European Journal of Pharmacology 761 (2015) 309–320

Contents lists available at ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo Sheau-Yun Yuan a,b, Chen-Li Cheng a, Hao-Chung Ho a, Shian-Shiang Wang a, Kun-Yuan Chiu a, Chung-Kuang Su a, Yen-Chuan Ou a,d,nn, Chi-Chen Lin c,d,n a

Division of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung 40705, Taiwan, ROC Department of Nursing, Hung Kung University, Taichung 43302, Taiwan, ROC Institute of Biomedical Science, National Chung-Hsing University, Taichung 40227, Taiwan, ROC d Department of Education and Research, Taichung Veterans General Hospital, Taichung 40705, Taiwan, ROC b c

art ic l e i nf o

a b s t r a c t

Article history: Received 9 December 2014 Received in revised form 30 May 2015 Accepted 2 June 2015 Available online 15 June 2015

Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans. & 2015 Elsevier B.V. All rights reserved.

Keywords: Nortriptyline Tricyclic antidepressant Bladder cancer Apoptosis Reactive oxygen species Mitochondria Chemical compounds studied in this article: 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium Bromide (MTT) (PubChem CID: 64965) DMSO (PubChem CID: 679) Propidium iodide (PubChem CID:104981) Tween 20 (PubChem CID:443314) JC-1 (PubChem CID:5492929) Dihydroethidium (DHE) (PubChem CID:128682) N-acetyl-L-cysteine (NAC) (PubChem CID:12035) Glutathione (GSH) (PubChem CID:124886)

1. Introduction Recently, the incidence and prevalence of bladder cancer (BCa) has been rising. It is the fourth and fifth most commonly diagnosed malignancy in men in Europe and the United States, n Corresponding author at: Institute of Biomedical Science, National Chung-Hsing University, Taichung 40227, Taiwan, ROC. Fax: þ886 4 23592705. nn Corresponding author at: Division of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung 40705, Taiwan, ROC. Fax: þ886 4 23593160. E-mail addresses: [email protected] (Y.-C. Ou), [email protected], [email protected] (C.-C. Lin).

http://dx.doi.org/10.1016/j.ejphar.2015.06.007 0014-2999/& 2015 Elsevier B.V. All rights reserved.

respectively (Rosser et al., 2009; van den Bosch and Alfred Witjes, 2011), and the seventh most common malignant neoplasm of the urinary tract in Taiwan (Peng et al., 2006; Yuan et al., 2011). Approximately 75% of all newly diagnosed cases of bladder cancer are non-muscle-invasive BCa (NMIBC), while the remaining cases are muscle-invasive BCa (MIBC) (van den Bosch and Alfred Witjes, 2011). The standard therapies for NMIBC include transurethral tumor resection and intravesical chemotherapy and immunotherapy; however, about 30% of all tumors are refractory and about 50% recur within five years (Carradori et al., 2012; Leliveld et al., 2011). Similarly, MIBC cases that are treated with

310

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

neoadjuvant chemotherapy plus radical cystectomy also tend to have poor prognosis and a mortality rate of about 50% (Dhawan et al., 2008). As a result, novel therapeutics for BCa are needed. Tricyclic antidepressants (TCAs), such as amitriptyline and desipramine, which are commonly used to treat depression and chronic pain (Kirino and Gitoh, 2011; Dell and Butrick, 2006), also have antineoplastic activity in a wide variety of cancer cells, such as human colon cancer HT-29 cells, human osteosarcoma MG63 cells, human prostate cancer PC3 cells, rat glioma C6 cells, mouse skin squamous carcinoma (Ca3/7), and human multiple myeloma MM cells (Kabolizadeh et al., 2012; Lu. et al., 2009; Chang et al., 2008; Ma et al., 2011; Kinjo et al., 2010; Mao et al., 2011). Other TCAs, such as imipramine and clomipramine, induce apoptosis in HL-60 human acute myeloid leukemia cells by increasing production of reactive oxygen species (ROS), activating caspase 3, and disrupting the mitochondrial membrane potential (Xia et al., 1999a, 1999b). Similarly, nortriptyline (NTP; Fig. 1a), also exhibits anticancer activity in several different types of cells. For example, in human cutaneous melanoma cells, NTP has a half maximal inhibitory concentration (IC50) of 9 mM compared with 27 mM and 33 mM for clomipramine and amitriptyline, respectively (Parker et al., 2012). In addition, NTP is cytotoxic to human osteosarcoma cells (IC50 E 35 μM) and induces apoptosis in PC3 cells (IC50 450 μM) by Ca2 þ -mediated mechanisms (Hsu et al., 2004; Chih-Chuan et al., 2010). However, the antitumour effects of NTP in bladder cancer and their underlying mechanisms are not known. Therefore, in this study, we determined the antitumor effects of NTP in both mouse MBT-2 and human TCCSUP bladder cancer cells. We also investigated the mechanisms responsible for these effects.

2. Materials and methods 2.1. Cell culture and reagents Human TCCSUP and mouse MBT-2 bladder cancer cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% antibiotic antimycotic solution (Gibco, Grand Island, New York). Cells were incubated at 37 °C in a 5% CO2 atmosphere. NTP (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mM and stored at  20 °C until use. 3-[4, 5-dimethylthiazol-2-yl]2, 5 diphenyltetrazolium Bromide (MTT) powder, Propidium Iodide, N-acetyl-L-cysteine (NAC) and glutathione (GSH) were obtained from sigma company (St. Louis, MO, USA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide (JC-1) and Dihydroethidium (DHE) were purchased from Invitrogen company(Carlsbad, CA, USA) and Setareh Biotech company (LLC, Eugene, OR, USA), respectively. 2.2. Animals Adult male C3H/HeN mice (25–30 g body weight; 2-3 months of age) were obtained from the animal center of National Cheng Kung University. The animals were maintained in an air conditioned procedures were conducted to the guidelines of the Committee of Ethics in Research of Taichung Veterans General of Hospital. To create a mouse model of bladder cancer, we subcutaneously injected MBT-2 cells (1  107) into the right flank of 16-week-old male C3H/HeN mice. Ten days later, fifteen tumorbearing mice (mean tumor volume ¼ 50 mm3) were divided into three groups of five and treated with either NTP (10 or 20 mg/kg) or vehicle control for three weeks. Every day, treated mice and control mice were injected intraperitoneally with 100 μl of NTP in 0.1% DMSO or vehicle only, respectively. Tumor volume was calculated as 0.5  length  width  thickness.

2.3. Cell viability assays The effect of NTP on the viability of TCCSUP and MBT-2 cells was determined visually by phase contrast microscopy and quantitatively by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium Bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). First, TCCSUP and MBT-2 cells (2  104) were seeded onto 24-well plates and treated with NTP at concentrations of 6.25, 12.5, 25, 50 and 100 μM or vehicle alone at 37 °C in a CO2 incubator for 24 h. Then, the RPMI culture medium with drug or vehicle control was removed and 200 μl from 1 mg/ml MTT solution was added to each well. Four hours later, the MTT solution was aspirated and the formazan product was solubilized in 600 μl of DMSO. Finally, a 150 μl aliquot was analyzed by using a microplate autoreader (PerkinElmer L225-0137, Taiwan) to measure the absorbance at 540 nm. All analyses were performed in triplicate. IC50 values were linearly interpolated from dose-response curves. 2.4. Cell cycle analysis Propidium iodide (PI) staining and flow cytometry were used to perform cell cycle analysis. First, TCCSUP and MBT-2 cells (1  106) were plated on 10-cm dishes and incubated with either NTP or 0.1% DMSO as the vehicle control for 24 h. Specifically, TCCSUP cells were treated with 25 μM, 50 μM, or 100 μM NTP, while MBT2 cells were treated with 12.5 μM, 25 μM, or 50 μM NTP. Subsequently, the floating cells and attaching cells, which were trypsinized, were combined, centrifuged at 450xg, washed with ice-cold phosphate-buffered saline (PBS) two times, and fixed with 70% ethanol at  20 °C overnight. The next day, the cells were washed with ice-cold PBS and then incubated with PI staining solution (0.2 mg/ml ribonuclease, 20 μg/ml propidium iodide, and 0.1% Triton X-100) for 30 min at room temperature in the dark. Finally, the cells were counted with a flow cytometer (BD, FACSCalibur San Jose, CA 95131, USA) and data were analyzed with WinMDI software (version 2.9). All experiments were performed in triplicate and 10,000 events were counted for each sample. 2.5. Annexin V assay Apoptosis was measured by an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BioVision, Milpitas, CA, USA). The manufacturer's protocol was followed. Briefly, TCCSUP and MBT-2 cells (1  106) were seeded onto 10-cm dishes for 24 h, and then TCCSUP cells were treated with 0 μM (vehicle only), 25 μM, 50 μM or 100 μM NTP for 24 h and MBT-2 cells were treated with 0 μM (vehicle only), 12.5 μM, 25 μM or 50 μM NTP for the same period of time. Subsequently, the floating cells were collected and the attaching cells were harvested by trypsinization. Both floating and attaching cells were combined and centrifuged, washed twice with PBS, and resuspended in 500 μl of binding buffer. Cell suspensions were then incubated with 5 μl of annexin V-FITC and 5 μl of PI for 10 min at room temperature in the dark. Finally, the cells were analyzed by flow cytometry. 2.6. Western blot analysis Western blotting was used to analyze protein expression. Specifically, cell lysates with equal amounts of protein, as determined by a Bradford assay (Biorad, Alfred Nobel Dr Hercules, CA, USA), were separated by 10–15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Then, proteins were electrophoretically transferred to polyvinylidene fluoride membranes, which were blocked with 5% nonfat milk in TBST buffer (20 mM Tris–HCl, 120 mM NaCl, and 0.1% Tween 20) for 1 h. Subsequently, the membranes were incubated with primary antibodies against

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

311

Fig. 1. Effects of nortriptyline (NTP) on human TCCSUP and mouse MBT-2 bladder cancer cells and normal human peripheral blood mononuclear cells (PBMC). (A) The chemical structure of NTP. (B) TCCSUP cells, MBT-2 cells, and PBMC were seeded at a density of 2  104 cells/well and then treated with NTP (6.25 μM, 12.5 μM, 25 μM, 50 μM, or 100 μM) or a vehicle control for 24 h. The MTT assay (described in the “Methods” section) was used to quantify cell viability. (C) TCCSUP and MBT-2 cells were also treated with the same concentrations of NTP for 24, 48, or 72 h, and then cell viability was assessed with the MTT assay. In (B) and (C), data points and error bars represent the mean 7 S.D. of three experiments, respectively. (D) Effect of NTP on the morphology of TCCSUP and MBT-2 cells. TCCSUP cells were treated with 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were treated with 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Cells were viewed by phase contrast microscopy and photographed at 200  magnification.

Fas, FasL, FADD (1:1000; Santa Crutz, Biotechnology, CA, USA) and Bcl-2, Bcl-xL, Bax, X-linked inhibitor of apoptosis protein (XIAP), survivin, caspase-3, caspase-8, caspase-9, poly(ADP-ribose) polymerase (PARP) (1:1000; Cell Signaling Technology, Boston, MA, USA) and caspase-9 (EMD Millipore, California, USA) for MBT-2 cells, cytochrome c (1 μg/ml; from BD Pharmingen, San Diego, CA, USA), Bak and β-actin and glyceraldehydes 3 phosphate dehydrogenase (Santa Cruz Biotechnology, CA, USA) at 4 °C overnight. After washing the membranes with TBST buffer, they were incubated with horseradish peroxidase-linked goat anti-rabbit or goat anti-mouse secondary antibodies (1:10,000; Merck Millipore, Germany) at room temperature for 2 h. Finally, the membranes were washed 10 min with TBST buffer for three times and visualized with enhanced chemiluminescence (Amersham Biosciences, San Francisco, CA, USA) in a LAS3000 imager (Fujifilm, Tokyo, Japan).

2.7. Mitochondrial membrane potential assay One of the hallmarks of apoptosis is mitochondrial disruption, which is characterized by changes in the mitochondrial membrane potential (ΔΨm). These changes can be detected by using JC-1 (Invitrogen, Carlsbad, CA, USA), a membrane-permeable dye which accumulates in mitochondria in a membrane potential-dependent manner. Specifically, aggregation of JC-1 monomers results in a shift in fluorescence emission from green (540 nm) when ΔΨm o 120 mV to red (590 nm) when ΔΨm 4120 mV. As a result, membrane depolarization is indicated by a reduction in the red: green fluorescence intensity ratio. For the assay, cells were seeded on a 10-cm dish, TCCSUP cells were treated with 0 μM (vehicle only), 25 μM, 50 μM or 100 μM NTP for 24 h and MBT-2 cells were treated with 0 μM (vehicle only), 12.5 μM, 25 μM or 50 μM NTP for the same period of time and then incubated with 5 μM JC-1 for 30 min at 37 °C. Subsequently, fluorescence was measured with a Flowcytometer (BD, FACSCalibur San Jose, CA 95131, USA).

312

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

Fig. 2. Cell-cycle analysis of NTP-treated bladder cancer cells. TCCSUP cells were treated with 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were treated with 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Subsequently, cells were stained with propidium iodide (PI), and then analyzed by flow cytometry.

Table 1 Nortriptyline affected cell cycle distribution in TCCSUP cells. μmole/l

0 25 50 100

24 h Sub-G1 (%)

G1 (%)

S (%)

G2/M (%)

0.5 7 0.1 0.4 7 0.3 0.7 7 0.3 12.8 7 1.4b

51.8 7 1.5 55.9 7 2.5 57.0 7 3.2a 6.0 7 1.8

22.0 7 4.3 18.5 7 4.4 21.3 7 2.4 14.17 2.5

29.7 7 3.5 30.8 7 5.4 19.4 7 2.7 49.97 1.9

TCCSUP cells were treated without or with nortriptyline (25, 50 and 100 μmole/l) for 24 h, and cell cycle distribution were determined with flow cytometry. Data denoted as mean 7S.D. of experiments (n¼ 3). a b

P o0.05 versus control. P o 0.01 versus control.

Table 2 Nortriptline affected cell cycle distribution in MBT-2 cells. μmole/l

0 12.5 25 50

24 h

b c

2.9. Statistical analyses Statistical comparisons were performed by using unpaired, two-tailed Student's t-tests. The tumor volumes in animals were measured and statistically evaluated using Kruskal–Wallis nonparametric analysis of variance followed by Mann–Whitney U test. The P-values less than 0.05 were considered to be statistically significant.

3. Results

Sub-G1 (%)

G1 (%)

S (%)

G2/M (%)

1.0 7 0.1 0.9 7 0.3 12.9 7 5.4a 78.2 7 0.3c

42.9 7 3.1 46.0 7 3.1 30.3 7 0.1 19.8 7 4.4

36.3 72.2 30.3 70.1 25.6 72.1 6.1 70.6

16.9 7 0.2 19.8 7 4.4 30.17 4.4b 1.3 7 0.3

MBT-2 cells were treated without or with nortriptyline (12.5, 25 and 50 μmole/l) for 24 h, and cell cycle distribution were determined with flow cytometry. Data denoted as mean7 S.D. of experiments (n ¼3). a

24 h. In some experiments, we also pretreated these cells with either 5 mM or 10 mM N-acetyl-L-cysteine (NAC) and glutathione (GSH) (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. Subsequently, these cells were incubated with 2 μM DHE in serum-free medium at 37 °C for 15 min, washed once with serum-free medium, and then centrifuged at 450xg to remove extracellular DHE. Finally, the cells were analyzed by flow cytometry.

P o 0.05 versus control. Po 0.01 versus control. Po 0.001 versus control.

2.8. Detection of reactive oxygen species A flow cytometric assay of intracellular ROS, which can trigger apoptosis, using dihydroethidium (DHE) (Setareh Biotech, LLC, Eugene, OR, USA) a fluorescent superoxide indicator, was described previously (Xia et al., 1999a, 1999b; Bindokas et al., 1996; Satoh et al., 1998). In this study, TCCSUP and MBT-2 cells were treated with 0 μM (vehicle only), 25 μM, or 50 μM NTP for 8, 16, or

3.1. Effect of nortriptyline on in vitro assays 3.1.1. Nortriptyline exhibits cytotoxic effects on TCCSUP and MBT-2 cells To determine the cytotoxic effect of NTP on bladder cancer cells, human TCCSUP and mouse MBT-2 cells were treated with various concentrations of NTP (6.2–100 μM) for 24, 48, or 72 h, and then cell viability was determined by using the MTT assay. As shown in Fig. 1B and C, NTP markedly reduced the viability of these cells in a concentration- and time-dependent manner. After 24, 48, and 72 h, the mean IC50 7standard deviation (S.D.) were 38.57 0.5 μM, 27.8 71.5 μM, and 24.9 70.9 μM in TCCSUP cells, respectively, and 18.5 70.3 μM, 15.6 71.2 μM, and 13.7 7 0.8 μM in MBT-2 cells, respectively. In addition, NTP was almost twice as cytotoxic in MBT-2 cells as in TCCSUP cells, as shown by the MTT assay (Fig. 1C). However, when normal human peripheral blood mononuclear cells (PBMC) were treated with 100 μM NTP for 24 h, more than 80% of the cells survived (Fig. 1B). The differences between the treated cells and the control cells were analyzed by

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

313

Fig. 3. Flow cytometry analysis of NTP-induced apoptosis in TCCSUP and MBT-2 cells. (A) TCCSUP cells were treated with 0, 25, 50 and 100 μM NTP for 24 h. (B) MBT-2 cells were treated with 0, 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Subsequently, treated cells were labeled with annexin V-fluorescein isothiocyanate and PI. In each flow cytometry plot, the lower right quadrant (annexin-V þ /PI  ) shows early apoptotic cells, while the upper right quadrant (annexin V þ /PI þ) depicts late apoptotic and necrotic cells. (C) The data of live cells, early apoptotic cells and late apoptotic cells represent the mean 7 S.D. of three independent experiments; bars, S.D., Statistical significance nP value o 0.05, nnPo 0.01, nnnP o0.001 as compared with control.

Student's t-test. A *P value o0.05, **P value o0.01 or ***P value o 0.001 was considered statistically significant. At concentrations of 50 μM or higher, NTP also caused marked morphological changes in TCCSUP and MBT-2 cells, such as shrinkage and rounding (Fig. 1D). These changes were more pronounced in MBT-2 cells than TCCSUP cells. This is consistent with the results obtained from the MTT assay (Fig. 1C). Taken together, these

results suggest that NTP induces apoptosis in these bladder cancer cells. 3.1.2. Nortriptyline induces cell cycle arrest and apoptosis in TCCSUP and MBT-2 cells To elucidate the underlying mechanism of NTP-induced cell death, we analyzed the distribution of cell cycles in TCCSUP and

314

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

Fig. 4. Effect of NTP on caspase activation in TCCSUP and MBT-2 cells. (A) TCCSUP cells were treated with 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were treated with 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Total cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies against the cleaved forms of caspase-8, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Data are representative of three independent experiments. (B) TCCSUP and MBT-2 cells were pretreated with 100 μM z-DEVD-FMK (a caspase-3 inhibitor), z-IETD-FMK (a caspase-8 inhibitor), or z-LEHDFMK (a caspase-9 inhibitor) for 1 h. Then, TCCSUP cells were further treated with 50 μM NTP, and MBT-2 cells were also further treated with 25 μM NTP. After 24 h, cell viability was determined by using the MTT assay. Solid bars and error bars indicate the mean and S.D., respectively, of three independent experiments. *P o 0.05; treatment with NTP plus caspase inhibitor versus NTP alone.

MBT-2 cells that were treated with various concentrations of NTP for 24 h. As shown in Fig. 2, the percentages of sub-G1 cells, which is an indicator of cell death, in TCCSUP and MBT-2 cells treated with the highest tested concentration of NTP (12.8 71.4% and 78.2 70.3%, respectively) were significantly higher than those in untreated (control) cells (0.5 70.1% and 1.0% 70.1%, respectively). In addition, the percentage of cells in G0/G1 transition in TCCSUP cells treated with 50 μM NTP (57.0 73.2%) was significantly higher than that in untreated cells (51.8 71.5%) (Table 1). Similarly, the percentage (30.1 74.4%) of cells in G2/M transition in MBT-2 cells treated with 25 μM NTP was significantly higher than that (16.9 70.2%) in untreated cells (Table 2). These results suggest that NTP causes cell cycle arrest in these bladder cancer cells. To further elucidate the type of cell death caused by NTP, we stained NTP-treated TCCSUP and MBT-2 cells with annexin V-FITC and PI to detect apoptosis and necrosis, respectively. The percentages of early apoptotic, late apoptotic, and necrotic cells in both cell lines increased in a concentration-dependent manner (Fig. 3). At 100 and 50 μM NTP for TCCSUP and MBT-2 cells, respectively, early apoptosis occurred in 22.2% of TCCSUP cells (Fig. 3A) and in 80.7% of MBT-2 cells (Fig. 3B). Thus, the percentages of live cells, early apoptotic cells and late apoptotic cells were calculated and subjected to statistical analysis (Fig 3C). These results show that NTP induces apoptosis in both TCCSUP and MBT-2 cells, but it has a stronger effect in MBT-2 cells than in TCCSUP cells

3.1.3. Nortriptyline induces caspase-dependent apoptosis in TCCSUP and MBT-2 cells Caspases, such as caspase-9, caspase-8, and caspase-3/7, can be activated by either an intrinsic mitochondria-mediated pathway or an extrinsic death receptor-mediated pathway (Wyllie et al., 1980). These activated caspases cause cleavages of poly(ADP-ribose) polymerase (PARP), which is a marker of apoptosis. Thus, to determine whether NTP induces apoptosis intrinsically or extrinsically, we performed Western blot analysis of the cleaved forms of caspase-8, caspase-9, caspase-3, and PARP. As illustrated in Fig. 4A, TCCSUP and MBT-2 cells which were treated with Z50 μM and Z 25 μM NTP, respectively, for 24 h exhibited elevated levels of the cleaved forms of caspase-8, caspase-9, caspase3 and PARP. It is important to note that the protein level of caspase-3 in 50 μM NTP-treated MBT-2 cells was lower than that in cells treated with 25 μM of NTP. We reason that many cells treated with 50 μM of NTP may be dead and detach from the wells. This is evidenced by decreased protein levels of the internal control (βactin) as compared to those in untreated and NTP-treated cells. In addition, the cleaved caspase-3 protein (19 and 17 kDa), which is the smallest proteins among all caspase-cleaved protein molecules, is easily further degraded and may not be detected by Western blot analysis. Thus, we perform the quantitative analysis of the caspase-3 protein level by BIO-1D software. The values of the caspase-3/β-actin ratio are significantly different among cells with 0 μM NTP (vehicle-only control) (0.045), 12.5 μM NTP

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

315

Fig. 5. Effect of NTP on the expression of proteins involved in death receptor-mediated apoptosis in TCCSUP and MBT-2 cells. (A) TCCSUP cells were treated with 0, 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were treated with 0, 12.5 μM, 25 μM or 50 μM NTP for the same time period. Whole-cell lysates were western blotted with antibodies against Fas, FasL, FADD, BH3 interacting domain death agonist (BID), and β-actin. (B) After incubation for 6 h and 24 h at 37 °C, Apoptotic cells was assayed with CCK8 kit. TCCSUP and MBT-2 cells were incubated with the blocking anti-FasL mAb, clone NOK1 and clone Kay-10 at 10 μg/ml or 5μg/ml, respectively, for 2 h at 37 °C before being co-cultured with NTP at (50 μM, 100 μM) and (25 μM, 50 μM). Solid bars and error bars indicate the mean and S.D., respectively, of three independent experiments. n P o0.05, nnP o 0.01; treatment with NTP plus anti-FasL mAb versus NTP alone.

(0.252), 25 μM NTP (0.778) and 50 μM NTP (0.358). We confirmed these findings by testing whether caspase inhibitors could increase the viability of NTP-treated TCCSUP and MBT-2 cells. As shown in Fig. 4B, TCCSUP and MBT-2 cells which were treated with NTP and z-IETD-FMK (a caspase-8 inhibitor), z-LEHD-FMK (a caspase-9 inhibitor), or z-DEVD-FMK (a caspase-3 inhibitor) (KAMIYA, Seattle, WA, USA) had significantly greater cell viability than those which were treated with NTP only (P o0.05). These results suggest that NTP induces both intrinsic and extrinsic apoptosis in these bladder cancer cells. 3.1.4. Nortriptyline induces death receptor-mediated apoptosis in TCCSUP and MBT-2 cells To confirm that NTP induces extrinsic apoptosis, we determined the effect of NTP on the expression of proteins in the death receptor pathway in TCCSUP and MBT-2 cells. As shown in Fig. 5A, treatment of TCCSUP cells with 100 μM NTP increased expression of the Fas death receptor but did not increase expression of Fas ligand (FasL) and Fas-Associated protein with Death Domain (FADD) whereas treatment with 50 μM NTP increased protein levels of Fas death receptor, FasL and FADD in MBT-2 cells. It is important to note that we were unable to detect FasL and FADD in the medium of TCCSUP cells treated with 100 μM NTP. This may be due to the specific inhibitory effects of 100 μM NTP on the expression of FasL, FADD, Bid and tBid in TCCSUP cells (Fig. 5A). In addition, BH3 interacting domain death agonist (Bid) decreased at higher concentrations (50 and 100 μM, and 25 and 50 μM) of NTP in TCCSUP and MBT-2 cells, respectively, along with that the cleaved form (15 kD) of t-Bid increased in TCCSUP cells treated

with 25 and 50 μM NTP and in MBT-2 cells treated with 12.5 and 25 μM NTP. It is important to note that the protein levels of Bid at the highest concentrations (100 and 50 μM) of NTP also decreased in TCCSUP and MBT-2 cells, respectively. To confirm the Fas death receptor is involved in NTP-induced apoptosis in both cell types, we performed the cytotoxicity assay using CCK8 assay by pretreating TCCSUP and MBT-2 cells with anti-FasL blocking antibodies NOK1(10 μg/ml) and Kay 10 (5 μg/ml), respectively, for 2 h and further treating these cells with NTP in the presence of the antibodies for 6 and 24 h. As shown in Fig. 5B, anti-FasL blocking antibody treatment significantly attenuated NTP-induced apoptosis in both types of bladder cancer cells. The differences in apoptosis between cells treated with anti-Fas L blocking antibody plus NTP and those treated with NTP only were analyzed by Student's ttest. A *P value o0.05 or **P value o 0.01 was considered statistically significant. These results support the notion that NTP induces death receptor-mediated apoptosis in bladder cancer cells. 3.1.5. Nortriptyline induces mitochondria-mediated apoptosis in TCCSUP and MBT-2 cells To confirm that NTP induces intrinsic mitochondria-mediated apoptosis, we determined the effect of NTP on the mitochondrial membrane potential (ΔΨm) in TCCSUP and MBT-2 cells. Loss of the mitochondrial membrane potential is a hallmark of intrinsic apoptosis, because it is associated with the release of pro-apoptotic proteins into the cytosol (Brunelle and Letai, 2009). When we assayed the mitochondrial membrane potential in NTP-treated TCCSUP and MBT-2 cells with JC-1 dye, we observed a concentration-dependent decrease in red fluorescence and increase in

316

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

Fig. 6. Effect of NTP on mitochondria-mediated apoptosis in TCCSUP and MBT-2 cells. (A) TCCSUP and MBT-2 cells were stained with JC-1 dye (R1: aggregated JC-1, red fluorescence; R2: monomeric JC-1, green fluorescence), and then the red:green fluorescence ratio, which indicates changes in the mitochondrial membrane potential, was measured by flow cytometry. (B) Measurement of depolarization of mitochondrial membrane potential using JC-1 green fluorescence assay in TCCSUP and MBT-2 cells treated with different concentrations of NTP. TCCSUP cells were treated with 0, 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were also treated with 0, 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Then, cells were stained with JC-1 dye. Intensities were compared at the centre of each peak. (C) Cytosolic lysates were resolved by SDS-PAGE and then Western blotted with anti-cytochrome c antibody. Data are representative of three independent experiments. (D) Effect of NTP on the expression of Bcl-2 and inhibitor of apoptosis family proteins in TCCSUP and MBT-2 cells. TCCSUP cells were treated with 0 μM, 25 μM, 50 μM, or 100 μM NTP for 24 h, and MBT-2 cells were treated with 0 μM, 12.5 μM, 25 μM, or 50 μM NTP for the same time period. Total cell lysates were resolved by SDS-PAGE, and then immunoblotted with antibodies against Bcl-2, Bcl-xL, Bax, Bak, X-linked inhibitor of apoptosis (XIAP), survivin, and glyceraldehype-3-phosphate dehydrogenase (GAPDH). Data are representative of three independent experiments.

green fluorescence in both cell lines (Fig. 6A and B). This suggests that NTP reduces ΔΨm. Since loss of ΔΨm promotes the release of cytochrome c into the cytosol (Wyllie et al., 1980), we determined the levels of cytochrome c in the cytosolic fractions of TCCSUP and MBT-2 cells that were treated with different concentrations of NTP. As shown in Fig. 6C, NTP increased cytosolic cytochrome c levels in a concentration-dependent manner in both TCCSUP and MBT-2 cells. These results support the notion that NTP induces mitochondria-mediated apoptosis in bladder cancer cells. To further elucidate the mechanism of NTP-induced mitochondria-mediated apoptosis, we determined the effect of NTP on the expression of several members of the Bcl-2 and inhibitor of

apoptosis (IAP) protein families, which regulate cytochrome c release and caspase activity, respectively (Brunelle and Letai, 2009; Wu et al., 2012). Specifically, we examined four Bcl-2 family proteins, namely, Bax and Bak, which promote cytochrome c release, and Bcl-2 and Bcl-xL, which inhibit cytochrome c release (Brunelle and Letai, 2009). In addition, we examined two IAP family proteins, namely, survivin and XIAP. As shown in Fig. 6D, NTP decreased the expression of Bcl-2, Bcl-xL, XIAP, and survivin and increased the expression of Bax and Bak in both TCCSUP and MBT2 cells in a concentration-dependent manner. Taken together, these results suggest that NTP induces mitochondria-mediated apoptosis in bladder cancer cells by disrupting proteins that down-

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

regulate cytochrome c release and relieve downstream inhibition of apoptosis.

317

3.1.6. Nortriptyline-induced apoptosis is associated with reactive oxygen species production Since ROS can alter the cellular redox state and mitochondrial membrane potential (Simon et al., 2000; Cheng et al., 2012), we investigated whether NTP-treated TCCSUP and MBT-2 cells produce ROS. DHE staining revealed that red fluorescence intensity (Fig. 7A) and ROS production (Fig. 7B) increased in both cell lines as the duration of NTP treatment increased from 8 to 24 h. In addition, JC-1 staining showed that green fluorescence intensity increased in both cell lines during the same time period (Fig. 7C), which indicates a reduction in the mitochondrial membrane potential. Moreover, pretreatment of these cells with two antioxidants, NAC and GSH, reduced NTP-induced ROS production and increased cell survival rate (roughly 10%) (Fig. 7D and E and G). Furthermore, pretreatment with NAC decreased NTP-induced apoptosis in both cell lines, as shown by the annexin V assay (Fig. 7F). Collectively, these results imply that NTP-induced mitochondria-mediated apoptosis is associated with ROS production in bladder cancer cells. In addition, whether ROS induces Fas and subsequently up-regulates Fas is directly linked to apoptotic cell death, we perform immunoblotting assay of the protein levels of Fas, FasL, FADD, caspase-8 and PARP by pretreating cells with NAC (10 mM) for 2 h and then further treating the cells with NTP in the presence of NAC. In general, there were no significant differences in these bladder cancer cells treated with NTP plus NAC and NTP only (Fig 7H and Fig. 7I). Moreover, we had performed the cytotoxicity assay in these bladder cancer cells. NAC appeared to have mild protective effects (roughly 10%) in NTP-induced apoptosis of these cells. Therefore, we hypothesize that ROS- and Fas-induced apoptosis pathways should be independent in these NTP-treated bladder cancer cells 3.2. In vivo assays 3.2.1. Nortriptyline inhibits tumor growth in mice inoculated with MBT-2 cells Since NTP inhibits murine MBT-2 cell proliferation in vitro, we tested whether NTP can suppress bladder tumor growth in vivo. In C3H/HeN mice which were inoculated with MBT-2 cells, NTPtreated mice (n ¼5) exhibited significantly slower tumor growth and smaller tumors compared to untreated mice (n ¼5). The size differences between tumors grown in mice treated with and without NTP were analyzed by Mann–Whitney U test (Fig. 8A and Fig. 7. Association with reactive oxygen species (ROS) production in NTP-induced mitochondria-mediated apoptosis in TCCSUP and MBT-2 cells. TCCSUP cells were treated with 50 μM NTP for 8, 16, or 24 h, and MBT-2 cells were treated with 25 μM NTP for the same time periods. (A) NTP-treated cells and vehicle only-treated (control) cells were stained with Dihydroethidine (DHE), and then the intensity of red fluorescence was measured by flow cytometry. (B) Amount of ROS production in NTP-treated cells relative to control cells, as determined by DHE staining. Control cells were set as one fold. (C) Green fluorescence intensity in NTP-treated cells relative to control cells was determined by [FL1-H intensity]. Green fluorescence is a measure of the mitochondrial membrane potential. In (B) and (C), solid bars and error bars indicate the mean and SD, respectively, of three independent experiments (nPo 0.05, nnPo 0.01). (D, E) Effects of N-acetyl-L-cysteine (NAC) and glutathione (GSH) on ROS production in TCCSUP and MBT-2 cells. Exponentially growing cells were pretreated with 10 mM NAC or 5 mM GSH for 1 h. Subsequently, TCCSUP cells were treated with 50 μM NTP or DMSO (vehicle control) for 24 h and MBT-2 cells were treated with 25 μM NTP or DMSO for the same time period. (F, G) Effect of NAC on NTP-induced apoptosis in TCCSUP and MBT-2 cells. Cells were pretreated with 10 mM NAC, and then TCCSUP cells were treated with 100 μM NTP, while MBT-2 cells were treated with 50 μM NTP for 16 h. Finally, the percentage of apoptotic cells was determined by annexin V staining and flow cytometry. In addition, apoptotic cells were determined with MTT assay at 24 h. Data are expressed as the mean 7S.D. from three independent experiment (*, Po 0.05). (H, I) Effect of NAC on the expression of proteins involved in death receptor-mediated apoptosis in TCCSUP and MBT-2 cells. TCCSUP cells were pretreated with 10 mM NAC, and then further treated with 0, 50 and 100 μM NTP, while MBT-2 cells were treated with 0, 25 and 50 μM NTP for 24 h. Whole-cell lysates were Western blotted with antibodies against Fas, FasL, FADD, caspase-8, PARP and β-actin.

318

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

Fig. 7. (continued)

8B; *Po 0.05, **Po0.01, ***Po 0.001). These results suggest that NTP can inhibit the growth of bladder tumors in vivo. 4. Discussion Previous studies have shown that NTP has beneficial effects

both in vitro and in vivo. For example, low concentrations of NTP (0.1–5 μM) confer neuroprotective effects, downregulate cytosolic phospholipase A2, and prevent mitochondrial depolarization with minimal toxicity in both astrocytes and mice (Zhang et al., 2008). In contrast, higher concentrations of NTP (10–50 μM) have antitumor effects on human osteosarcoma and cutaneous melanoma cells (Parker et al., 2012; Hsu et al., 2004). In this study, we demonstrated for the first time that NTP induces apoptosis in human bladder cancer cells in vitro and inhibits bladder tumor growth in vivo. Our results showed that NTP is cytotoxic to both human TCCSUP and mouse MBT-2 bladder cancer cells (IC50s: 38.57 0.5 μM and 18.5 70.3 μM, respectively). However, this effect is more pronounced in the mouse cell line. Nevertheless, The IC50 of NTP in human TCCSUP cells is similar to that reported for human osteosarcoma cells (35 μM) (Parker et al., 2012). In addition, NTP causes cell cycle arrest at G0/G1 transition of TCCSUP cells and G2/M transition of MBT-2 cells. These differential effects of NTP in TCCSUP and MBT-2 cells may be due to differences between cell lines or species, such as differences in cell doubling time. However, additional experiments involving NTP and cell cycle regulators, such as cyclin-dependent kinases, would be needed to elucidate the molecular base of these differences. We also established that NTP induces caspase-dependent apoptosis in TCCSUP and MBT-2 cells, and that both mitochondriamediated and death receptor-mediated pathways are involved. Specifically, we showed that NTP increases the percentage of early apoptotic cells (Fig. 3A, B and 3C), decreases the mitochondrial membrane potential (Fig. 6A and B), and promotes mitochondrial release of cytochrome c (Fig. 6C). We also demonstrated that NTP alters the expression levels of several proteins that are important in apoptosis (Brunelle and Letai, 2009; Wyllie et al., 1980; Wu et al., 2012); it decreases the expression of anti-apoptotic proteins, such as Bcl-2, Bcl-xL, XIAP, and survivin, and increases the expression of proapoptotic protein, such as Fas, FasL, FADD, t-Bid, Bax, and Bak (Figs. 5A and 6D), as well as downstream proteins, such as the cleaved forms of PARP and caspases 3, 8, and 9 (Fig. 4A). Collectively, these results imply that the cytotoxicity of NTP in bladder cancer cells results from a multi-pronged disruption of both extrinsic and intrinsic apoptotic pathways. As noted, we are unable to detect FADD and t-Bid protein in the cell lysates using immunoblotting assay in TCCSUP and MBT-2 cells treated with 100 or 50 μM NTP, respectively. This may be due to proteolytic degradation under the experimental conditions. Furthermore, we demonstrated that NTP-induced mitochondria-mediated apoptosis in TCCSUP and MBT-2 cells is associated with the production of ROS, which can be inhibited by antioxidants (Fig. 7A–G). This finding is consistent with several previous studies that have shown that other TCAs, such as imipramine, clomipramine, and amitriptyline, also induce production of ROS, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, and caspase-dependent apoptosis in human cancer cells, such as acute myeloid leukemia HL-60 cells (Xia et al., 1999a, 1999b), non-small cell lung carcinoma H460 cells, cervix carcinoma HeLa cells, and hepatocellular carcinoma HepG2 cells (Cordero et al., 2010). However, this effect may not be specific to TCAs, because there is evidence that many chemotherapeutic drugs increase ROS production in almost all cancer cells (Cordero et al., 2010), due to their low levels of enzymes which protect cells from oxidative damage, such as catalase, superoxide dismutase, and glutathione peroxidase (Pelicano et al., 2004). It is important to know whether ROS production can upregulate the Fas death receptor to induce apoptosis pathways. Thus, we have demonstrated that the protein levels of Fas, FasL, FADD, caspase-8 and PARP have no significant differences in bladder cancer cells treated with NTP plus NAC and NTP only (Fig 7H and I). Based on these

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

319

as a chemotherapeutic agent for human patients with bladder cancer in a phase I clinical trial.

5. Conclusion We have shown that NTP induces caspase-dependent apoptosis in human TCCSUP and mouse MBT-2 bladder cancer cells by both mitochondria-mediated and death receptor-mediated mechanisms. Furthermore, NTP inhibits the growth of bladder tumors in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP may be as a clinically useful drug for human bladder cancer.

Declaration of conflicting interests The authors declare no potential conflicts of interests with respect to the authorship and/or publication of this article

Acknowledgments The work was supported by Taichung Veterans General Hospital (TCVGH-1025001B). We would like to thank Miss. Chia-Chiao Shih and Yu-Chia Huang for their help with Western blot assay.

References Fig. 8. Effect of NTP on tumor growth in vivo. C3H/HeN mice (n¼ 5) were inoculated with 1  107 MBT-2 cells, and then treated with 10 or 20 mg/kg NTP or 0.1% DMSO as a control, as described in the Methods section. (A) Subsequently, tumor volumes were measured every three days. Data points and error bars indicate the mean and SD, respectively, of two experiments. (B) Similar results were obtained from duplicate experiments.

results, we hypothesize that ROS- and Fas-induced apoptosis pathways should be independent in these NTP-treated bladder cancer cells Finally, we show that NTP can inhibit the growth of bladder tumors in mice inoculated with MBT-2 cells according to the criteria of tumor volume (Fig. 8). It is important to note that we did not determine the survival rates in the tumor-bearing mice treated with vehicle only (control group) and with NTP (10 mg/kg and 20 mg/kg NTP) because no death of mice in either group was found in the 23-day experimental period. We expect that, if the long term ( 42-3 month) survival of these mice was investigated, the survival rate in tumor-bearing mice treated with NTP should be significantly higher than that in tumor-bearing mice treated with vehicle only. This in vivo result, together with the finding of the ability of NTP to induce apoptosis by two different pathways, are significant because these suggest that human bladder cancer cells may be not only susceptible to NTP but also less likely to develop resistance to NTP than other drugs with more specific cellular targets. Another advantage of using NTP is that it has low toxicity in human cells. In this study, we demonstrated that NTP has a minimal effect on the viability of normal human PBMCs (Fig. 1B). Furthermore, NTP has a long history of use as an antidepressant without many detectable adverse effects (Menza et al., 2009; Osborne et al., 2014). Typically, NTP dosages are 25–75 mg/ day (Krymchantowski et al., 2011; Maizels and McCarberg, 2005; Yeragani et al., 2002), which correspond to plasma concentrations up to 10 μM (Yeragani et al., 2002). Although somewhat higher plasma concentrations may be needed to achieve the antitumour effects shown in this study, our results warrant further tests of NTP

Bindokas, V.P., Jordan, J., Lee, D.C., Miller, R., 1996. Superoxide production in rat hippocampal neuron: selective imaging with hydroethidine. J. Neurosci. 16, 1324–1336. Brunelle, J.K., Letai, A., 2009. Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell Sci. 122, 437–441. Carradori, S., Cristini, C., Secci, D., Gulia, C., Gentile, V., Di Pierro, G.B., 2012. Current and emerging strategies in bladder cancer. Anticancer Agents Med. Chem. 12, 589–603. Chang, H.C., Huang, C.C., Huang, C.J., Cheng, J.S., Liu, S.I., Tsai, J.Y., Chang, H.T., Huang, J.K., Chou, C.T., Jan, C.R., 2008. Desipramine-induced apoptosis in human PC3 prostate cancer cells: activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2 þ ]i elevation. Toxicology 250, 9–14. Cheng, S.B., Wu, L.C., Hsieh, Y.C., Wu, C.H., Chan, Y.J., Chang, L.H., Chang, C.M., Hsu, S. L., Teng, C.L., Wu, C.C., 2012. Supercritical carbon dioxide extraction of aromatic turmerone from Curcuma longa Linn. induces apoptosis through reactive oxygen species-triggered intrinsic and extrinsic pathways in human hepatocellular carcinoma HepG2 cells. J. Agric. Food Chem. 60, 9620–9630. Chih-Chuan, P., Shaw, C.F., Huang, J.K., Kuo, C.C., Kuo, D.H., Shieh, P., Lu, T., Chen, W. C., Ho, C.M., Jan, C.R., 2010. Effect of nortriptyline on cytosolic Ca2 þ regulation and viability in PC3 human prostate cancer cells. Drug Dev. Res. 71, 323–330. Cordero, M.D., Sánchez-Alcázar, J.A., Bautista-Ferrufino, M.R., Carmona-López, M.I., Illanes, M., Ríos, M.J., Garrido-Maraver, J., Alcudia, A., Navas, P., de Miguel, M., 2010. Acute oxidant damage promoted on cancer cells by amitriptyline in comparison with some common chemotherapeutic drugs. Anticancer Drugs 21, 932–944. Dell, J.R., Butrick, C.W., 2006. Multimodal therapy for painful bladder syndrome/ interstitial cystitis. J. Reprod. Med. 51 (3 Suppl), 253–260. Dhawan, D., Jeffreys, A.B., Zheng, R., Stewart, J.C., Knapp, D.W., 2008. Cyclooxygenase-2 dependent and independent antitumor effects induced by celecoxib in urinary bladder cancer cells. Mol. Cancer Ther. 7, 897–904. Hsu, S.S., Huang, C.J., Chen, J.S., Cheng, H.H., Chang, H.T., Jiann, B.P., Lin, K.L., Wang, J. L., Ho, C.M., Jan, C.R., 2004. Effect of nortriptyline on intracellular Ca2 þ handling and proliferation in human osteosarcoma cells. Basic Clin. Pharmacol. Toxicol. 95, 124–130. Kabolizadeh, P., Engelmann, B.J., Pullen, N., Stewart, J.K., Ryan, J.J., Farrell, N.P., 2012. Platinum anticancer agents and antidepressants: desipramine enhances platinum-based cytotoxicity in human colon cancer cells. J. Biol. Inorg. Chem. 17, 123–132. Kinjo, T., Kowalczyk, P., Kowalczyk, M., Walaszek, Z., Slaga, T.J., Hanausek, M., 2010. Effects of desipramine on the cell cycle and apoptosis in Ca3/7 mouse skin squamous carcinoma cells. Int. J. Mol. Med. 25, 861–867. Kirino, E., Gitoh, M., 2011. Rapid improvement of depressive symptoms in suicide attempters following treatment with milnacipran and tricyclic antidepressants: a case series. Neuropsychiatr. Dis. Treat. 7, 723–728. Krymchantowski, A.V., da Cunha Jevoux, C., Bigal, M.E., 2011. Topiramate plus nortriptyline in the preventive treatment of migraine: a controlled study for

320

S.-Y. Yuan et al. / European Journal of Pharmacology 761 (2015) 309–320

nonresponders. J. Headache Pain 13, 53–59. Leliveld, A.M., Bastiaannet, E., Doornweerd, B.H., Schaapveld, M., de Jong, I.J., 2011. High risk bladder cancer: current management and survival. Int. Braz. J. Urol. 37, 203–210, discussion 210–212. Lu., T., Huang, C.C., Lu, Y.C., Lin, K.L., Liu, S.I., Wang, B.W., Chang, P.M., Chen, I.S., Chen., S.S., Tsai, J.Y., Chou, C.T., Jan, C.R., 2009. Desipramine-induced Ca-independent apoptosis in Mg63 human osteosarcoma cells: dependence on P38 mitogen-activated protein kinase-regulated activation of caspase 3. Clin. Exp. Pharmacol. Physiol. 36, 297–303. Ma, J., Qiu, Y., Yang, L., Peng, L., Xia, Z., Hou, L.N., Fang, C., Qi, H., Chen, H.Z., 2011. Desipramine induces apoptosis in rat glioma cells via endoplasmic reticulum stress-dependent CHOP pathway. J. Neuro-Oncol. 101, 41–48. Maizels, M., McCarberg, B., 2005. Antidepressants and antiepileptic drugs for chronic non-cancer pain. Am. Fam. Physician 71, 483–490. Mao, X., Hou, T., Cao, B., Wang, W., Li, Z., Chen, S., Fei, M., Hurren, R., Gronda, M., Wu, D., Trudel, S., Schimmer, A.D., 2011. The tricyclic antidepressant amitriptyline inhibits D-cyclin transactivation and induces myeloma cell apoptosis by inhibiting histone deacetylases: in vitro and in silico evidence. Mol. Pharmacol. 79, 672–680. Menza, M., Dobkin, R.D., Marin, H., Mark, M.H., Gara, M., Buyske, S., Bienfait, K., Dicke, A., 2009. A controlled trial of antidepressants in patients with Parkinson disease and depression. Neurology 72, 886–892. Osborne, L.M., Birndorf, C.A., Szkodny, L.E., Wisner, K.L., 2014. Returning to tricyclic antidepressants for depression during childbearing: clinical and dosing challenges. Arch. Womens Ment. Health 17, 239–246. Parker, K.A., Glaysher, S., Hurren, J., Knight, L.A., McCormick, D., Suovouri, A., Amberger-Murphy, V., Pilkington, G.J., Cree, I.A., 2012. The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures. Anticancer Drugs 23, 65–69. Pelicano, H., Carney, D., Huang, P., 2004. ROS stress in cancer cells and therapeutic implications. Drug Resist. Updat. 7, 97–110. Peng, C.C., Chen, K.C., Peng, R.Y., Su, C.H., Hsieh-Li, H.M., 2006. Human urinary bladder cancer T24 cells are susceptible to the Antrodia camphorata extracts. Cancer Lett. 243, 109–119. Rosser, C.J., Liu, L., Sun, Y., Villicana, P., McCullers, M., Porvasnik, S., Young, P.R., Parker, A.S., Goodison, S., 2009. Bladder cancer-associated gene expression signatures identified by profiling of exfoliated urothelia. Cancer Epidemiol.

Biomark. Prev. 18, 444–453. Satoh, T., Numakawa, T., Abiru, Y., Yamagata, T., Ishikawa, Y., Enokido, Y., et al., 1998. Production of reactive oxygen species and release of L-glutamate during superoxide anion-induced cell death of cerebellar granule neurons. J. Neurochem. 70, 316–324. Simon, H.U., Haj-Yehia, A., Levi-Schaffer, F., 2000. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415–418. van den Bosch, S., Alfred Witjes, J., 2011. Long-term cancer-specific survival in patients with high-risk, non-muscle-invasive bladder cancer and tumour progression: a systematic review. Eur. Urol. 60, 493–500. Wu, C.S., Chen, Y.J., Chen, J.J., Shieh, J.J., Huang, C.H., Lin, P.S., Chang, G.C., Chang, J.T., Lin, C.C., 2012. Terpinen-4-ol induces apoptosis in human nonsmall cell lung cancer in vitro and in vivo. Evid. Based Complement. Altern. Med. 2012, 818261. Wyllie, A.H., Kerr, J.F., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306. Xia, Z., Bergstrand, A., DePierre, J.W., Nässberger, L., 1999a. The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation. J. Biochem. Mol. Toxicol 13, 338–347. Xia, Z., Lundgren, B., Bergstrand, A., DePierre, J.W., Nässberger, L., 1999b. Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and BclxL. Biochem. Pharmacol. 57, 1199–1208. Yeragani, V.K., Roose, S., Mallavarapu, M., Radhakrishna, R.K., Pesce, V., 2002. Major depression with ischemic heart disease: effects of paroxetine and nortriptyline on measures of nonlinearity and chaos of heart rate. Neuropsychobiology 46, 125–135. Yuan, S.Y., Lin, C.C., Hsu, S.L., Cheng, Y.W., Wu, J.H., Cheng, C.L., Yang, C.R., 2011. Leaf extracts of Calocedrus formosana (Florin) induce G2/M cell cycle arrest and apoptosis in human bladder cancer cells. Evid. Based Complement. Altern. Med. 2011, 380923. Zhang, W.H., Wang, H., Wang, X., Narayanan, M.V., Stavrovskaya, I.G., Kristal, B.S., Friedlander, R.M., 2008. Nortriptyline protects mitochondria and reduces cerebral ischemia/hypoxia injury. Stroke 39, 455–462.