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Desipramine-induced apoptosis in human PC3 prostate cancer cells: Activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2+]i elevation
Toxicology 250 (2008) 9–14
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Toxicology journal homepage: www.elsevier.com/locate/toxicol
Desipramine-induced apoptosis in human PC3 prostate cancer cells: Activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2+ ]i elevation Hong-Chiang Chang a , Chorng-Chih Huang b , Chun-Jen Huang c,d , Jin-Shiung Cheng e , Shiuh-In Liu f , Jeng-Yu Tsai f , Hong-Tai Chang f , Jong-Khing Huang f , Chiang-Ting Chou g , Chung-Ren Jan g,∗ a
Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan Department of Nursery, Tzu Hui Institute of Technology, Pingtung 926, Taiwan Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Department of Psychiatry, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan e Department of Medicine, Yongkang Veterans Hospital, Tainan 710, Taiwan f Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan g Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan b c
a r t i c l e
i n f o
Article history: Received 11 March 2008 Received in revised form 15 May 2008 Accepted 15 May 2008 Available online 27 May 2008 Keywords: Apoptosis Caspase-3 Desipramine MAPKs PC3 cells Prostate
a b s t r a c t The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+ ]i ) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+ ]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+ ]i rises may slow down or alleviate desipramine-induced cytotoxicity. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In addition to its clinical effect in treating psychological disorders (Mayers and Baldwin, 2005), desipramine has been shown to exert different effects on many cell types in vitro, such as induction of Ca2+ influx and cytotoxicity in PC3 human prostate cancer cells (Huang et al., 2007), inhibition of activity-dependent sodium channel (Lenkey et al., 2006), and inhibition of Na+ /H+ exchanger in human submandibular cells (Choi et al., 2006). Desipramine is commonly used as an inhibitor for acid ceramidase (Elojeimy et al., 2006). Furthermore, desipramine was shown to cause apoptosis in human HT29 colon carcinoma cells (Arimochi and Morita, 2006), and to increase cytosolic free Ca2+ levels ([Ca2+ ]i ) and kill cells in renal tubular cells (Ho et al., 2005). Other actions of desipramine include decreasing tumor necrosis
∗ Corresponding author. Tel.: +886 7 3422121/1509; fax: +886 7 3468056. E-mail address: [email protected] (C.-R. Jan). 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.05.010
factor-alpha production (Reynolds et al., 2005), downregulating the cortical alpha1- and alpha2-adrenoceptors (Subhash et al., 2003), inhibiting cytochrome P450 enzymes (Shin et al., 2002) and neuronal/skeletal ATP-Ca2+ pump of endoplasmic reticulum (Couture et al., 2001; Soler et al., 2000), suppressing plasma membrane Ca2+ pump (Plenge-Tellechea et al., 1999), activating steroid transporters (Pariante et al., 2001) and GABAergic systems (Asahi and Yonehara, 2001), and altering protein kinase C activity (Morishita and Watanabe, 1997), etc. The term “apoptosis” was coined in 1972 by John Kerr who observed that certain dying cells shared several morphologic properties. All the criteria used to describe apoptotic cells were morphological and included condensation and margination of chromatin, cytoplasmic vacuolization, cellular shrinkage, increase in cellular density, nuclear fragmentation, and apoptotic body formation (Kerr et al., 1972). Cysteine aspartate specific proteases (caspases) families, which have highly specific proteases activity and can cleave proteins exclusively after aspartate residues, are well-known main actors of the apoptotic proteolytic cascades. Such
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as caspase-8 is an initiator or activator in caspase cascades; caspase3 is an effector in caspase cascades; and caspase-12 is a cytokine processor in caspase cascades (Coffey et al., 2001; Hengartner, 2001; Thornberry and Lazebnik, 1998). Caspase-3 is a key protease activated during the early stages of apoptosis (Nakagawa and Yuan, 2000). Alteration in [Ca2+ ]i is a crucial regulator of many cellular events (Berridge, 2006). To allow a precise regulation of [Ca2+ ]i and many signaling pathways by Ca2+ , cells have numerous mechanisms by which to modulate [Ca2+ ]i both globally and at the subcellular level (Berridge, 2005). Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved serine-threonine kinases that participate in cell differentiation, cell growth and survival (Sharma et al., 2005). Extracellular signal-regulated kinases (ERK), stress activated protein kinase c-Jun/N-terminal kinases (JNK) and p38 are three major components of MAPKs. Signal transduction involves sequential phosphorylation of a tripartite kinase module, culminating in an activated MAPK. Activated MAPK may stay in the cytoplasm to phosphorylate structural proteins or translocate to the nucleus, where it can activate transcription factors involved in DNA synthesis and cell division. MAPKs have been suggested to play a pivotal role in apoptosis (Cross et al., 2000; Reddy et al., 2003). Therefore, a [Ca2+ ]i rise and subsequent apoptosis is tightly related. However, not all types of apoptosis are caused by a Ca2+ signal. We have previously reported that in PC3 human prostate cancer cells desipramine-induced cell death that was worsened by chelating cytosolic Ca2+ with BAPTA/AM (Huang et al., 2007). The goal of this study was (1) to explore whether apoptosis accounted for desipramine-induced cell death, (2) to examine the involvement of ERK, JNK and p38 mitogen-activated protein kinases, and (3) to investigate the mechanism underlying the cytotoxic effect of BAPTA/AM. 2. Materials and methods 2.1. Materials The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). BAPTA/AM was from Molecular Probes (Eugene, OR, USA). Desipramine, propidium iodide (PI), dimethyl sulfoxide (DMSO) and other reagents were from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Cell culture PC3 cells were obtained from American Type Culture Collection and were cultured in Dulbeco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were kept at 37 ◦ C in 5% CO2 -containing humidified air.
2.3. Solutions Ca2+ -containing medium (pH 7.4) had (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; and glucose 5. Ca2+ -free medium contained similar components as Ca2+ containing medium except that CaCl2 was substituted with 0.3 mM EGTA. Agents were dissolved in water, ethanol or dimethyl sulfoxide (DMSO) as concentrated stocks.
2.5. Cell viability assays The measurement of viability was based on the ability of viable cells to cleave tetrazolium salts by dehydrogenases. Augmentation in the amount of developed color directly correlated with the number of metabolically active cells. Assays were performed according to manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a density of 50,000 cells/well in culture medium for 4 h to allow attachment. Then the culture medium was added with 10 l of serum-free medium containing different concentrations of treatments. The cell viability detecting reagent 4-[3-[4lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate (WST-1; 10 l pure solution) was added to each sample 24 h after various concentrations of desipramine, and cells were incubated for additional 2 h in a humidified atmosphere (37 ◦ C). In experiments using BAPTA/AM to chelate intracellular Ca2+ , cells were treated with 20 M BAPTA/AM for 1 h. The cells were washed once with Ca2+ -containing medium and incubated with or without 100 M desipramine for 24 h. The absorbance of samples (A450 ) was determined using a scanning multiwell spectrophotometer. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control value. Experiments were repeated five times in six replicates (wells).
2.6. Assessment of MAPKs and caspase-3 activation by immunoblotting Cell concentrations were adjusted to 3 × 106 cells/dish and were seeded to 6 cm culture dishes. After 2 h of incubation, the culture medium was replaced by serumfree medium supplemented with 1 mg/ml bovine serum albumin (USBTM , Cleveland, OH, USA) and serum starvation was continued for 4 h, followed by various treatments. The treatments were terminated after indicated time intervals by aspirating the supernatant and washing the dishes with a physiological saline. After washing, the cells were lysed on ice for 5 min with 70 l of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3 VO4 , 1 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). The lysed cells were scraped off the dish using a rubber policeman, transferred to microcentrifuge tubes, and vortexed for 10 s. The cell lysates were then centrifuged to remove insoluble materials and the protein concentration of each sample was measured. Approximately, 50 g of supernatant protein from each sample was used for gel electrophoresis analysis on a 10% SDSpolyacrylamide gel. After electrophoresis, the fractionated proteins on gel were transferred to PVDF membranes (NENTM Life Science Products Inc., Boston, MA, USA). For immuoblotting, the membranes were blocked with 5% non-fat milk in a buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20, and incubated overnight with the primary antibody (rabbit anti-human Bcl-2, rabbit anti-human phospho-ERK antibody, rabbit anti-human ERK antibody, rabbit antihuman phospho-JNK antibody, rabbit anti-human JNK antibody, rabbit anti-human phospho-p38 MAPK antibody, rabbit anti-human p38 MAPK antibody, rabbit-anti human cleaved caspase-3 or rabbit-anti human  tubulin; all from Cell Signaling Technology, Beverly, MA, USA). Then the membranes were extensively washed with the same buffer and incubated for 1 h with the secondary antibody (goat anti-rabbit antibody, Transduction Laboratories, Lexington, KY, USA). After extensive washing, the immune complexes were detected by chemiluminescence using the RenaissanceTM Western Blot Chemiluminescence Reagent Plus kit (NENTM Life Science Products Inc., Boston, MA, USA).
2.7. Measurements of subdiploidy nuclei by flow cytometry After treatments, cells were collected from the media, and were washed with ice-cold physiological saline twice and resuspended in 3 ml of 70% ethanol at −20 ◦ C. The cells were centrifuged for 5 min at 200 × g. Ethanol was decanted and the cell pellet was washed with ice-cold saline twice, and was suspended in 1 ml propidium iodide (PI) solution (1% Triton X-100, 20 g PI, 0.1 mg/ml RNase). The cell pellet was incubated in the dark for 30 min at room temperature. Cell fluorescence was measured in the FACScan flow cytometer (Becton Dickinson immunocytometry systems, San Jose, CA, USA) and the data were analyzed using the MODFIT software.
2.8. Detection of released cytochrome-c 2.4. [Ca2+ ]i measurements Trypsinized cells (106 ml−1 ) were loaded with 2 M fura-2/AM for 30 min at 25 C in culture medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 ◦ C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 (plus 5 mM CaCl2 ) and 10 mM EGTA sequentially at the end of each experiment. [Ca2+ ]i was calculated as previously described (Grynkiewicz et al., 1985). ◦
Preparation for cytosolic fraction has been previously described (Oyama et al., 2000). PC3 cells were plated onto a 10 cm dish (7.5 × 105 cells) 1 day before treatment. Various concentrations of desipramine were added to the medium for 24 h. The cells were washed twice with Ca2+ -containing medium, and suspended in icecold buffer (20 mM HEPES-KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitor), followed by homogenization with a dounce homogenizer (50 strokes) on ice. The supernatant was recovered by centrifugation (14,000 × g, 15 min, 4 ◦ C), and subjected to Western blot analysis using anti-cytochrome-c antibody (Cell Signaling Technology, Beverly, MA, USA).
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Fig. 1. Desipramine-induced apoptosis. (A) After treatment with various concentrations of desipramine for 18 h, cells were examined for apoptosis by using flow cytometry. Data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control. (B) Caspase-3 cysteine proteases were activated in desipramine-induced apoptosis. Protein extracts were prepared 8 h after exposure to various concentrations of desipramine. Data are typical of five experiments.
2.9. Statistics Data are reported as mean ± S.E.M. of five experiments and were analyzed by two-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS® , SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s HSD (honestly significant difference) procedure. A P-value less than 0.05 was considered significant.
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Fig. 2. Effect of desipramine on the phosphorylation of ERK, JNK, and p38 MAPKs. Cells were treated with 100 M desipramine for indicated time periods. Activated ERK (phospho-ERK), JNK (phospho-JNK), and p38 MAPK (phospho-p38) were detected in immunoblots using antibodies specific for the phosphorylated form of each kinase. The same blot was stripped and used to determine the amount of each kinase. Data are typical of five experiments.
stand whether JNK was also involved in desipramine-induced apoptosis, 50 M SP600125 (based on the data from Fig. 3A) was administered 2 h prior to the addition of 100 M desipramine. Fig. 3B shows that pretreatment with SP600125 did not alter basal value but significantly rescued cells from desipramine-induced apoptosis by 12 ± 2%. 3.3. JNK regulated caspase-3 activation Based on the above results, it appeared that activation of JNK could regulate desipramine-induced apoptosis of PC3 cells, but
3. Results 3.1. Effect of desipramine on apoptosis of PC3 cells Previous data showed that desipramine-induced concentrationdependent inhibition of viability of PC3 cells at concentrations between 10 and 800 M (Huang et al., 2007). The possibility of involvement of apoptosis in the cell death was explored. As shown in Fig. 1A, apoptosis (measuring the increase in subdiploidy nuclei appeared in cells) occurred in cells treated with 10–800 M desipramine in a concentration-dependent manner. Caspase-3 is thought to function as an executioner of apoptosis and its activation could reflect the percentage of apoptotic cells. Caspase-3 activation was determined by immunoblotting, and it was found that the expression of active caspase-3 increased in the presence of 100 M desipramine at 360–720 min in a time-dependent manner (Fig. 1B). 3.2. Involvement of MAPKs in desipramine-induced apoptosis MAPKs are activated by phosphorylation of specific tyrosine and threonine residues and the relative levels of phosphorylated MAPKs in total MAPKs represent the degree of MAPK activation. Fig. 2A and C shows that the level of phosphorylated ERK (phospho-ERK) and p38 did not increase after addition of 100 M desipramine. Exposure to desipramine increased the intensity of phosphorylated JNK (phospho-JNK) between 1 and 120 min (Fig. 2B). To further examine whether JNK phosphorylation was indeed involved in desipramine-induced cell death, 10–50 M SP600125 (a specific JNK inhibitor) was administered 2 h prior to the addition of 100 M desipramine. WST-1 metabolism was measured 24 h after treatment. SP600125 (10–50 M) did not alter basal viability. While 10–20 M SP600125 did not inhibit desipramineinduced cell death, 50 M SP600125 significantly rescued cells from desipramine-induced cell death by 12 ± 1% (Fig. 3A). To under-
Fig. 3. Effect of SP600125 on desipramine-induced cell death. (A) Cells were pretreated with 10 M, 20 M, 50 M SP600125 and DMSO (vehicle control) for 2 h before 24 h-treatment with 100 M desipramine. Cell viability was determined by WST-1 assays. (B) Cells were pretreated with 50 M SP600125 and DMSO (vehicle control) for 2 h and then were treated with or without 100 M desipramine for 24 h. Apoptosis was assessed by flow cytometry. Data are mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
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Fig. 4. Effect of SP600125 on activation of caspase-3. (A) Protein extracts were prepared after treating with/without 50 M SP600125 for 2 h and the cells were then treated with 100 M desipramine for 8 h. Immunoblotting assays were performed with antibodies against cleaved caspase-3 and  tubulin. Data are typical of five experiments. (B) The effect of SP600125 on activation of caspase-3 was quantified by densitometry. The figure normalized intensities of the bands of cleaved caspase3 against the bands of  tubulin using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
the relationship between JNK phosphorylation and caspase-3 activation was unclear. To understand whether phosphorylation of JNK could regulate caspase-3 activation, SP600125 (50 M) was added for 2 h followed by addition of desipramine for 8 h. It was found that pretreatment with SP600125 partly reduced the activation of caspase-3 (by 18 ± 1 folds compared with treatment with desipramine alone) (Fig. 4A and B). 3.4. Enhancing effect of chelating Ca2+ with BAPTA on desipramine-induced cell death Previous evidence (Huang et al., 2007) shows that in the presence of 100 M desipramine, cell viability was reduced to 57 ± 3%. In the presence of BAPTA, the desipramine-induced decrease in cell viability was further decreased to 47 ± 3%. Thus experiments were designed to examine whether the BAPTA-induced further decrease of cell death was also mediated by JNK phosphorylation. Fig. 5A shows that pretreatment with 20 M BAPTA did not significantly alter basal JNK phosphorylation, but significantly increased desipramine-induced JNK phosphorylation by 2.8 ± 2 folds. Consistently, Fig. 5B shows that BAPTA/AM pretreatment did not change basal caspase activation, but significantly increased desipramineinduced caspase activation by 55 ± 2 folds. 3.5. Lack of involvement of mitochondrial pathway of apoptosis in desipramine-induced cell death The cytochrome-c release and expression of Bcl-2 in desipramine-induced apoptosis of human PC3 prostate cancer cells were explored by Western blotting. No significant release of cytochrome-c or down-expression of Bcl-2 in the cytoplasmic fraction of desipramine-induced apoptosis was found (not shown). 4. Discussion The major novel finding of the present study was that desipramine caused death of human PC3 prostate cancer cells
via evoking apoptosis with the participation of JNK pathway, and [Ca2+ ]i rises played a preventive role in the process. We studied the effect of desipramine on cytotoxic markers such as cell viability, subdiploidy nuclei and activation of caspase-3 (Hengartner, 2001), and found that desipramine-induced cell death was accompanied by DNA fragmentation as detected by propidium iodide staining, and the activation of caspase-3. The previous reported concentration range (10–800 M) of desipramine for inducing cell death (Huang et al., 2007) was consistent with the range that evoked apoptosis in the present study. Thus it appears that desipramineinduced cell death via evoking apoptosis in PC3 cells. In other studies, desipramine was shown to induce apoptosis in different cell lines (Arimochi and Morita, 2006; Ho et al., 2005; Qi et al., 2002). To further investigate the mechanisms and the signaling pathways in desipramine-induced apoptosis of PC3 cells, immunoblotting was used to explore the alternation of phosphorylation of three members of the MAPK family (ERK, JNK, p38), because recent research suggests that phosphorylation of MAPKs plays an important role in the processes of apoptosis (Cross et al., 2000; Leger et al., 2006; Nusuetrong et al., 2005). MAPKs are activated by many stimuli and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses (Bost et al., 2005). Desipramine was shown to activate the phosphorylation of JNK, but not ERK and p38, and the JNK specific inhibitor SP600125 inhibited desipramine-induced cell death and apoptosis by the same percentage. Therefore, it appears that desipramineinduced apoptosis via JNK/caspase-3-dependent signaling pathway in PC3 cells. JNK has been shown to be involved in apoptosis in many cell types stimulated with different ligands. For instance, Yang et al. showed that JNK mediated proliferation and tumor growth of human prostate carcinoma (Yang et al., 2003). Wanpen et al. showed that salsolinol, an endogenous neurotoxin, induced apoptosis via activating JNK and NF-kappaB signaling pathways in human neuroblastoma cells (Wanpen et al., 2007). CerezoGuisado et al. demonstrated that JNK signaling pathway mediated lovastatin-induced rat brain neuroblast apoptosis (Cerezo-Guisado et al., 2007). Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induced apoptosis in malignant glioma cells via the JNK pathway (Iwamaru et al., 2007). Altiok et al. reported that JNK pathway regulated estradiol-induced apoptosis in hormone-dependent human breast cancer cells (Altiok et al., 2007). Our previous report showed that desipramine-induced cell death that was worsened by chelating cytosolic Ca2+ with the Ca2+ chelator BAPTA/AM, however the mechanism was unexplored (Huang et al., 2007). Here we have clarified the mechanism by showing that BAPTA/AM treatment enhanced desipramineinduced activation of caspases and JNK phosphorylation. Thus it appears that [Ca2+ ]i rises may slow down or alleviate desipramineinduced cell death, suggesting a Ca2+ -dependent anti-apoptotic mechanism. Chin et al. have shown that thapsigargin-induced cell death in porcine aortic smooth muscle cells is also enhanced by BAPTA/AM (Chin et al., 2007). Ragel et al. demonstrated that Ca2+ channel antagonists augmented hydroxyurea- and ru486-induced apoptosis of meningioma in vivo and in vitro, indirectly suggesting that [Ca2+ ]i rises may prevent apoptosis (Ragel et al., 2006). Furthermore, Bickler and Fahlman reported that the inhaled anesthetic, isoflurane, enhanced Ca2+ -dependent survival signaling in cortical neurons and modulated MAPKs, apoptosis proteins and transcription factors during hypoxia (Bickler and Fahlman, 2006). Ca2+ plays a key role in both apoptotic and necrotic cell death. Emptying of intracellular Ca2+ stores and/or influx of extracellular Ca2+ can modulate cell death in many cell types (Saris and Carafoli,
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Fig. 5. Interaction between desipramine-induced cell death and [Ca2+ ]i rises. (A) Cells were pretreated with 20 M BAPTA/AM for 1 h before incubation with 100 M desipramine for 24 h. *P < 0.05 compared to control. #P < 0.05 compared to the third bar. (B) Effect of BAPTA on desipramine-induced phosphorylation of JNK. Cells were treated with 100 M desipramine for indicated time periods. Activated JNKs (phospho-JNK) were detected in immunoblots using antibodies specific for the phosphorylated form of JNKs. The same blot was stripped and used to determine the amount of kinases. Data are typical of five experiments. (C) The effect of BAPTA/AM on activation of JNK was quantified by densitometry. The figure normalized intensities of the bands of phospho JNKs against the bands of JNKs using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 compared to control; #P < 0.05 compared to treatment with desipramine alone. (D) Effect of BAPTA on activation of caspase-3. Protein extracts were prepared after treating with/without 20 M BAPTA/AM for 2 h and the cells were then treated with 100 M desipramine for 8 h. Immunoblotting assays were performed with antibodies against cleaved caspase-3 and  tubulin. Data are typical of five experiments. (E) The effect of BAPTA on activation of caspase-3 was quantified by densitometry. The figure normalized intensities of the bands of cleaved caspase-3 against the bands of  tubulin using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
2005; Waring, 2005). However, Ca2+ -independent apoptosis could be found in some cell types such as thymic lymphoma cells, neutrophil, and pancreatic beta cells (Matuszyk et al., 1998; Das et al., 1999; Barbosa et al., 2002). Recent evidence (Arimochi and Morita, 2008) suggests that desipramine induces apoptotic cell death through nonmitochondrial and mitochondrial pathways in different types of human colon carcinoma cells. In our study, no significant release of cytochromec or down-expression of Bcl-2 in the cytoplasmic fraction of desipramine-induced apoptosis was found. Thus it appears that desipramine-induced apoptosis in PC3 cells is via nonmitochondrial pathways. Contradictory reports have shown the anti-apoptotic effects of desipramine in other cell types (Li and Luo, 2002; Tang et al., 2005; Huang et al., 2007). Li and Luo (2002) reported that desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells. However, these results are observed at low concentrations of desipramine (<5 M). Similarly, although Tang et al. demonstrated that preincubation with 2 M desipramine protected YAC128 medium spiny neurons from glutamate-induced apoptosis, this treatment failed to rescue wild type medium spiny neurons from glutamate-induced apoptosis. Furthermore, in their model, desipramine was only tested at 2 M. In contrast, our data
show that at concentrations ≥10 M, desipramine causes toxicity in PC3 cells. Although Huang et al. (2007) have reported that desipramine inhibited lipopolysaccharide-induced inflammatory process in neural stem cells via the modulation of Bcl-2 expression, these results were observed under a low concentration condition (2 M desipramine). Notably, their study shows that the viability of neural stem cells was decreased by incubation with desipramine at concentrations ≥50 M, which was similar to our results. Together, we have demonstrated that in human PC3 cells, desipramine-induced cell death via a JNK-dependent, apoptotic pathway; and [Ca2+ ]i rises may slow down or alleviate this apoptotic pathway. Acknowledgement This work was supported by grants from Veterans General Hospital Kaohsiung (VGHKS97-071) to CR Jan. References Altiok, N., Koyuturk, M., Altiok, S., 2007. JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells. Breast Cancer Res. Treat. 105, 247–254.
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Desipramine-induced apoptosis in human PC3 prostate cancer cells: Activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2+ ]i elevation Hong-Chiang Chang a , Chorng-Chih Huang b , Chun-Jen Huang c,d , Jin-Shiung Cheng e , Shiuh-In Liu f , Jeng-Yu Tsai f , Hong-Tai Chang f , Jong-Khing Huang f , Chiang-Ting Chou g , Chung-Ren Jan g,∗ a
Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan Department of Nursery, Tzu Hui Institute of Technology, Pingtung 926, Taiwan Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan d Department of Psychiatry, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan e Department of Medicine, Yongkang Veterans Hospital, Tainan 710, Taiwan f Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan g Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan b c
a r t i c l e
i n f o
Article history: Received 11 March 2008 Received in revised form 15 May 2008 Accepted 15 May 2008 Available online 27 May 2008 Keywords: Apoptosis Caspase-3 Desipramine MAPKs PC3 cells Prostate
a b s t r a c t The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+ ]i ) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+ ]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+ ]i rises may slow down or alleviate desipramine-induced cytotoxicity. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In addition to its clinical effect in treating psychological disorders (Mayers and Baldwin, 2005), desipramine has been shown to exert different effects on many cell types in vitro, such as induction of Ca2+ influx and cytotoxicity in PC3 human prostate cancer cells (Huang et al., 2007), inhibition of activity-dependent sodium channel (Lenkey et al., 2006), and inhibition of Na+ /H+ exchanger in human submandibular cells (Choi et al., 2006). Desipramine is commonly used as an inhibitor for acid ceramidase (Elojeimy et al., 2006). Furthermore, desipramine was shown to cause apoptosis in human HT29 colon carcinoma cells (Arimochi and Morita, 2006), and to increase cytosolic free Ca2+ levels ([Ca2+ ]i ) and kill cells in renal tubular cells (Ho et al., 2005). Other actions of desipramine include decreasing tumor necrosis
∗ Corresponding author. Tel.: +886 7 3422121/1509; fax: +886 7 3468056. E-mail address: [email protected] (C.-R. Jan). 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.05.010
factor-alpha production (Reynolds et al., 2005), downregulating the cortical alpha1- and alpha2-adrenoceptors (Subhash et al., 2003), inhibiting cytochrome P450 enzymes (Shin et al., 2002) and neuronal/skeletal ATP-Ca2+ pump of endoplasmic reticulum (Couture et al., 2001; Soler et al., 2000), suppressing plasma membrane Ca2+ pump (Plenge-Tellechea et al., 1999), activating steroid transporters (Pariante et al., 2001) and GABAergic systems (Asahi and Yonehara, 2001), and altering protein kinase C activity (Morishita and Watanabe, 1997), etc. The term “apoptosis” was coined in 1972 by John Kerr who observed that certain dying cells shared several morphologic properties. All the criteria used to describe apoptotic cells were morphological and included condensation and margination of chromatin, cytoplasmic vacuolization, cellular shrinkage, increase in cellular density, nuclear fragmentation, and apoptotic body formation (Kerr et al., 1972). Cysteine aspartate specific proteases (caspases) families, which have highly specific proteases activity and can cleave proteins exclusively after aspartate residues, are well-known main actors of the apoptotic proteolytic cascades. Such
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as caspase-8 is an initiator or activator in caspase cascades; caspase3 is an effector in caspase cascades; and caspase-12 is a cytokine processor in caspase cascades (Coffey et al., 2001; Hengartner, 2001; Thornberry and Lazebnik, 1998). Caspase-3 is a key protease activated during the early stages of apoptosis (Nakagawa and Yuan, 2000). Alteration in [Ca2+ ]i is a crucial regulator of many cellular events (Berridge, 2006). To allow a precise regulation of [Ca2+ ]i and many signaling pathways by Ca2+ , cells have numerous mechanisms by which to modulate [Ca2+ ]i both globally and at the subcellular level (Berridge, 2005). Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved serine-threonine kinases that participate in cell differentiation, cell growth and survival (Sharma et al., 2005). Extracellular signal-regulated kinases (ERK), stress activated protein kinase c-Jun/N-terminal kinases (JNK) and p38 are three major components of MAPKs. Signal transduction involves sequential phosphorylation of a tripartite kinase module, culminating in an activated MAPK. Activated MAPK may stay in the cytoplasm to phosphorylate structural proteins or translocate to the nucleus, where it can activate transcription factors involved in DNA synthesis and cell division. MAPKs have been suggested to play a pivotal role in apoptosis (Cross et al., 2000; Reddy et al., 2003). Therefore, a [Ca2+ ]i rise and subsequent apoptosis is tightly related. However, not all types of apoptosis are caused by a Ca2+ signal. We have previously reported that in PC3 human prostate cancer cells desipramine-induced cell death that was worsened by chelating cytosolic Ca2+ with BAPTA/AM (Huang et al., 2007). The goal of this study was (1) to explore whether apoptosis accounted for desipramine-induced cell death, (2) to examine the involvement of ERK, JNK and p38 mitogen-activated protein kinases, and (3) to investigate the mechanism underlying the cytotoxic effect of BAPTA/AM. 2. Materials and methods 2.1. Materials The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). BAPTA/AM was from Molecular Probes (Eugene, OR, USA). Desipramine, propidium iodide (PI), dimethyl sulfoxide (DMSO) and other reagents were from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Cell culture PC3 cells were obtained from American Type Culture Collection and were cultured in Dulbeco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were kept at 37 ◦ C in 5% CO2 -containing humidified air.
2.3. Solutions Ca2+ -containing medium (pH 7.4) had (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; and glucose 5. Ca2+ -free medium contained similar components as Ca2+ containing medium except that CaCl2 was substituted with 0.3 mM EGTA. Agents were dissolved in water, ethanol or dimethyl sulfoxide (DMSO) as concentrated stocks.
2.5. Cell viability assays The measurement of viability was based on the ability of viable cells to cleave tetrazolium salts by dehydrogenases. Augmentation in the amount of developed color directly correlated with the number of metabolically active cells. Assays were performed according to manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a density of 50,000 cells/well in culture medium for 4 h to allow attachment. Then the culture medium was added with 10 l of serum-free medium containing different concentrations of treatments. The cell viability detecting reagent 4-[3-[4lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate (WST-1; 10 l pure solution) was added to each sample 24 h after various concentrations of desipramine, and cells were incubated for additional 2 h in a humidified atmosphere (37 ◦ C). In experiments using BAPTA/AM to chelate intracellular Ca2+ , cells were treated with 20 M BAPTA/AM for 1 h. The cells were washed once with Ca2+ -containing medium and incubated with or without 100 M desipramine for 24 h. The absorbance of samples (A450 ) was determined using a scanning multiwell spectrophotometer. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control value. Experiments were repeated five times in six replicates (wells).
2.6. Assessment of MAPKs and caspase-3 activation by immunoblotting Cell concentrations were adjusted to 3 × 106 cells/dish and were seeded to 6 cm culture dishes. After 2 h of incubation, the culture medium was replaced by serumfree medium supplemented with 1 mg/ml bovine serum albumin (USBTM , Cleveland, OH, USA) and serum starvation was continued for 4 h, followed by various treatments. The treatments were terminated after indicated time intervals by aspirating the supernatant and washing the dishes with a physiological saline. After washing, the cells were lysed on ice for 5 min with 70 l of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3 VO4 , 1 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). The lysed cells were scraped off the dish using a rubber policeman, transferred to microcentrifuge tubes, and vortexed for 10 s. The cell lysates were then centrifuged to remove insoluble materials and the protein concentration of each sample was measured. Approximately, 50 g of supernatant protein from each sample was used for gel electrophoresis analysis on a 10% SDSpolyacrylamide gel. After electrophoresis, the fractionated proteins on gel were transferred to PVDF membranes (NENTM Life Science Products Inc., Boston, MA, USA). For immuoblotting, the membranes were blocked with 5% non-fat milk in a buffer containing 25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20, and incubated overnight with the primary antibody (rabbit anti-human Bcl-2, rabbit anti-human phospho-ERK antibody, rabbit anti-human ERK antibody, rabbit antihuman phospho-JNK antibody, rabbit anti-human JNK antibody, rabbit anti-human phospho-p38 MAPK antibody, rabbit anti-human p38 MAPK antibody, rabbit-anti human cleaved caspase-3 or rabbit-anti human  tubulin; all from Cell Signaling Technology, Beverly, MA, USA). Then the membranes were extensively washed with the same buffer and incubated for 1 h with the secondary antibody (goat anti-rabbit antibody, Transduction Laboratories, Lexington, KY, USA). After extensive washing, the immune complexes were detected by chemiluminescence using the RenaissanceTM Western Blot Chemiluminescence Reagent Plus kit (NENTM Life Science Products Inc., Boston, MA, USA).
2.7. Measurements of subdiploidy nuclei by flow cytometry After treatments, cells were collected from the media, and were washed with ice-cold physiological saline twice and resuspended in 3 ml of 70% ethanol at −20 ◦ C. The cells were centrifuged for 5 min at 200 × g. Ethanol was decanted and the cell pellet was washed with ice-cold saline twice, and was suspended in 1 ml propidium iodide (PI) solution (1% Triton X-100, 20 g PI, 0.1 mg/ml RNase). The cell pellet was incubated in the dark for 30 min at room temperature. Cell fluorescence was measured in the FACScan flow cytometer (Becton Dickinson immunocytometry systems, San Jose, CA, USA) and the data were analyzed using the MODFIT software.
2.8. Detection of released cytochrome-c 2.4. [Ca2+ ]i measurements Trypsinized cells (106 ml−1 ) were loaded with 2 M fura-2/AM for 30 min at 25 C in culture medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 ◦ C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 (plus 5 mM CaCl2 ) and 10 mM EGTA sequentially at the end of each experiment. [Ca2+ ]i was calculated as previously described (Grynkiewicz et al., 1985). ◦
Preparation for cytosolic fraction has been previously described (Oyama et al., 2000). PC3 cells were plated onto a 10 cm dish (7.5 × 105 cells) 1 day before treatment. Various concentrations of desipramine were added to the medium for 24 h. The cells were washed twice with Ca2+ -containing medium, and suspended in icecold buffer (20 mM HEPES-KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitor), followed by homogenization with a dounce homogenizer (50 strokes) on ice. The supernatant was recovered by centrifugation (14,000 × g, 15 min, 4 ◦ C), and subjected to Western blot analysis using anti-cytochrome-c antibody (Cell Signaling Technology, Beverly, MA, USA).
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Fig. 1. Desipramine-induced apoptosis. (A) After treatment with various concentrations of desipramine for 18 h, cells were examined for apoptosis by using flow cytometry. Data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control. (B) Caspase-3 cysteine proteases were activated in desipramine-induced apoptosis. Protein extracts were prepared 8 h after exposure to various concentrations of desipramine. Data are typical of five experiments.
2.9. Statistics Data are reported as mean ± S.E.M. of five experiments and were analyzed by two-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS® , SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s HSD (honestly significant difference) procedure. A P-value less than 0.05 was considered significant.
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Fig. 2. Effect of desipramine on the phosphorylation of ERK, JNK, and p38 MAPKs. Cells were treated with 100 M desipramine for indicated time periods. Activated ERK (phospho-ERK), JNK (phospho-JNK), and p38 MAPK (phospho-p38) were detected in immunoblots using antibodies specific for the phosphorylated form of each kinase. The same blot was stripped and used to determine the amount of each kinase. Data are typical of five experiments.
stand whether JNK was also involved in desipramine-induced apoptosis, 50 M SP600125 (based on the data from Fig. 3A) was administered 2 h prior to the addition of 100 M desipramine. Fig. 3B shows that pretreatment with SP600125 did not alter basal value but significantly rescued cells from desipramine-induced apoptosis by 12 ± 2%. 3.3. JNK regulated caspase-3 activation Based on the above results, it appeared that activation of JNK could regulate desipramine-induced apoptosis of PC3 cells, but
3. Results 3.1. Effect of desipramine on apoptosis of PC3 cells Previous data showed that desipramine-induced concentrationdependent inhibition of viability of PC3 cells at concentrations between 10 and 800 M (Huang et al., 2007). The possibility of involvement of apoptosis in the cell death was explored. As shown in Fig. 1A, apoptosis (measuring the increase in subdiploidy nuclei appeared in cells) occurred in cells treated with 10–800 M desipramine in a concentration-dependent manner. Caspase-3 is thought to function as an executioner of apoptosis and its activation could reflect the percentage of apoptotic cells. Caspase-3 activation was determined by immunoblotting, and it was found that the expression of active caspase-3 increased in the presence of 100 M desipramine at 360–720 min in a time-dependent manner (Fig. 1B). 3.2. Involvement of MAPKs in desipramine-induced apoptosis MAPKs are activated by phosphorylation of specific tyrosine and threonine residues and the relative levels of phosphorylated MAPKs in total MAPKs represent the degree of MAPK activation. Fig. 2A and C shows that the level of phosphorylated ERK (phospho-ERK) and p38 did not increase after addition of 100 M desipramine. Exposure to desipramine increased the intensity of phosphorylated JNK (phospho-JNK) between 1 and 120 min (Fig. 2B). To further examine whether JNK phosphorylation was indeed involved in desipramine-induced cell death, 10–50 M SP600125 (a specific JNK inhibitor) was administered 2 h prior to the addition of 100 M desipramine. WST-1 metabolism was measured 24 h after treatment. SP600125 (10–50 M) did not alter basal viability. While 10–20 M SP600125 did not inhibit desipramineinduced cell death, 50 M SP600125 significantly rescued cells from desipramine-induced cell death by 12 ± 1% (Fig. 3A). To under-
Fig. 3. Effect of SP600125 on desipramine-induced cell death. (A) Cells were pretreated with 10 M, 20 M, 50 M SP600125 and DMSO (vehicle control) for 2 h before 24 h-treatment with 100 M desipramine. Cell viability was determined by WST-1 assays. (B) Cells were pretreated with 50 M SP600125 and DMSO (vehicle control) for 2 h and then were treated with or without 100 M desipramine for 24 h. Apoptosis was assessed by flow cytometry. Data are mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
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Fig. 4. Effect of SP600125 on activation of caspase-3. (A) Protein extracts were prepared after treating with/without 50 M SP600125 for 2 h and the cells were then treated with 100 M desipramine for 8 h. Immunoblotting assays were performed with antibodies against cleaved caspase-3 and  tubulin. Data are typical of five experiments. (B) The effect of SP600125 on activation of caspase-3 was quantified by densitometry. The figure normalized intensities of the bands of cleaved caspase3 against the bands of  tubulin using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
the relationship between JNK phosphorylation and caspase-3 activation was unclear. To understand whether phosphorylation of JNK could regulate caspase-3 activation, SP600125 (50 M) was added for 2 h followed by addition of desipramine for 8 h. It was found that pretreatment with SP600125 partly reduced the activation of caspase-3 (by 18 ± 1 folds compared with treatment with desipramine alone) (Fig. 4A and B). 3.4. Enhancing effect of chelating Ca2+ with BAPTA on desipramine-induced cell death Previous evidence (Huang et al., 2007) shows that in the presence of 100 M desipramine, cell viability was reduced to 57 ± 3%. In the presence of BAPTA, the desipramine-induced decrease in cell viability was further decreased to 47 ± 3%. Thus experiments were designed to examine whether the BAPTA-induced further decrease of cell death was also mediated by JNK phosphorylation. Fig. 5A shows that pretreatment with 20 M BAPTA did not significantly alter basal JNK phosphorylation, but significantly increased desipramine-induced JNK phosphorylation by 2.8 ± 2 folds. Consistently, Fig. 5B shows that BAPTA/AM pretreatment did not change basal caspase activation, but significantly increased desipramineinduced caspase activation by 55 ± 2 folds. 3.5. Lack of involvement of mitochondrial pathway of apoptosis in desipramine-induced cell death The cytochrome-c release and expression of Bcl-2 in desipramine-induced apoptosis of human PC3 prostate cancer cells were explored by Western blotting. No significant release of cytochrome-c or down-expression of Bcl-2 in the cytoplasmic fraction of desipramine-induced apoptosis was found (not shown). 4. Discussion The major novel finding of the present study was that desipramine caused death of human PC3 prostate cancer cells
via evoking apoptosis with the participation of JNK pathway, and [Ca2+ ]i rises played a preventive role in the process. We studied the effect of desipramine on cytotoxic markers such as cell viability, subdiploidy nuclei and activation of caspase-3 (Hengartner, 2001), and found that desipramine-induced cell death was accompanied by DNA fragmentation as detected by propidium iodide staining, and the activation of caspase-3. The previous reported concentration range (10–800 M) of desipramine for inducing cell death (Huang et al., 2007) was consistent with the range that evoked apoptosis in the present study. Thus it appears that desipramineinduced cell death via evoking apoptosis in PC3 cells. In other studies, desipramine was shown to induce apoptosis in different cell lines (Arimochi and Morita, 2006; Ho et al., 2005; Qi et al., 2002). To further investigate the mechanisms and the signaling pathways in desipramine-induced apoptosis of PC3 cells, immunoblotting was used to explore the alternation of phosphorylation of three members of the MAPK family (ERK, JNK, p38), because recent research suggests that phosphorylation of MAPKs plays an important role in the processes of apoptosis (Cross et al., 2000; Leger et al., 2006; Nusuetrong et al., 2005). MAPKs are activated by many stimuli and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses (Bost et al., 2005). Desipramine was shown to activate the phosphorylation of JNK, but not ERK and p38, and the JNK specific inhibitor SP600125 inhibited desipramine-induced cell death and apoptosis by the same percentage. Therefore, it appears that desipramineinduced apoptosis via JNK/caspase-3-dependent signaling pathway in PC3 cells. JNK has been shown to be involved in apoptosis in many cell types stimulated with different ligands. For instance, Yang et al. showed that JNK mediated proliferation and tumor growth of human prostate carcinoma (Yang et al., 2003). Wanpen et al. showed that salsolinol, an endogenous neurotoxin, induced apoptosis via activating JNK and NF-kappaB signaling pathways in human neuroblastoma cells (Wanpen et al., 2007). CerezoGuisado et al. demonstrated that JNK signaling pathway mediated lovastatin-induced rat brain neuroblast apoptosis (Cerezo-Guisado et al., 2007). Eupalmerin acetate, a novel anticancer agent from Caribbean gorgonian octocorals, induced apoptosis in malignant glioma cells via the JNK pathway (Iwamaru et al., 2007). Altiok et al. reported that JNK pathway regulated estradiol-induced apoptosis in hormone-dependent human breast cancer cells (Altiok et al., 2007). Our previous report showed that desipramine-induced cell death that was worsened by chelating cytosolic Ca2+ with the Ca2+ chelator BAPTA/AM, however the mechanism was unexplored (Huang et al., 2007). Here we have clarified the mechanism by showing that BAPTA/AM treatment enhanced desipramineinduced activation of caspases and JNK phosphorylation. Thus it appears that [Ca2+ ]i rises may slow down or alleviate desipramineinduced cell death, suggesting a Ca2+ -dependent anti-apoptotic mechanism. Chin et al. have shown that thapsigargin-induced cell death in porcine aortic smooth muscle cells is also enhanced by BAPTA/AM (Chin et al., 2007). Ragel et al. demonstrated that Ca2+ channel antagonists augmented hydroxyurea- and ru486-induced apoptosis of meningioma in vivo and in vitro, indirectly suggesting that [Ca2+ ]i rises may prevent apoptosis (Ragel et al., 2006). Furthermore, Bickler and Fahlman reported that the inhaled anesthetic, isoflurane, enhanced Ca2+ -dependent survival signaling in cortical neurons and modulated MAPKs, apoptosis proteins and transcription factors during hypoxia (Bickler and Fahlman, 2006). Ca2+ plays a key role in both apoptotic and necrotic cell death. Emptying of intracellular Ca2+ stores and/or influx of extracellular Ca2+ can modulate cell death in many cell types (Saris and Carafoli,
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Fig. 5. Interaction between desipramine-induced cell death and [Ca2+ ]i rises. (A) Cells were pretreated with 20 M BAPTA/AM for 1 h before incubation with 100 M desipramine for 24 h. *P < 0.05 compared to control. #P < 0.05 compared to the third bar. (B) Effect of BAPTA on desipramine-induced phosphorylation of JNK. Cells were treated with 100 M desipramine for indicated time periods. Activated JNKs (phospho-JNK) were detected in immunoblots using antibodies specific for the phosphorylated form of JNKs. The same blot was stripped and used to determine the amount of kinases. Data are typical of five experiments. (C) The effect of BAPTA/AM on activation of JNK was quantified by densitometry. The figure normalized intensities of the bands of phospho JNKs against the bands of JNKs using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 compared to control; #P < 0.05 compared to treatment with desipramine alone. (D) Effect of BAPTA on activation of caspase-3. Protein extracts were prepared after treating with/without 20 M BAPTA/AM for 2 h and the cells were then treated with 100 M desipramine for 8 h. Immunoblotting assays were performed with antibodies against cleaved caspase-3 and  tubulin. Data are typical of five experiments. (E) The effect of BAPTA on activation of caspase-3 was quantified by densitometry. The figure normalized intensities of the bands of cleaved caspase-3 against the bands of  tubulin using NIH image 1.61. The data are presented as a mean ± S.E.M. of five experiments. *P < 0.05 as compared with control; #P < 0.05 as compared with treatment with desipramine alone.
2005; Waring, 2005). However, Ca2+ -independent apoptosis could be found in some cell types such as thymic lymphoma cells, neutrophil, and pancreatic beta cells (Matuszyk et al., 1998; Das et al., 1999; Barbosa et al., 2002). Recent evidence (Arimochi and Morita, 2008) suggests that desipramine induces apoptotic cell death through nonmitochondrial and mitochondrial pathways in different types of human colon carcinoma cells. In our study, no significant release of cytochromec or down-expression of Bcl-2 in the cytoplasmic fraction of desipramine-induced apoptosis was found. Thus it appears that desipramine-induced apoptosis in PC3 cells is via nonmitochondrial pathways. Contradictory reports have shown the anti-apoptotic effects of desipramine in other cell types (Li and Luo, 2002; Tang et al., 2005; Huang et al., 2007). Li and Luo (2002) reported that desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells. However, these results are observed at low concentrations of desipramine (<5 M). Similarly, although Tang et al. demonstrated that preincubation with 2 M desipramine protected YAC128 medium spiny neurons from glutamate-induced apoptosis, this treatment failed to rescue wild type medium spiny neurons from glutamate-induced apoptosis. Furthermore, in their model, desipramine was only tested at 2 M. In contrast, our data
show that at concentrations ≥10 M, desipramine causes toxicity in PC3 cells. Although Huang et al. (2007) have reported that desipramine inhibited lipopolysaccharide-induced inflammatory process in neural stem cells via the modulation of Bcl-2 expression, these results were observed under a low concentration condition (2 M desipramine). Notably, their study shows that the viability of neural stem cells was decreased by incubation with desipramine at concentrations ≥50 M, which was similar to our results. Together, we have demonstrated that in human PC3 cells, desipramine-induced cell death via a JNK-dependent, apoptotic pathway; and [Ca2+ ]i rises may slow down or alleviate this apoptotic pathway. Acknowledgement This work was supported by grants from Veterans General Hospital Kaohsiung (VGHKS97-071) to CR Jan. References Altiok, N., Koyuturk, M., Altiok, S., 2007. JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells. Breast Cancer Res. Treat. 105, 247–254.
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