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Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon carcinoma cells
European Journal of Pharmacology 541 (2006) 17 – 23 www.elsevier.com/locate/ejphar
Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon carcinoma cells Hideki Arimochi a , Kyoji Morita b,⁎ a
Department of Molecular Bacteriology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan b Department of Pharmacology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan Received 12 September 2005; received in revised form 20 April 2006; accepted 28 April 2006 Available online 20 May 2006
Abstract Preclinical studies have suggested that the long-term use of antidepressants may result in the initiation and/or promotion of tumor in the gastrointestinal tract. However, a possible relationship between the use of antidepressants and the production of colon cancer has not yet been confirmed, and hence requires to be further investigated. To address this issue, the effects of antidepressants on the proliferation of colorectal tumor cells were examined using human HT29 colon carcinoma cells, and tricyclic antidepressant, such as imipramine, desipramine and amitriptyline, were shown to reduce the cell viability in a manner dependent on the time exposing to these drugs. In addition to these drugs, a selective serotonin reuptake inhibitor fluoxetine, but not a monoamine oxidase inhibitor tranylcypromine, caused the reduction of cell viability, similar in extent to that caused by imipramine. Further studies showed that desipramine caused the apoptotic cell death, which could be prevented by neither catalase, reduced-form glutathione (GSH), nor N-acetylcysteine (NAC), without accompanying the disruption of mitochondrial membrane potential within the cells and the release of cytochrome c into the cell cytoplasm. Moreover, desipramine caused the arrest of cell-cycle progression at either G0/G1-phase or G2/M-phase, which might be depending upon the drug concentration. Thus, these results suggest that tricyclic antidepressants may be cytotoxic, and induce the non-oxidative apoptotic death of human HT29 colon carcinoma cells probably through a non-mitochondrial pathway associated with the cell-cycle progression. © 2006 Elsevier B.V. All rights reserved. Keywords: Tricyclic antidepressant; HT29 colorectal tumor cell; Apoptotic cell death; Non-mitochondrial pathway; Cell-cycle arrest
1. Introduction Tricyclic antidepressants, such as imipramine, desipramine and amitriptyline, are commonly prescribed for depression, and selective serotonin reuptake inhibitors, fluoxetine and paroxetine, are recently introduced as effective drugs to the medication for depressive mood disorders. In such treatments, patients are usually having medication for long period, and hence there is great risk of these drugs inducing unexpected adverse effects during their long-term use. In particular, the question of whether antidepressants can initiate and/or promote tumor growth in patients and experimental animals has been raised, and the clinical and basic studies on a possible connection between antidepressant use and cancer risk have ⁎ Corresponding author. Tel.: +81 886 33 7061; fax: +81 886 33 7062. E-mail address: [email protected] (K. Morita). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.04.053
been carried out. Consequently, the case-control studies have suggested that the increased risk of breast cancer may be associated with the use of antidepressants, particularly genotoxic tricyclic antidepressants (Cotterchio et al., 2000; Sharpe et al., 2002). On the contrary, several studies have provided evidence against the association of antidepressant use with cancer risk (Coogan et al., 2000; Dalton et al., 2000; Dublin et al., 2002; Wang et al., 2001; Weiss et al., 1998). On the other hand, preclinical studies have shown that antidepressants have either no effect, stimulatory effect, or inhibitory effect on the growth of tumors implanted in rodents (Abdul et al., 1995; Bendele et al., 1992; Brandes et al., 1992; Freire-Garabal et al., 1998; Lin et al., 1999). Thus, these previous studies have only provided inconsistent or contradictory evidence, and hence the question regarding the association between antidepressant use and cancer risk still remains to be elucidated. More recently, the analyses of scientific reports in oncology and epidemiology
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have also shown that the link is questionable but deserving of further investigation on their genotoxic actions (see review; Sternbach, 2003). Cells have been isolated from different-types of tumors originated from various tissues and organs of different species, and widely used as an in vitro model system for the assessment of tumor growth, and hence the cytotoxic actions of various chemicals and natural substances on tumor cells in culture have been studied as a primary screening for their antitumor activities. As such studies, the direct actions of antidepressants on the growth of various tumor cells have recently been examined, and antidepressants have been shown to cause the apoptotic death of malignant tumor cells (Gavrilova-Ruch et al., 2002; Koch et al., 2003; Serafeim et al., 2003; Slamon and Pentreath, 1998, 2000; Slamon et al., 2001; Xia et al., 1999, 1998). In contrast, these drugs have been reported to protect tumor cells against the apoptotic death, and also shown to cause either the stimulatory effect or no notable effect on the proliferation of tumor cells in vitro (Li et al., 2003; Li and Luo, 2002; Volpe et al., 2003). These studies have been carefully carried out, and hence it seems possible that their inconsistent results may be mainly attributed to the difference in tumor cell lines used. Then, we anticipate that it is meaningful as a preclinical study to investigate a potential effects of antidepressants on particular tumor using an appropriate cell line. Since antidepressants are orally administered to depressive patients, it seems conceivable that these drugs may directly contact with epithelial cells or tumor cells in the gastrointestinal tract for long period, thus resulting in the initiation and/or promotion of tumors with a considerably large probability. Previously, tricyclic antidepressants have been shown to cause the stimulation of cell proliferation in rat intestinal crypt epithelium and the promotion of experimental carcinogenesis in rat colon (Iishi et al., 1993; Tutton and Barkla, 1989), and these studies are thought to suggest that the long-term use of antidepressants may be associated somewhat with the initiation of tumors in the gastrointestinal tract. However, the relationship between the antidepressant use and the risk of colorectal tumors has not yet been fully elucidated because of the inconsistent results of clinical and preclinical studies. Even the direct actions of these drugs on tumor cell growth have been still uncertain. Therefore, it seemed necessary to further investigate the effects of antidepressants on tumor cells, particularly the direct actions of these drugs on the growth of colon cancer cells in culture. Then, to obtain a clue to understanding somewhat the contradiction and inconsistency of the experimental results reported previously, we carried out the in vitro model experiments using human HT29 colon carcinoma cells. 2. Materials and methods 2.1. Materials Human HT29 colon adenocarcinoma cells (HTB-38) were obtained from the American Type Culture Collection (Rockville, MD, USA). Antidepressants, neutral red solution,
reduced-form glutathione (GSH), N-acetylcysteine (NAC), catalase, rhodamine-123, digitonin, and carbonyl cyanide 3chlorophenylhydrazone (CCCP) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). TACS Annexin V-FITC Apoptosis Detection Kit was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Monoclonal mouse anti-human cytochrome c antibody was from Genzyme-Techne (Minneapolis, MN, USA). ECL Western-blotting kit and Hyperfilm ECL were from Amersham Biosciences Corp. (Piscataway, NJ, USA). Other chemicals were commercially available reagent grade or ultrapure grade. 2.2. Cell culture HT29 cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin, 50 μg/ml of streptomycin and 50 μg/ml of gentamycin sulfate at 37 °C in a humidified incubator containing 95% air − 5% CO2 atmosphere. The cells were harvested from subconfluent cultures by trypsinization, and seeded onto a 24-well cluster plate or a 35-mm and a 60-mm culture dish at an appropriate density for each experiment, and maintained for at least 24 h to allow the cells to attach to the bottom of a plastic plate and dish. 2.3. Cell viability Cells were plated on a 24-well cluster plate at a density of 2 × 104 cells/well, and exposed to the drugs for 24 h. The cell viability was determined by measuring the amount of neutral red taken up into the cells as described previously (Fautz et al., 1991; Morita et al., 1999; Morita and Wong, 2000). Briefly, the cells were washed with saline solution, and incubated in 0.5 ml of McCoy's 5A medium containing neutral red (50 μg/ml) for 2 h in a humidified incubator. Then, the cells were rinsed with saline solution, and extracted with 1 ml of acidified ethanol solution (50% ethanol − 1% acetic acid) for 20 min at room temperature with constant gentle shaking. The amount of neutral red in the extracts was spectrophotometrically determined by measuring the optical density at 540 nm. 2.4. Characterization of apoptotic cell death For a fluorescence cytochemical study, the cells were plated on a poly-D-lysine-coated 35-mm culture dish at a density of 1 × 105 cells/dish, and exposed to the drugs for 24 h. The cells were rinsed with PBS, and fixed with methanol–acetic acid solution (3:1) for 1 h at room temperature. The fixed cells were stained with acridine orange (50 μg/ml) and propidium iodide (5 μg/ml) in PBS for 1 h at room temperature, and examined by an inverted fluorescence microscope. For a DNA fragmentation analysis, the cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 24 h. The cells attached at the bottom of the dish were scraped off, and collected together with unattached cells by centrifuging at 1500×g for 5 min at 4 °C. The DNA was prepared from the pelleted cells, and applied to a 2% agarose gel containing 0.5 μg/ml of ethidium bromide for an
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electrophoretic analysis as described previously (Morita et al., 1999; Morita and Wong, 2000). For a flow cytometric analysis, the cells were plated on a 60mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h. The cells were harvested by trypsinization, and rinsed with phosphate-buffered saline (PBS). The cells were stained with FITC-labeled annexin V and propidium iodide (PI) using TACS Annexin V-FITC Apoptosis Detection Kit according to the manufacture's instruction, and a flow cytometric analysis was then carried out using a flow cytometer EPICS XL-MCL System II (Beckman Coulter, Inc., Fullerton, CA, USA).
with PBS, and then fixed with 70% ethanol for overnight. The fixed cells were incubated with propidium iodide (10 μg/ml) and RNase A (10 μg/ml) in PBS at room temperature for 30 min in the dark, and the DNA contents of these cells were analyzed using a flow cytometry.
2.5. Measurement of mitochondrial membrane potential
3. Results
Cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h, and then harvested by trypsinization. The collected cells were rinsed with PBS, and incubated with 1 μM rhodamine-123 in PBS at 37 °C for 60 min in the dark. The cells stained with rhodamine-123 were then analyzed as described previously (Koubi et al., 2005) using a flow cytometer EPICS XL-MCL System II (Beckman Coulter, Inc., Fullerton, CA, USA).
3.1. Cytotoxic actions of antidepressants on HT29 cells
2.6. Protein extraction and immunoblotting analysis Cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h. The cells were rinsed with PBS, and permeabilized with 20 μM digitonin in 500 μl of permeabilizing buffer [HEPES-KOH (pH 7.4), 210 mM manitol, 70 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml of aprotinin and 1 μg/ml of leupeptin] as reported previously (Agarwal et al., 2003). The solution covering the cells was collected, and centrifuged at 18,000×g for 20 min to obtain the soluble fraction. The cells were lysed with 500 μl of lysis buffer [20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml of aprotinin and 1 μg/ ml of leupeptin] followed by subjecting them to a freeze–thaw cycle, and the cell extracts were obtained for an immunoblotting analysis of cytochrome c. The soluble fraction (cytoplasmic fraction) and the cell extracts (mitochondrial fraction) were analyzed by a sodium dodecylsulfate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, and the immunoblotting was carried out using a monoclonal mouse anti-human cytochrome c antibody and the ECL Westernblotting starter kit following the manufacturer's instructions. The amounts of total protein in the cell extracts were determined by a dye-binding method (Bradford, 1976) using bovine immunoglobulin G as a standard. 2.7. Flow cytometric analysis of cell-cycle Cells were plated on a 60-mm culture dish at a density of 1 × 106 cells/dish, and maintained for 24 h. After exposing to the drug for 24 h, the cells were harvested by trypsinization, rinsed
2.8. Data analysis Results were presented as the mean ± S.E.M., and the data were analyzed using an analysis of variance (ANOVA) followed by Tukey's post hoc test. A P value of <0.05 was accepted as a statistically significant difference.
To examine the direct effects of antidepressants on malignant colorectal tumor cells, human HT29 colon carcinoma cells were exposed to these drugs at the concentrations of 50 μM for 24 h, and the cell viability was determined by measuring the uptake of neutral red into these cells. As shown in Fig. 1, the viability of HT29 cells was reduced by exposing to tricyclic antidepressants, such as imipramine, desipramine and amitriptyline. In addition to these tricyclic antidepressants, a selective serotonin reuptake inhibitor fluoxetine also caused the reduction of cell viability, similar in extent to that caused by imipramine. Tranylcypromine, a monoamine oxidase inhibitor, failed to cause any significant change in the cell viability under the same conditions. The concentrations of these drugs required for the 50% reduction of cell viability were approximately 53, 31, 38, and 48 μM for imipramine, desipramine, amitriptyline, and fluoxetine, respectively. Furthermore, the cells were exposed to
Fig. 1. Effects of different-type antidepressants on proliferation of human colon carcinoma cells. HT29 cells were exposed to 50 μM of either imipramine (IMP), desipramine (DMI), amitriptyline (AMT), fluoxetine (FLX), or tranylcypromine (TCP) for 24 h, and their viability was determined as described in the text. Results were expressed as the percentage of control. Values are the mean ± S.E.M. (⁎P < 0.05, n = 6).
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the drugs for up to 4 days, and the reduction of cell viability was observed in a manner dependent on the time of drug exposure (data not shown). These results showed that tricyclic antidepressants and fluoxetine caused the cytotoxic actions on human colon cancer cells. Desipramine was shown to be the most potent among the drugs tested here, and therefore used for further investigation. 3.2. Tricyclic antidepressants cause apoptotic damage to HT29 cells To characterize the cytotoxic actions of tricyclic antidepressants, HT29 cells were exposed to desipramine, and the fluorescence cytochemical study was carried out using a double-staining with acridine orange and propidium iodide. As shown in Fig. 2, the chromatin condensation was observed in the nuclei of the cells treated with 50 μM desipramine for 24 h (panel A). The DNA fragmentation was also caused by exposing the cells to various concentrations of desipramine for 24 h (Fig. 2, panel B). Furthermore, the flow cytometric analysis indicated that the number of the cells stained with FITC-labeled annexin V (apoptotic cells) increased from 24% to 57% of total cells after exposing to 100 μM desipramine for 12 h (data not shown). Thus, it seemed obvious that the direct challenge of tricyclic antidepressants to HT29 cells caused the apoptotic damage to the cells, resulting in the reduction of their viability under the conditions used here. 3.3. Characterization of tricyclic antidepressant-induced apoptosis and cell-cycle arrest To further investigate the property of apoptotic cell death induced by tricyclic antidepressants, the effects of antioxidants
Fig. 2. Characterization of cytotoxic action of desipramine on human colon carcinoma cells. The fluorescence cytochemistry was carried out after exposing HT29 cells to 50 μM drug for 24 h [A], and the DNA fragmentation was determined after exposing to various concentrations of the drug for 24 h [B] as described in the text.
Fig. 3. Effect of desipramine on mitochondrial function in human colon carcinoma cells. HT29 cells were exposed to desipramine (DMI) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 12 h, and the mitochondrial function was assessed by analyzing the mitochondrial membrane potential [A] and the leakage of cytochrome c [B] as described in the text.
on the cytotoxic action of desipramine was examined. The reduction of cell viability induced by desipramine was not significantly affected by adding 1 mg/ml of catalase, 1 mM GSH, or 1 mM NAC to the culture medium (data not shown). Therefore, it seemed unlikely that tricyclic antidepressants might cause the oxidative damage to the cells under these conditions. Moreover, the influence of desipramine treatment on the mitochondrial function was investigated by determining the mitochondrial membrane potential. A considerable decrease in rhodamine-123 fluorescence was observed in the cells exposed to CCCP, but not in the cells exposed to 100 μM desipramine for 12 h (Fig. 3A). Furthermore, the immunoblotting analysis indicated that, although the amount of cytochrome c in the cytoplasmic fraction was markedly increased by exposing the cells to 100 μM CCCP for 12 h, the exposure to desipramine failed to significantly increase the amount of cytochrome c in the cytoplasm under the same conditions (Fig. 3B). Thus, it seemed unlikely that desipramine might cause the apoptotic death of HT29 cells as a result of disturbing the mitochondrial function. To further characterize the effects of tricyclic antidepressants on the cell-cycle progression, HT29 cells were exposed to different concentrations of desipramine, and then subjected to a flow cytometric analysis of the cell-cycle distribution. As shown in Fig. 4, the low concentration of desipramine (25 μM) increased the number of cells at G0/G1-phase with a concomitant decrease in the cells at S-phase, while the high concentration of the drug (75 μM) significantly decreased the cells at G0/G1-phase with a marked increase in the cells at G2/ M-phase, and the moderate concentration of desipramine (50 μM) increased the cell numbers at G0/G1- and G2/Mphase with a significant decrease in the cells at S-phase. Thus, it
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Fig. 4. Effect of desipramine on cell-cycle distribution in human colon carcinoma cells. HT29 cells were exposed to various concentrations of desipramine (DMI) for 24 h, and the cell-cycle distribution was analyzed as described in the text. Results were expressed as the percentage of total cell number. Values are the mean ± S.E.M. (⁎P < 0.05, n = 6).
seemed conceivable that exposure of HT29 cells to desipramine caused the apoptotic cell death, which might be largely attributed to the arrest of cell-cycle progression. 4. Discussion Direct actions of antidepressants on human HT29 colon carcinoma cells were examined as an in vitro model experiment for investigating their possible influence on the growth of malignant colorectal tumors. Tricyclic antidepressants, such as imipramine, desipramine and amitriptyline, were shown to cause their cytotoxic actions on these cells in culture (Fig. 1). In addition to these tricyclic antidepressants, fluoxetine, one of the selective serotonin reuptake inhibitors, was shown to cause the cytotoxic action on HT29 cells, almost similar in extent to the action of imipramine, and hence the cytotoxic actions of tricyclic antidepressants observed here are considered to be not necessarily specific to their chemical structures. Previously, fluoxetine has been shown to increase the extracellular concentrations of norepinephrine and dopamine as well as serotonin in different parts of the brain (Jordan et al., 1994; Perry and Fuller, 1997; Pozzi et al., 1999). Recent study has furthermore shown that, among selective serotonin reuptake inhibitors, only fluoxetine can induce the elevation of extracellular norepinephrine and dopamine levels in prefrontal cortex, suggesting that fluoxetine is not completely selective to the serotonin reuptake system (Bymaster et al., 2002), and interacts with the norepinephrine reuptake system in the brain. Therefore, it seems likely that fluoxetine causes the toxic damage to HT29 cells probably through interacting with the common site associated with the cytotoxic actions of tricyclic antidepressants. Cytotoxic actions of tricyclic antidepressants on HT29 cells were furthermore characterized by a fluorescence cytochemical study and an electrophoretic DNA analysis, and desipramine
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was shown to induce the chromatin condensation and the DNA fragmentation (Fig. 2). The apoptosis of HT29 cells induced by desipramine was confirmed by a flow cytometric analysis. Therefore, the direct challenge of desipramine to HT29 cells is considered to cause the apoptotic damage to these cells under the experimental conditions used here. Moreover, the effect of desipramine on HT29 cells was assessed again in the medium containing either catalase, GSH, or NAC, and none of them was shown to effectively prevent the cytotoxic action of desipramine on the cells. Thus, it seems reasonable to conclude that desipramine induces the apoptotic death of HT29 cells through its non-oxidative action on the cells. Desipramine was shown to cause neither the disruption of mitochondrial membrane potential within the cells nor the release of cytochrome c into the cytoplasmic space of the cells (Fig. 3). In previous study, the deprivation of glial cell linederived neurotrophic factor, a neurotrophic factor derived from glial cells, has been shown to cause the apoptotic damage to sympathetic neurons in culture without inducing the release of cytochrome c and the activation of caspase-9 and -3, thus suggesting that the deprivation of this neurotrophic factor may cause neuronal cell death probably through a novel nonmitochondrial pathway (Yu et al., 2003). Recent study has also shown that the inhibition of choline kinase in human tumor cells may cause the promotion of apoptosis by activating caspase-3 prior to the release of cytochrome c and the loss of mitochondrial potential, suggesting that the activation of caspase-3 may be independent of the mitochondrial dysfunction, and hence, even though both cytochrome c release and membrane potential disruption occur, it seems possible that the apoptotic cell death induced by choline kinase inhibition is not necessarily mediated by a mitochondrial pathway (RodriguezGonzalez et al., 2005). Based on these findings, it seems possible that desipramine may cause the non-oxidative damage to HT29 cells, resulting in the apoptotic cell death probably through a non-mitochondrial pathway. Several antitumor agents have been shown to cause the apoptotic cell death by interfering with the progression of cellcycle in different types of tumor cells. For instance, the inhibitors of DNA topoisomerase I and II, such as camptothecin, CPT-11 and its metabolite SN-38, imidazoacridone C-1311, and indenoindole derivatives, have been shown to arrest the cell-cycle progression at G2/M-phase, promoting the apoptotic death of human leukemia cells, testicular, ovarian, and colorectal carcinoma cells (Bal et al., 2004; Hyzy et al., 2005; Magrini et al., 2002; McDonald and Brown, 1998; Ueno et al., 2002). Furthermore, silibinin, a bioactive flavonone and a major compound of milk thistle extract, has also been shown to arrest the cell-cycle progression at G2/M-phase by acting the cellcycle regulation system, promoting the apoptotic death of HT29 cells without caspase activation, PARP cleavage, and cytochrome c release in the cells (Agarwal et al., 2003). Based on these findings, the effect of desipramine on the cell-cycle distribution was examined, and the cell-cycle progression was shown to be arrested at G2/M-phase by exposing the cells to desipramine (Fig. 4). Therefore, it seems possible that desipramine may cause the arrest of cell-cycle progression at
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G2/M-phase, resulting in the apoptotic death of human HT29 colon carcinoma cells. Tricyclic antidepressants was shown to cause the nonoxidative cytotoxic actions on human HT29 colon carcinoma cells through a non-mitochondrial pathway associated with the cell-cycle progression, suggesting that these drugs are probably capable of causing their cytotoxic actions on malignant colorectal tumors, and may be applicable for the clinical treatment of gastrointestinal tumors in combination with other anticancer agents. However, the critical question of whether the concentrations of these drugs in tumor tissues can reach the effective cytotoxic levels still remains to be answered. Although the serum concentration of free desipramine has previously been shown to reach approximately 9.5 μM (Hursting et al., 1992), there has been no data available to indicate the drug concentrations in colon and rectum. However, antidepressants are considered to come into contact with the inner surface of gastrointestinal tract, and assumed to directly act on tumor cells inside colon and rectum during the long-term oral administration of these drugs. It therefore seems conceivable that the concentrations of these drugs within colon and rectum can possibly be high enough to cause their cytotoxic actions on colorectal tumor cells in vivo. In our preliminary studies on the direct action of imipramine on the different types of colon cancer cells, such as HT29 cells and HCT116 cells, we have found that these cells clearly show their different sensitivities to the drug. Therefore, it seemed important to choose an appropriate cell line as an in vitro experimental system. In the present study, the cells more sensitive to imipramine, which were HT29 cells, were considered to be better and suitable for the experiments. However, it seems necessary to compare the direct actions of antidepressants on different-type cells for further characterizing the cytotoxic actions of these drugs on colon cancer cells. In fact, we have continued to further characterize the cytotoxic action of desipramine on different colon cancer cells for elucidating a possible mechanism of their different sensitivities to the drug. In addition, it still remains to elucidate the detailed mechanism underlying the cytotoxic actions of tricyclic antidepressants, and hence studies on the effects of these drugs on the mechanism of the cell-cycle progression, particularly focusing on the cyclin/ cyclin-dependent kinase system, are in progress. References Abdul, M., Logothetis, C.J., Hoosein, N.M., 1995. Growth-inhibitory effects of serotonin uptake inhibitors on human prostate carcinoma cell lines. J. Urol. 154, 247–250. Agarwal, C., Singh, R.P., Dhanalakshmi, S., Tyagi, A.K., Tecklenburg, M., Sclafani, R.A., Agarwal, R., 2003. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 22, 8271–8282. Bal, C., Baldeyrou, B., Moz, F., Lansiaux, A., Colson, P., Kraus-Berthier, L., Leonce, S., Pierre, A., Boussard, M.F., Rousseau, A., Wierzbicki, M., Bailly, C., 2004. Novel antitumor indenoindole derivatives targeting DNA and topoisomerase II. Biochem. Pharmacol. 68, 1911–1922. Bendele, R.A., Adams, E.R., Hoffman, W.P., Gries, C.L., Morton, D.M., 1992. Carcinogenicity studies of fluoxetine hydrochloride in rats and mice. Cancer Res. 52, 6931–6935.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brandes, L.J., Arron, R.J., Bogdanovic, R.P., Tong, J., Zaborniak, C.L., Hogg, G.R., Warrington, R.C., Fang, W., LaBella, F.S., 1992. Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses. Cancer Res. 52, 3796–3800. Bymaster, F.P., Zhang, W., Carter, P.A., Shaw, J., Chernet, E., Phebus, L., Wong, D.T., Perry, K.W., 2002. Fluoxetine, but not other selective serotonin uptake inhibitors, increases norepinephrine and dopamine extracellular levels in prefrontal cortex. Psychopharmacology 160, 353–361. Coogan, P.F., Rosenberg, L., Palmer, J.R., Strom, B.L., Stolley, P.D., Zauber, A. G., Shapiro, S., 2000. Risk of ovarian cancer according to use of antidepressants, phenothiazines, and benzodiazepines (United States). Cancer Causes Control 11, 839–845. Cotterchio, M., Kreiger, N., Darlington, G., Steingart, A., 2000. Antidepressant medication use and breast cancer risk. Am. J. Epidemiol. 151, 951–957. Dalton, S.O., Johansen, C., Mellemkjaer, L., Sorensen, H.T., McLaughlin, J.K., Olsen, J., Olsen, J.H., 2000. Antidepressant medications and risk for cancer. Epidemiology 11, 171–176. Dublin, S., Rossing, M.A., Heckbert, S.R., Goff, B.A., Weiss, N.S., 2002. Risk of epithelial ovarian cancer in relation to use of antidepressants, benzodiazepines, and other centrally acting medications. Cancer Causes Control 13, 35–45. Fautz, R., Husein, B., Hechenberger, C., 1991. Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). Mutat. Res. 253, 173–179. Freire-Garabal, M., Nunez, M.J., Pereiro, D., Riveiro, P., Losada, C., FernandezRial, J.C., Garcia-Iglesias, E., Prizmic, J., Mayan, J.M., Rey-Mendez, M., 1998. Effects of fluoxetine on the development of lung metastases induced by operative stress in rats. Life Sci. 63, PL31–PL38. Gavrilova-Ruch, O., Schonherr, K., Gessner, G., Schonherr, R., Klapperstuck, T., Wohlrab, W., Heinemann, S.H., 2002. Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells. J. Membr. Biol. 188, 137–149. Hursting, M.J., Clark, G.D., Raisys, V.A., Miller, S.J., Opheim, K.E., 1992. Measurement of free desipramine in serum by ultrafiltration with immunoassay. Clin. Chem. 38, 2468–2471. Hyzy, M., Bozko, P., Konopa, J., Skladanowski, A., 2005. Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells. Biochem. Pharmacol. 69, 801–809. Iishi, H., Tatsuta, M., Baba, M., Taniguchi, H., 1993. Enhancement by the tricyclic antidepressant, desipramine, of experimental carcinogenesis in rat colon induced by azoxymethane. Carcinogenesis 14, 1837–1840. Jordan, S., Kramer, G.L., Zukas, P.K., Moeller, M., Petty, F., 1994. In vivo biogenic amine efflux in medial prefrontal cortex with imipramine, fluoxetine, and fluvoxamine. Synapse 18, 294–297. Koch, J.M., Kell, S., Aldenhoff, J.B., 2003. Differential effects of fluoxetine and imipramine on the phosphorylation of the transcription factor CREB and cell-viability. J. Psychiatr. Res. 37, 53–59. Koubi, D., Jiang, H., Zhang, L., Tang, W., Kuo, J., Rodriguez, A.I., Hunter, T.J., Seidman, M.D., Corcoran, G.B., Levine, R.A., 2005. Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation. Neurochem. Int. 46, 73–81. Li, Y.F., Luo, Z.P., 2002. Desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells. Acta Pharmacol. Sin. 23, 311–314. Li, Y.F., Liu, Y.Q., Huang, W.C., Luo, Z.P., 2003. Cytoprotective effect is one of common action pathways for antidepressants. Acta Pharmacol. Sin. 24, 996–1000. Lin, X., Levitsky, D.A., King, J.M., Campbell, T.C., 1999. The promotion effect of anorectic drugs on aflatoxin B(1)-induced hepatic preneoplastic foci. Carcinogenesis 20, 1793–1799. Magrini, R., Bhonde, M.R., Hanski, M.L., Notter, M., Scherubl, H., Boland, C.R., Zeitz, M., Hanski, C., 2002. Cellular effects of CPT-11 on colon carcinoma cells: dependence on p53 and hMLH1 status. Int. J. Cancer 101, 23–31. McDonald, A.C., Brown, R., 1998. Induction of p53-dependent and p53independent cellular responses by topoisomerase 1 inhibitors. Br. J. Cancer 78, 745–751.
H. Arimochi, K. Morita / European Journal of Pharmacology 541 (2006) 17–23 Morita, K., Wong, D.L., 2000. Glucocorticoids enhance serum deprivation- but not calcium-induced cytotoxicity in rat C6 glioma cells. Cell Biol. Int. 24, 223–228. Morita, K., Ishimura, K., Tsuruo, Y., Wong, D.L., 1999. Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors. Brain Res. 816, 309–316. Perry, K.W., Fuller, R.W., 1997. Fluoxetine increases norepinephrine release in rat hypothalamus as measured by tissue levels of MHPG-SO4 and microdialysis in conscious rats. J. Neural Transm. 104, 953–966. Pozzi, L., Invernizzi, R., Garavaglia, C., Samanin, R., 1999. Fluoxetine increases extracellular dopamine in the prefrontal cortex by a mechanism not dependent on serotonin: a comparison with citalopram. J. Neurochem. 73, 1051–1057. Rodriguez-Gonzalez, A., Ramirez De Molina, A., Banez-Coronel, M., Megias, D., Lacal, J.C., 2005. Inhibition of choline kinase renders a highly selective cytotoxic effect in tumor cells through a mitochondrial independent mechanism. Int. J. Oncol. 26, 999–1008. Serafeim, A., Holder, M.J., Grafton, G., Chamba, A., Drayson, M.T., Luong, Q.T., Bunce, C.M., Gregory, C.D., Barnes, N.M., Gordon, J., 2003. Selective serotonin reuptake inhibitors directly signal for apoptosis in biopsy-like Burkitt lymphoma cells. Blood 101, 3212–3219. Sharpe, C.R., Collet, J.P., Belzile, E., Hanley, J.A., Boivin, J.F., 2002. The effects of tricyclic antidepressants on breast cancer risk. Br. J. Cancer 86, 92–97. Slamon, N.D., Pentreath, V.W., 1998. A comparison of the acute and chronic effects of antidepressants in cultured C6 and 1321N1 cells. ATLA 26, 303–319. Slamon, N.D., Pentreath, V.W., 2000. Antioxidant defense against antidepressants in C6 and 1321N1 cells. Chem.-Biol. Interact. 127, 181–199.
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Slamon, N.D., Ward, T.H., Butler, J., Pentreath, V.W., 2001. Assessment of DNA damage in C6 glioma cells after antidepressant treatment using an alkaline comet assay. Arch. Toxicol. 75, 243–250. Sternbach, H., 2003. Are antidepressants carcinogenic? A review of preclinical and clinical studies. J. Clin. Psychiatry 64, 1153–1162. Tutton, P.J., Barkla, D.H., 1989. Effect of an inhibitor of noradrenaline uptake, desipramine, on cell proliferation in the intestinal crypt epithelium. Virchows Arch., B Cell Pathol. 57, 349–352. Ueno, M., Nonaka, S., Yamazaki, R., Deguchi, N., Murai, M., 2002. SN-38 induces cell cycle arrest and apoptosis in human testicular cancer. Eur. Urol. 42, 390–397. Volpe, D.A., Ellison, C.D., Parchment, R.E., Grieshaber, C.K., Faustino, P.J., 2003. Effects of amitriptyline and fluoxetine upon the in vitro proliferation of tumor cell lines. J. Exp. Ther. Oncol. 3, 169–184. Wang, P.S., Walker, A.M., Tsuang, M.T., Orav, E.J., Levin, R., Avorn, J., 2001. Antidepressant use and the risk of breast cancer: a non-association. J. Clin. Epidemiol. 54, 728–734. Weiss, S.R., McFarland, B.H., Burkhart, G.A., Ho, P.T., 1998. Cancer recurrences and secondary primary cancers after use of antihistamines or antidepressants. Clin. Pharmacol. Ther. 63, 594–599. Xia, Z., DePierre, J.W., Nassberger, L., 1998. Modulation of apoptosis induced by tricyclic antidepressants in human peripheral lymphocytes. J. Biochem. Mol. Toxicol. 12, 115–123. Xia, Z., Bergstrand, A., DePierre, J.W., Nassberger, L., 1999. 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. Yu, L.Y., Jokitalo, E., Sun, Y.F., Mehlen, P., Lindholm, D., Saarma, M., Arumae, U., 2003. GDNF-deprived sympathetic neurons die via a novel nonmitochondrial pathway. J. Cell Biol. 163, 987–997.
Characterization of cytotoxic actions of tricyclic antidepressants on human HT29 colon carcinoma cells Hideki Arimochi a , Kyoji Morita b,⁎ a
Department of Molecular Bacteriology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan b Department of Pharmacology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan Received 12 September 2005; received in revised form 20 April 2006; accepted 28 April 2006 Available online 20 May 2006
Abstract Preclinical studies have suggested that the long-term use of antidepressants may result in the initiation and/or promotion of tumor in the gastrointestinal tract. However, a possible relationship between the use of antidepressants and the production of colon cancer has not yet been confirmed, and hence requires to be further investigated. To address this issue, the effects of antidepressants on the proliferation of colorectal tumor cells were examined using human HT29 colon carcinoma cells, and tricyclic antidepressant, such as imipramine, desipramine and amitriptyline, were shown to reduce the cell viability in a manner dependent on the time exposing to these drugs. In addition to these drugs, a selective serotonin reuptake inhibitor fluoxetine, but not a monoamine oxidase inhibitor tranylcypromine, caused the reduction of cell viability, similar in extent to that caused by imipramine. Further studies showed that desipramine caused the apoptotic cell death, which could be prevented by neither catalase, reduced-form glutathione (GSH), nor N-acetylcysteine (NAC), without accompanying the disruption of mitochondrial membrane potential within the cells and the release of cytochrome c into the cell cytoplasm. Moreover, desipramine caused the arrest of cell-cycle progression at either G0/G1-phase or G2/M-phase, which might be depending upon the drug concentration. Thus, these results suggest that tricyclic antidepressants may be cytotoxic, and induce the non-oxidative apoptotic death of human HT29 colon carcinoma cells probably through a non-mitochondrial pathway associated with the cell-cycle progression. © 2006 Elsevier B.V. All rights reserved. Keywords: Tricyclic antidepressant; HT29 colorectal tumor cell; Apoptotic cell death; Non-mitochondrial pathway; Cell-cycle arrest
1. Introduction Tricyclic antidepressants, such as imipramine, desipramine and amitriptyline, are commonly prescribed for depression, and selective serotonin reuptake inhibitors, fluoxetine and paroxetine, are recently introduced as effective drugs to the medication for depressive mood disorders. In such treatments, patients are usually having medication for long period, and hence there is great risk of these drugs inducing unexpected adverse effects during their long-term use. In particular, the question of whether antidepressants can initiate and/or promote tumor growth in patients and experimental animals has been raised, and the clinical and basic studies on a possible connection between antidepressant use and cancer risk have ⁎ Corresponding author. Tel.: +81 886 33 7061; fax: +81 886 33 7062. E-mail address: [email protected] (K. Morita). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.04.053
been carried out. Consequently, the case-control studies have suggested that the increased risk of breast cancer may be associated with the use of antidepressants, particularly genotoxic tricyclic antidepressants (Cotterchio et al., 2000; Sharpe et al., 2002). On the contrary, several studies have provided evidence against the association of antidepressant use with cancer risk (Coogan et al., 2000; Dalton et al., 2000; Dublin et al., 2002; Wang et al., 2001; Weiss et al., 1998). On the other hand, preclinical studies have shown that antidepressants have either no effect, stimulatory effect, or inhibitory effect on the growth of tumors implanted in rodents (Abdul et al., 1995; Bendele et al., 1992; Brandes et al., 1992; Freire-Garabal et al., 1998; Lin et al., 1999). Thus, these previous studies have only provided inconsistent or contradictory evidence, and hence the question regarding the association between antidepressant use and cancer risk still remains to be elucidated. More recently, the analyses of scientific reports in oncology and epidemiology
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have also shown that the link is questionable but deserving of further investigation on their genotoxic actions (see review; Sternbach, 2003). Cells have been isolated from different-types of tumors originated from various tissues and organs of different species, and widely used as an in vitro model system for the assessment of tumor growth, and hence the cytotoxic actions of various chemicals and natural substances on tumor cells in culture have been studied as a primary screening for their antitumor activities. As such studies, the direct actions of antidepressants on the growth of various tumor cells have recently been examined, and antidepressants have been shown to cause the apoptotic death of malignant tumor cells (Gavrilova-Ruch et al., 2002; Koch et al., 2003; Serafeim et al., 2003; Slamon and Pentreath, 1998, 2000; Slamon et al., 2001; Xia et al., 1999, 1998). In contrast, these drugs have been reported to protect tumor cells against the apoptotic death, and also shown to cause either the stimulatory effect or no notable effect on the proliferation of tumor cells in vitro (Li et al., 2003; Li and Luo, 2002; Volpe et al., 2003). These studies have been carefully carried out, and hence it seems possible that their inconsistent results may be mainly attributed to the difference in tumor cell lines used. Then, we anticipate that it is meaningful as a preclinical study to investigate a potential effects of antidepressants on particular tumor using an appropriate cell line. Since antidepressants are orally administered to depressive patients, it seems conceivable that these drugs may directly contact with epithelial cells or tumor cells in the gastrointestinal tract for long period, thus resulting in the initiation and/or promotion of tumors with a considerably large probability. Previously, tricyclic antidepressants have been shown to cause the stimulation of cell proliferation in rat intestinal crypt epithelium and the promotion of experimental carcinogenesis in rat colon (Iishi et al., 1993; Tutton and Barkla, 1989), and these studies are thought to suggest that the long-term use of antidepressants may be associated somewhat with the initiation of tumors in the gastrointestinal tract. However, the relationship between the antidepressant use and the risk of colorectal tumors has not yet been fully elucidated because of the inconsistent results of clinical and preclinical studies. Even the direct actions of these drugs on tumor cell growth have been still uncertain. Therefore, it seemed necessary to further investigate the effects of antidepressants on tumor cells, particularly the direct actions of these drugs on the growth of colon cancer cells in culture. Then, to obtain a clue to understanding somewhat the contradiction and inconsistency of the experimental results reported previously, we carried out the in vitro model experiments using human HT29 colon carcinoma cells. 2. Materials and methods 2.1. Materials Human HT29 colon adenocarcinoma cells (HTB-38) were obtained from the American Type Culture Collection (Rockville, MD, USA). Antidepressants, neutral red solution,
reduced-form glutathione (GSH), N-acetylcysteine (NAC), catalase, rhodamine-123, digitonin, and carbonyl cyanide 3chlorophenylhydrazone (CCCP) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). TACS Annexin V-FITC Apoptosis Detection Kit was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Monoclonal mouse anti-human cytochrome c antibody was from Genzyme-Techne (Minneapolis, MN, USA). ECL Western-blotting kit and Hyperfilm ECL were from Amersham Biosciences Corp. (Piscataway, NJ, USA). Other chemicals were commercially available reagent grade or ultrapure grade. 2.2. Cell culture HT29 cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin, 50 μg/ml of streptomycin and 50 μg/ml of gentamycin sulfate at 37 °C in a humidified incubator containing 95% air − 5% CO2 atmosphere. The cells were harvested from subconfluent cultures by trypsinization, and seeded onto a 24-well cluster plate or a 35-mm and a 60-mm culture dish at an appropriate density for each experiment, and maintained for at least 24 h to allow the cells to attach to the bottom of a plastic plate and dish. 2.3. Cell viability Cells were plated on a 24-well cluster plate at a density of 2 × 104 cells/well, and exposed to the drugs for 24 h. The cell viability was determined by measuring the amount of neutral red taken up into the cells as described previously (Fautz et al., 1991; Morita et al., 1999; Morita and Wong, 2000). Briefly, the cells were washed with saline solution, and incubated in 0.5 ml of McCoy's 5A medium containing neutral red (50 μg/ml) for 2 h in a humidified incubator. Then, the cells were rinsed with saline solution, and extracted with 1 ml of acidified ethanol solution (50% ethanol − 1% acetic acid) for 20 min at room temperature with constant gentle shaking. The amount of neutral red in the extracts was spectrophotometrically determined by measuring the optical density at 540 nm. 2.4. Characterization of apoptotic cell death For a fluorescence cytochemical study, the cells were plated on a poly-D-lysine-coated 35-mm culture dish at a density of 1 × 105 cells/dish, and exposed to the drugs for 24 h. The cells were rinsed with PBS, and fixed with methanol–acetic acid solution (3:1) for 1 h at room temperature. The fixed cells were stained with acridine orange (50 μg/ml) and propidium iodide (5 μg/ml) in PBS for 1 h at room temperature, and examined by an inverted fluorescence microscope. For a DNA fragmentation analysis, the cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 24 h. The cells attached at the bottom of the dish were scraped off, and collected together with unattached cells by centrifuging at 1500×g for 5 min at 4 °C. The DNA was prepared from the pelleted cells, and applied to a 2% agarose gel containing 0.5 μg/ml of ethidium bromide for an
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electrophoretic analysis as described previously (Morita et al., 1999; Morita and Wong, 2000). For a flow cytometric analysis, the cells were plated on a 60mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h. The cells were harvested by trypsinization, and rinsed with phosphate-buffered saline (PBS). The cells were stained with FITC-labeled annexin V and propidium iodide (PI) using TACS Annexin V-FITC Apoptosis Detection Kit according to the manufacture's instruction, and a flow cytometric analysis was then carried out using a flow cytometer EPICS XL-MCL System II (Beckman Coulter, Inc., Fullerton, CA, USA).
with PBS, and then fixed with 70% ethanol for overnight. The fixed cells were incubated with propidium iodide (10 μg/ml) and RNase A (10 μg/ml) in PBS at room temperature for 30 min in the dark, and the DNA contents of these cells were analyzed using a flow cytometry.
2.5. Measurement of mitochondrial membrane potential
3. Results
Cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h, and then harvested by trypsinization. The collected cells were rinsed with PBS, and incubated with 1 μM rhodamine-123 in PBS at 37 °C for 60 min in the dark. The cells stained with rhodamine-123 were then analyzed as described previously (Koubi et al., 2005) using a flow cytometer EPICS XL-MCL System II (Beckman Coulter, Inc., Fullerton, CA, USA).
3.1. Cytotoxic actions of antidepressants on HT29 cells
2.6. Protein extraction and immunoblotting analysis Cells were plated on a 60-mm culture dish at a density of 2 × 106 cells/dish, and exposed to the drugs for 12 h. The cells were rinsed with PBS, and permeabilized with 20 μM digitonin in 500 μl of permeabilizing buffer [HEPES-KOH (pH 7.4), 210 mM manitol, 70 mM sucrose, 50 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml of aprotinin and 1 μg/ml of leupeptin] as reported previously (Agarwal et al., 2003). The solution covering the cells was collected, and centrifuged at 18,000×g for 20 min to obtain the soluble fraction. The cells were lysed with 500 μl of lysis buffer [20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml of aprotinin and 1 μg/ ml of leupeptin] followed by subjecting them to a freeze–thaw cycle, and the cell extracts were obtained for an immunoblotting analysis of cytochrome c. The soluble fraction (cytoplasmic fraction) and the cell extracts (mitochondrial fraction) were analyzed by a sodium dodecylsulfate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, and the immunoblotting was carried out using a monoclonal mouse anti-human cytochrome c antibody and the ECL Westernblotting starter kit following the manufacturer's instructions. The amounts of total protein in the cell extracts were determined by a dye-binding method (Bradford, 1976) using bovine immunoglobulin G as a standard. 2.7. Flow cytometric analysis of cell-cycle Cells were plated on a 60-mm culture dish at a density of 1 × 106 cells/dish, and maintained for 24 h. After exposing to the drug for 24 h, the cells were harvested by trypsinization, rinsed
2.8. Data analysis Results were presented as the mean ± S.E.M., and the data were analyzed using an analysis of variance (ANOVA) followed by Tukey's post hoc test. A P value of <0.05 was accepted as a statistically significant difference.
To examine the direct effects of antidepressants on malignant colorectal tumor cells, human HT29 colon carcinoma cells were exposed to these drugs at the concentrations of 50 μM for 24 h, and the cell viability was determined by measuring the uptake of neutral red into these cells. As shown in Fig. 1, the viability of HT29 cells was reduced by exposing to tricyclic antidepressants, such as imipramine, desipramine and amitriptyline. In addition to these tricyclic antidepressants, a selective serotonin reuptake inhibitor fluoxetine also caused the reduction of cell viability, similar in extent to that caused by imipramine. Tranylcypromine, a monoamine oxidase inhibitor, failed to cause any significant change in the cell viability under the same conditions. The concentrations of these drugs required for the 50% reduction of cell viability were approximately 53, 31, 38, and 48 μM for imipramine, desipramine, amitriptyline, and fluoxetine, respectively. Furthermore, the cells were exposed to
Fig. 1. Effects of different-type antidepressants on proliferation of human colon carcinoma cells. HT29 cells were exposed to 50 μM of either imipramine (IMP), desipramine (DMI), amitriptyline (AMT), fluoxetine (FLX), or tranylcypromine (TCP) for 24 h, and their viability was determined as described in the text. Results were expressed as the percentage of control. Values are the mean ± S.E.M. (⁎P < 0.05, n = 6).
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the drugs for up to 4 days, and the reduction of cell viability was observed in a manner dependent on the time of drug exposure (data not shown). These results showed that tricyclic antidepressants and fluoxetine caused the cytotoxic actions on human colon cancer cells. Desipramine was shown to be the most potent among the drugs tested here, and therefore used for further investigation. 3.2. Tricyclic antidepressants cause apoptotic damage to HT29 cells To characterize the cytotoxic actions of tricyclic antidepressants, HT29 cells were exposed to desipramine, and the fluorescence cytochemical study was carried out using a double-staining with acridine orange and propidium iodide. As shown in Fig. 2, the chromatin condensation was observed in the nuclei of the cells treated with 50 μM desipramine for 24 h (panel A). The DNA fragmentation was also caused by exposing the cells to various concentrations of desipramine for 24 h (Fig. 2, panel B). Furthermore, the flow cytometric analysis indicated that the number of the cells stained with FITC-labeled annexin V (apoptotic cells) increased from 24% to 57% of total cells after exposing to 100 μM desipramine for 12 h (data not shown). Thus, it seemed obvious that the direct challenge of tricyclic antidepressants to HT29 cells caused the apoptotic damage to the cells, resulting in the reduction of their viability under the conditions used here. 3.3. Characterization of tricyclic antidepressant-induced apoptosis and cell-cycle arrest To further investigate the property of apoptotic cell death induced by tricyclic antidepressants, the effects of antioxidants
Fig. 2. Characterization of cytotoxic action of desipramine on human colon carcinoma cells. The fluorescence cytochemistry was carried out after exposing HT29 cells to 50 μM drug for 24 h [A], and the DNA fragmentation was determined after exposing to various concentrations of the drug for 24 h [B] as described in the text.
Fig. 3. Effect of desipramine on mitochondrial function in human colon carcinoma cells. HT29 cells were exposed to desipramine (DMI) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 12 h, and the mitochondrial function was assessed by analyzing the mitochondrial membrane potential [A] and the leakage of cytochrome c [B] as described in the text.
on the cytotoxic action of desipramine was examined. The reduction of cell viability induced by desipramine was not significantly affected by adding 1 mg/ml of catalase, 1 mM GSH, or 1 mM NAC to the culture medium (data not shown). Therefore, it seemed unlikely that tricyclic antidepressants might cause the oxidative damage to the cells under these conditions. Moreover, the influence of desipramine treatment on the mitochondrial function was investigated by determining the mitochondrial membrane potential. A considerable decrease in rhodamine-123 fluorescence was observed in the cells exposed to CCCP, but not in the cells exposed to 100 μM desipramine for 12 h (Fig. 3A). Furthermore, the immunoblotting analysis indicated that, although the amount of cytochrome c in the cytoplasmic fraction was markedly increased by exposing the cells to 100 μM CCCP for 12 h, the exposure to desipramine failed to significantly increase the amount of cytochrome c in the cytoplasm under the same conditions (Fig. 3B). Thus, it seemed unlikely that desipramine might cause the apoptotic death of HT29 cells as a result of disturbing the mitochondrial function. To further characterize the effects of tricyclic antidepressants on the cell-cycle progression, HT29 cells were exposed to different concentrations of desipramine, and then subjected to a flow cytometric analysis of the cell-cycle distribution. As shown in Fig. 4, the low concentration of desipramine (25 μM) increased the number of cells at G0/G1-phase with a concomitant decrease in the cells at S-phase, while the high concentration of the drug (75 μM) significantly decreased the cells at G0/G1-phase with a marked increase in the cells at G2/ M-phase, and the moderate concentration of desipramine (50 μM) increased the cell numbers at G0/G1- and G2/Mphase with a significant decrease in the cells at S-phase. Thus, it
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Fig. 4. Effect of desipramine on cell-cycle distribution in human colon carcinoma cells. HT29 cells were exposed to various concentrations of desipramine (DMI) for 24 h, and the cell-cycle distribution was analyzed as described in the text. Results were expressed as the percentage of total cell number. Values are the mean ± S.E.M. (⁎P < 0.05, n = 6).
seemed conceivable that exposure of HT29 cells to desipramine caused the apoptotic cell death, which might be largely attributed to the arrest of cell-cycle progression. 4. Discussion Direct actions of antidepressants on human HT29 colon carcinoma cells were examined as an in vitro model experiment for investigating their possible influence on the growth of malignant colorectal tumors. Tricyclic antidepressants, such as imipramine, desipramine and amitriptyline, were shown to cause their cytotoxic actions on these cells in culture (Fig. 1). In addition to these tricyclic antidepressants, fluoxetine, one of the selective serotonin reuptake inhibitors, was shown to cause the cytotoxic action on HT29 cells, almost similar in extent to the action of imipramine, and hence the cytotoxic actions of tricyclic antidepressants observed here are considered to be not necessarily specific to their chemical structures. Previously, fluoxetine has been shown to increase the extracellular concentrations of norepinephrine and dopamine as well as serotonin in different parts of the brain (Jordan et al., 1994; Perry and Fuller, 1997; Pozzi et al., 1999). Recent study has furthermore shown that, among selective serotonin reuptake inhibitors, only fluoxetine can induce the elevation of extracellular norepinephrine and dopamine levels in prefrontal cortex, suggesting that fluoxetine is not completely selective to the serotonin reuptake system (Bymaster et al., 2002), and interacts with the norepinephrine reuptake system in the brain. Therefore, it seems likely that fluoxetine causes the toxic damage to HT29 cells probably through interacting with the common site associated with the cytotoxic actions of tricyclic antidepressants. Cytotoxic actions of tricyclic antidepressants on HT29 cells were furthermore characterized by a fluorescence cytochemical study and an electrophoretic DNA analysis, and desipramine
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was shown to induce the chromatin condensation and the DNA fragmentation (Fig. 2). The apoptosis of HT29 cells induced by desipramine was confirmed by a flow cytometric analysis. Therefore, the direct challenge of desipramine to HT29 cells is considered to cause the apoptotic damage to these cells under the experimental conditions used here. Moreover, the effect of desipramine on HT29 cells was assessed again in the medium containing either catalase, GSH, or NAC, and none of them was shown to effectively prevent the cytotoxic action of desipramine on the cells. Thus, it seems reasonable to conclude that desipramine induces the apoptotic death of HT29 cells through its non-oxidative action on the cells. Desipramine was shown to cause neither the disruption of mitochondrial membrane potential within the cells nor the release of cytochrome c into the cytoplasmic space of the cells (Fig. 3). In previous study, the deprivation of glial cell linederived neurotrophic factor, a neurotrophic factor derived from glial cells, has been shown to cause the apoptotic damage to sympathetic neurons in culture without inducing the release of cytochrome c and the activation of caspase-9 and -3, thus suggesting that the deprivation of this neurotrophic factor may cause neuronal cell death probably through a novel nonmitochondrial pathway (Yu et al., 2003). Recent study has also shown that the inhibition of choline kinase in human tumor cells may cause the promotion of apoptosis by activating caspase-3 prior to the release of cytochrome c and the loss of mitochondrial potential, suggesting that the activation of caspase-3 may be independent of the mitochondrial dysfunction, and hence, even though both cytochrome c release and membrane potential disruption occur, it seems possible that the apoptotic cell death induced by choline kinase inhibition is not necessarily mediated by a mitochondrial pathway (RodriguezGonzalez et al., 2005). Based on these findings, it seems possible that desipramine may cause the non-oxidative damage to HT29 cells, resulting in the apoptotic cell death probably through a non-mitochondrial pathway. Several antitumor agents have been shown to cause the apoptotic cell death by interfering with the progression of cellcycle in different types of tumor cells. For instance, the inhibitors of DNA topoisomerase I and II, such as camptothecin, CPT-11 and its metabolite SN-38, imidazoacridone C-1311, and indenoindole derivatives, have been shown to arrest the cell-cycle progression at G2/M-phase, promoting the apoptotic death of human leukemia cells, testicular, ovarian, and colorectal carcinoma cells (Bal et al., 2004; Hyzy et al., 2005; Magrini et al., 2002; McDonald and Brown, 1998; Ueno et al., 2002). Furthermore, silibinin, a bioactive flavonone and a major compound of milk thistle extract, has also been shown to arrest the cell-cycle progression at G2/M-phase by acting the cellcycle regulation system, promoting the apoptotic death of HT29 cells without caspase activation, PARP cleavage, and cytochrome c release in the cells (Agarwal et al., 2003). Based on these findings, the effect of desipramine on the cell-cycle distribution was examined, and the cell-cycle progression was shown to be arrested at G2/M-phase by exposing the cells to desipramine (Fig. 4). Therefore, it seems possible that desipramine may cause the arrest of cell-cycle progression at
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G2/M-phase, resulting in the apoptotic death of human HT29 colon carcinoma cells. Tricyclic antidepressants was shown to cause the nonoxidative cytotoxic actions on human HT29 colon carcinoma cells through a non-mitochondrial pathway associated with the cell-cycle progression, suggesting that these drugs are probably capable of causing their cytotoxic actions on malignant colorectal tumors, and may be applicable for the clinical treatment of gastrointestinal tumors in combination with other anticancer agents. However, the critical question of whether the concentrations of these drugs in tumor tissues can reach the effective cytotoxic levels still remains to be answered. Although the serum concentration of free desipramine has previously been shown to reach approximately 9.5 μM (Hursting et al., 1992), there has been no data available to indicate the drug concentrations in colon and rectum. However, antidepressants are considered to come into contact with the inner surface of gastrointestinal tract, and assumed to directly act on tumor cells inside colon and rectum during the long-term oral administration of these drugs. It therefore seems conceivable that the concentrations of these drugs within colon and rectum can possibly be high enough to cause their cytotoxic actions on colorectal tumor cells in vivo. In our preliminary studies on the direct action of imipramine on the different types of colon cancer cells, such as HT29 cells and HCT116 cells, we have found that these cells clearly show their different sensitivities to the drug. Therefore, it seemed important to choose an appropriate cell line as an in vitro experimental system. In the present study, the cells more sensitive to imipramine, which were HT29 cells, were considered to be better and suitable for the experiments. However, it seems necessary to compare the direct actions of antidepressants on different-type cells for further characterizing the cytotoxic actions of these drugs on colon cancer cells. In fact, we have continued to further characterize the cytotoxic action of desipramine on different colon cancer cells for elucidating a possible mechanism of their different sensitivities to the drug. In addition, it still remains to elucidate the detailed mechanism underlying the cytotoxic actions of tricyclic antidepressants, and hence studies on the effects of these drugs on the mechanism of the cell-cycle progression, particularly focusing on the cyclin/ cyclin-dependent kinase system, are in progress. References Abdul, M., Logothetis, C.J., Hoosein, N.M., 1995. Growth-inhibitory effects of serotonin uptake inhibitors on human prostate carcinoma cell lines. J. Urol. 154, 247–250. Agarwal, C., Singh, R.P., Dhanalakshmi, S., Tyagi, A.K., Tecklenburg, M., Sclafani, R.A., Agarwal, R., 2003. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 22, 8271–8282. Bal, C., Baldeyrou, B., Moz, F., Lansiaux, A., Colson, P., Kraus-Berthier, L., Leonce, S., Pierre, A., Boussard, M.F., Rousseau, A., Wierzbicki, M., Bailly, C., 2004. Novel antitumor indenoindole derivatives targeting DNA and topoisomerase II. Biochem. Pharmacol. 68, 1911–1922. Bendele, R.A., Adams, E.R., Hoffman, W.P., Gries, C.L., Morton, D.M., 1992. Carcinogenicity studies of fluoxetine hydrochloride in rats and mice. Cancer Res. 52, 6931–6935.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brandes, L.J., Arron, R.J., Bogdanovic, R.P., Tong, J., Zaborniak, C.L., Hogg, G.R., Warrington, R.C., Fang, W., LaBella, F.S., 1992. Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses. Cancer Res. 52, 3796–3800. Bymaster, F.P., Zhang, W., Carter, P.A., Shaw, J., Chernet, E., Phebus, L., Wong, D.T., Perry, K.W., 2002. Fluoxetine, but not other selective serotonin uptake inhibitors, increases norepinephrine and dopamine extracellular levels in prefrontal cortex. Psychopharmacology 160, 353–361. Coogan, P.F., Rosenberg, L., Palmer, J.R., Strom, B.L., Stolley, P.D., Zauber, A. G., Shapiro, S., 2000. Risk of ovarian cancer according to use of antidepressants, phenothiazines, and benzodiazepines (United States). Cancer Causes Control 11, 839–845. Cotterchio, M., Kreiger, N., Darlington, G., Steingart, A., 2000. Antidepressant medication use and breast cancer risk. Am. J. Epidemiol. 151, 951–957. Dalton, S.O., Johansen, C., Mellemkjaer, L., Sorensen, H.T., McLaughlin, J.K., Olsen, J., Olsen, J.H., 2000. Antidepressant medications and risk for cancer. Epidemiology 11, 171–176. Dublin, S., Rossing, M.A., Heckbert, S.R., Goff, B.A., Weiss, N.S., 2002. Risk of epithelial ovarian cancer in relation to use of antidepressants, benzodiazepines, and other centrally acting medications. Cancer Causes Control 13, 35–45. Fautz, R., Husein, B., Hechenberger, C., 1991. Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS). Mutat. Res. 253, 173–179. Freire-Garabal, M., Nunez, M.J., Pereiro, D., Riveiro, P., Losada, C., FernandezRial, J.C., Garcia-Iglesias, E., Prizmic, J., Mayan, J.M., Rey-Mendez, M., 1998. Effects of fluoxetine on the development of lung metastases induced by operative stress in rats. Life Sci. 63, PL31–PL38. Gavrilova-Ruch, O., Schonherr, K., Gessner, G., Schonherr, R., Klapperstuck, T., Wohlrab, W., Heinemann, S.H., 2002. Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells. J. Membr. Biol. 188, 137–149. Hursting, M.J., Clark, G.D., Raisys, V.A., Miller, S.J., Opheim, K.E., 1992. Measurement of free desipramine in serum by ultrafiltration with immunoassay. Clin. Chem. 38, 2468–2471. Hyzy, M., Bozko, P., Konopa, J., Skladanowski, A., 2005. Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells. Biochem. Pharmacol. 69, 801–809. Iishi, H., Tatsuta, M., Baba, M., Taniguchi, H., 1993. Enhancement by the tricyclic antidepressant, desipramine, of experimental carcinogenesis in rat colon induced by azoxymethane. Carcinogenesis 14, 1837–1840. Jordan, S., Kramer, G.L., Zukas, P.K., Moeller, M., Petty, F., 1994. In vivo biogenic amine efflux in medial prefrontal cortex with imipramine, fluoxetine, and fluvoxamine. Synapse 18, 294–297. Koch, J.M., Kell, S., Aldenhoff, J.B., 2003. Differential effects of fluoxetine and imipramine on the phosphorylation of the transcription factor CREB and cell-viability. J. Psychiatr. Res. 37, 53–59. Koubi, D., Jiang, H., Zhang, L., Tang, W., Kuo, J., Rodriguez, A.I., Hunter, T.J., Seidman, M.D., Corcoran, G.B., Levine, R.A., 2005. Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation. Neurochem. Int. 46, 73–81. Li, Y.F., Luo, Z.P., 2002. Desipramine antagonized corticosterone-induced apoptosis in cultured PC12 cells. Acta Pharmacol. Sin. 23, 311–314. Li, Y.F., Liu, Y.Q., Huang, W.C., Luo, Z.P., 2003. Cytoprotective effect is one of common action pathways for antidepressants. Acta Pharmacol. Sin. 24, 996–1000. Lin, X., Levitsky, D.A., King, J.M., Campbell, T.C., 1999. The promotion effect of anorectic drugs on aflatoxin B(1)-induced hepatic preneoplastic foci. Carcinogenesis 20, 1793–1799. Magrini, R., Bhonde, M.R., Hanski, M.L., Notter, M., Scherubl, H., Boland, C.R., Zeitz, M., Hanski, C., 2002. Cellular effects of CPT-11 on colon carcinoma cells: dependence on p53 and hMLH1 status. Int. J. Cancer 101, 23–31. McDonald, A.C., Brown, R., 1998. Induction of p53-dependent and p53independent cellular responses by topoisomerase 1 inhibitors. Br. J. Cancer 78, 745–751.
H. Arimochi, K. Morita / European Journal of Pharmacology 541 (2006) 17–23 Morita, K., Wong, D.L., 2000. Glucocorticoids enhance serum deprivation- but not calcium-induced cytotoxicity in rat C6 glioma cells. Cell Biol. Int. 24, 223–228. Morita, K., Ishimura, K., Tsuruo, Y., Wong, D.L., 1999. Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors. Brain Res. 816, 309–316. Perry, K.W., Fuller, R.W., 1997. Fluoxetine increases norepinephrine release in rat hypothalamus as measured by tissue levels of MHPG-SO4 and microdialysis in conscious rats. J. Neural Transm. 104, 953–966. Pozzi, L., Invernizzi, R., Garavaglia, C., Samanin, R., 1999. Fluoxetine increases extracellular dopamine in the prefrontal cortex by a mechanism not dependent on serotonin: a comparison with citalopram. J. Neurochem. 73, 1051–1057. Rodriguez-Gonzalez, A., Ramirez De Molina, A., Banez-Coronel, M., Megias, D., Lacal, J.C., 2005. Inhibition of choline kinase renders a highly selective cytotoxic effect in tumor cells through a mitochondrial independent mechanism. Int. J. Oncol. 26, 999–1008. Serafeim, A., Holder, M.J., Grafton, G., Chamba, A., Drayson, M.T., Luong, Q.T., Bunce, C.M., Gregory, C.D., Barnes, N.M., Gordon, J., 2003. Selective serotonin reuptake inhibitors directly signal for apoptosis in biopsy-like Burkitt lymphoma cells. Blood 101, 3212–3219. Sharpe, C.R., Collet, J.P., Belzile, E., Hanley, J.A., Boivin, J.F., 2002. The effects of tricyclic antidepressants on breast cancer risk. Br. J. Cancer 86, 92–97. Slamon, N.D., Pentreath, V.W., 1998. A comparison of the acute and chronic effects of antidepressants in cultured C6 and 1321N1 cells. ATLA 26, 303–319. Slamon, N.D., Pentreath, V.W., 2000. Antioxidant defense against antidepressants in C6 and 1321N1 cells. Chem.-Biol. Interact. 127, 181–199.
23
Slamon, N.D., Ward, T.H., Butler, J., Pentreath, V.W., 2001. Assessment of DNA damage in C6 glioma cells after antidepressant treatment using an alkaline comet assay. Arch. Toxicol. 75, 243–250. Sternbach, H., 2003. Are antidepressants carcinogenic? A review of preclinical and clinical studies. J. Clin. Psychiatry 64, 1153–1162. Tutton, P.J., Barkla, D.H., 1989. Effect of an inhibitor of noradrenaline uptake, desipramine, on cell proliferation in the intestinal crypt epithelium. Virchows Arch., B Cell Pathol. 57, 349–352. Ueno, M., Nonaka, S., Yamazaki, R., Deguchi, N., Murai, M., 2002. SN-38 induces cell cycle arrest and apoptosis in human testicular cancer. Eur. Urol. 42, 390–397. Volpe, D.A., Ellison, C.D., Parchment, R.E., Grieshaber, C.K., Faustino, P.J., 2003. Effects of amitriptyline and fluoxetine upon the in vitro proliferation of tumor cell lines. J. Exp. Ther. Oncol. 3, 169–184. Wang, P.S., Walker, A.M., Tsuang, M.T., Orav, E.J., Levin, R., Avorn, J., 2001. Antidepressant use and the risk of breast cancer: a non-association. J. Clin. Epidemiol. 54, 728–734. Weiss, S.R., McFarland, B.H., Burkhart, G.A., Ho, P.T., 1998. Cancer recurrences and secondary primary cancers after use of antihistamines or antidepressants. Clin. Pharmacol. Ther. 63, 594–599. Xia, Z., DePierre, J.W., Nassberger, L., 1998. Modulation of apoptosis induced by tricyclic antidepressants in human peripheral lymphocytes. J. Biochem. Mol. Toxicol. 12, 115–123. Xia, Z., Bergstrand, A., DePierre, J.W., Nassberger, L., 1999. 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. Yu, L.Y., Jokitalo, E., Sun, Y.F., Mehlen, P., Lindholm, D., Saarma, M., Arumae, U., 2003. GDNF-deprived sympathetic neurons die via a novel nonmitochondrial pathway. J. Cell Biol. 163, 987–997.