causes activation of caspase-8 and of the downstream effector caspase-3, to induce apoptosis of NCI-H2052 MPM cells. Naftopidil Suppresses the Proliferation of NCI-H2052 MPM Cells We finally examined the effect of naftopidil on NCIH2052 cell proliferation using mice inoculated with NCIH2052 cells. The IC50 of naftopidil against LNCaP and PC-3 prostate cancer cell proliferation is shown to be 22 and 33 mmol/l, respectively [10]. In addition, naftopidil at a dose of 10 mg/kg was administered in an in vivo experiment grafted with prostate cancer cells [11]. In the light of these facts, naftopidil was intraperitoneally injected at a dose of 9.8 mg/kg, corresponding to approximately 25 μmol/l, twice a week in the present experiments. Intraperitoneal injection of naftopidil significantly inhibited NCI-H2052 cell growth as compared with that for control mice (p < 0.001, Fisher’s PLSD test; fig. 6a). All the mice injected with naftopidil survived 8 weeks after the first injection, and naftopidil had no effect on mouse weight (fig.  6b). These results indicate that naftopidil could exert an antitumor action on MPM. Mikami /Nagaya /Gotoh /Kanno / Tsuchiya /Nakano /Nishizaki  

 

 

 

 

 

 

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TUNEL-positive cells (% of total cells)

100

0

4

0

3

6

9

b

i

TNFR2 mRNA

8 4 0

0

3

6

9

DR6 mRNA

6

9

c

f

4 0

0

3

6

9

d

12

8 4 0

8

0

3

6

9

g

4 0

4

0

3

6

9

h

0

8

8

8

8

0

3

6

0

9

j

0

3

6

Naftopidil treatment (h)

4 0

9

k

DR5 mRNA

12

0

6

9

0

3

6

9

3

6

9

4

12

4

3

8

12

4

0

12

8

0

8

12

12

0

3

6

0

9

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4

l

0

Naftopidil treatment (h)

8 4 0

m

3

DR4 mRNA

TWEAK mRNA

e

0

12

DR3 mRNA

TNFR1 mRNA

12

0

TRADD mRNA

0

4

TNF-į mRNA

8

12

TRAIL mRNA

8

12 FADD mRNA

12

Fas mRNA

FasL mRNA

a

12

0

3

6

9

Naftopidil treatment (h)

Fig. 4. Real-time RT-PCR analysis. NCI-H2052 cells were treated with naftopidil (100 μmol/l) for the indicated periods of time, and then

real-time RT-PCR was carried out. Each mRNA quantity was calculated from the standard curve made by amplifying different amounts of GAPDH mRNA and normalized by regarding the average of the independent basal mRNA quantity at 0 h as 1. Each point represents the mean ratio (± SEM) relative to the basal mRNA levels. n = 4 independent experiments.

The results of the present study clearly demonstrate that naftopidil activates caspase-3 and -8, but not caspase-9, and induces apoptosis of NCI-H2052 MPM cells. Caspase-8 is activated through DR. They include TNFR1, FAS/apoptosis antigen 1 (APO1)/CD95, DR3/ Naftopidil-Induced Anticancer Effect

APO3/WSL-1/lymphocyte-associated receptor of death (LARD)/TRAMP, DR4/TRAIL receptor 1 (TRAIL-R1), DR5/TRAIL-R2/TNF-related apoptosis-inducing ligand receptor inducer of cell killing-2 (TRICK2)/KILLER, and DR6 [15]. TNF-α, alternatively, activates TNFR1, which forms a complex of TRADD/receptor interacting protein 1 Pharmacology 2014;94:163–169 DOI: 10.1159/000368050

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Discussion

Naftopidil – + FasL

Fig. 5. Naftopidil-stimulated FasL secretion. a NCI-H2052 cells were treated with

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a

p = 0.009

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0

b

300

30

250

25

200 150 100 50

3 6 Naftopidil treatment (h)

1

2 3 4 5 6 Treatment time (weeks)

7

8

15 10

0

b

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5 0

p = 0.020

2

Body weight (g)

Tumor volume (mm3)

a

FasL

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naftopidil (100 μmol/l) for 6 h followed by Western blotting. The signal intensity for FasL was normalized by that for β-actin. In the graph, each value represents the mean normalized FasL intensity (± SEM). n = 4 independent experiments. b Cells were treated with naftopidil (100 μmol/l) for the indicated periods of time, and the culture media were collected. Then, Western blotting was carried out in the extraction from extracellular solution using an anti-FasL antibody. The signal intensity for FasL was normalized by regarding the signal at 0 h as 1. In the graph, each value represents the mean normalized FasL intensity (± SEM). n = 4 independent experiments. The p values as compared with the intensity at 0 h were calculated with Dunnett’s test.

0

Naftopidil treatment (h) 3 6 9

0

0

1

2 3 4 5 Treatment time (weeks)

6

7

Fig. 6. Naftopidil-induced suppression of NCI-H2052 tumor growth in mice. NCI-H2052 cells were subcutaneously inoculated into the flank of the mice, and 1 week later, a physiological salt solution (Control) or naftopidil (9.8 mg/kg) was intraperitoneally injected twice a week. a Tumor volume (mean ± SEM). n = 6 independent experiments. b Body weight (mean ± SEM). n = 2–6 independent experiments.

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Pharmacology 2014;94:163–169 DOI: 10.1159/000368050

vates Fas, involving the recruitment of procaspase-8 through FADD to activate caspase-8 [17]. Intriguingly, naftopidil stimulated FasL secretion from NCI-H2052 cells, although the expression of FasL mRNA and protein was not affected. This implies that naftopidil promotes Fas activation in response to increased FasL secretion to activate caspases-8 and, in turn, caspase-3 in NCI-H2052 Mikami /Nagaya /Gotoh /Kanno / Tsuchiya /Nakano /Nishizaki  

 

 

 

 

 

 

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(RIP1)/FADD/procaspase-8 to activate caspase-8 [16]. In the present study, naftopidil upregulated the expression of TNF-α mRNA in NCI-H2052 cells, while the expression of mRNAs for TRADD and FADD was not affected. Naftopidil, accordingly, could increase the expression of TNF-α protein and activate caspase-8 through TNFR1 and the effector caspase-3 in NCI-H2052 cells. FasL acti-

cells. Overall, these results indicate that naftopidil induces apoptosis of NCI-H2052 cells by upregulating the expression of TNF-α and stimulating FasL secretion to activate caspase-8 and the effector caspase-3. Naftopidil completely suppressed tumor growth in mice inoculated with NCI-H2052 cells throughout 8 weeks after injection. This raises the possibility that naftopidil is useful for the treatment of MPM. In conclusion, the results of the present study show that naftopidil induces apoptosis of NCI-H2052 MPM cells by activating caspase-8 in association with upregulated TNF-α and increased FasL secretion followed by the effector caspase-3. The results also show that naftopidil clearly suppresses tumor growth in mice inoculated with

NCI-H2052 cells. Naftopidil, thus, may have promising characteristics to be developed as a drug for the treatment of MPM.

Acknowledgements This study was supported by research grants from the Takeda Science Foundation and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2012–2016.

Disclosure Statement The authors have no conflicts of interest to disclose.

References

Naftopidil-Induced Anticancer Effect

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Naftopidil is useful for the treatment of malignant pleural mesothelioma.

Naftopidil, an α1-adrenoceptor blocker, induced apoptosis of human malignant pleural mesothelioma NCI-H2052 cells. Naftopidil upregulated the expressi...
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