- Email: [email protected]
Novel neuronal targets for the acetylcholinesterase inhibitor donepezil
Neuropharmacology 47 (2004) 1198–1204 www.elsevier.com/locate/neuropharm
Novel neuronal targets for the acetylcholinesterase inhibitor donepezil M.A. Sortino a,, G. Frasca a, M. Chisari a, P. Platania a, S. Chiechio b, C. Vancheri c, A. Copani b, P.L. Canonico d a
Department of Experimental and Clinical Pharmacology, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy b Department of Pharmaceutical Sciences, University of Catania, Catania, Italy c Department of Internal and Specialistic Medicine, University of Catania, Catania, Italy d DISCAFF, University of Piemonte Orientale, Novara, Italy Received 24 October 2003; received in revised form 16 July 2004; accepted 17 August 2004
Abstract The effects of the acetylcholinesterase inhibitor donepezil on cell viability and proliferation events have been analysed in SH-SY5Y human neuroblastoma cells. Short- (48 h) or long-term (7 days) exposure of SH-SY5Y cells to donepezil (100 nM–10 lM) induced a concentration-dependent inhibition of cell proliferation that was not modified by muscarinic and nicotinic receptor antagonists, or mimicked by galantamine, and was not related to induction of apoptosis. By analysing the distribution profile of cell populations within the cell cycle following treatment with 10 lM donepezil, a reduction of cells in the S-G2/M phases of the cycle and a parallel increase of the G0/G1 population were observed. In addition, the expression of two cyclins of the G1/S and G2/M transitions, cyclin E and cyclin B, was significantly reduced in donepezil-treated cells. In contrast, the expression of the cell cycle inhibitor p21 rapidly (6 h) increased following exposure to the drug. Finally, donepezil increased the expression of the neuronal marker MAP-2 in selected subpopulations of SH-SY5Y cells, suggesting that the effect on cell proliferation by donepezil may correlate to a trend to neuronal differentiation. # 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cell cycle; Differentiation; Alzheimer’s disease
1. Introduction Re-expression of the complex machinery of cell cycle activation in neurons has been recently related to several neuropathologies including Alzheimer’s disease (AD) (Husseman et al., 2000; McShea et al., 1999; Yang et al., 2003). An attempt to re-enter a mitotic cycle precedes neuronal death as demonstrated by the expression of several cell cycle-related proteins in neurons at risk for death (Busser et al., 1998; McShea et al., 1997; Nagy et al., 1997; Vincent et al., 1996, 1997). Thus, cyclins D, E and B as well as the regulators of cell cycle progression, cyclin dependent kinases (CDK) 1 and 4 are expressed in neurons from Corresponding author. Tel.: +39-095-7384086; fax: +39-0957384228. E-mail address: [email protected] (M.A. Sortino).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.08.011
AD brain (Busser et al., 1998; McShea et al., 1997; Nagy et al., 1997; Vincent et al., 1996, 1997). In addition, in post-mitotic cortical neurons in vitro, b-amyloid induces expression of specific markers of the G1-S phases such as cyclins D, E and A, whereas blockade of CDK4/6 and CDK2 protects against bAPinduced neuronal death (Copani et al., 1999). Interestingly, in these neurons, an attempt to duplicate DNA precedes death as demonstrated by the appearance of an unexpected S phase by cell cycle analysis (Copani et al., 1999). All these findings have contributed to provide a novel view of neuronal death by highlighting a link between cell division and neurodegeneration. Although the precise sequence of intracellular events driving neurons to death has not been identified, cell cycle regulators have been suggested as possible, novel therapeutic targets for the treatment of neurodegenerative disorders (Copani et al., 2003).
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Donepezil belongs to the class of acetylcholinesterase inhibitors and represents at present one of the most used drug for the treatment of early and/or mild stages of AD (Giacobini, 2000; Nordberg and Svensson, 1998). Its clinical efficacy has been related to the ability of the drug to inhibit acetylcholinesterase activity (Snape et al., 1999), thus increasing acetylcholine extracellular levels. However, it still remains uncertain whether the therapeutic benefits of this class of compounds can be entirely explained by the increased availability of acetylcholine at the synapse. Certainly this action will contribute to the amelioration of memory and cognition, but other mechanisms may underlie the efficacy of these drugs. With all this in mind, we have asked whether these alternative mechanisms for donepezil may include modifications of cell cycle profiles. In the present study, we have used the SH-SY5Y human neuroblastoma cell line that is sensitive to changes in cell replication rates and differentiation (Ross et al., 1983). We here demonstrate that donepezil modifies proliferation and cell cycle profiles of SH-SY5Y neuroblastoma cells suggesting potential, additional molecular targets for the clinical efficacy of this drug.
1199
scraper, washed with phosphate buffer (PBS), and fixed v in 70% ethanol at 20 C overnight. Cells were repeatedly washed, incubated with 100 lg/ml RNase in PBS v for 1 h at 37 C, and stained with the nuclear dye propidium iodide (final concentration, 50 lg/ml PBS). Analysis was carried out on a Coulter Elite flow cytometer. Cell cycle distribution was analysed by the Multicycle AV software program (Phoenix Flow System, San Diego, CA, USA). 2.4. Flow cytometry SH-SY5Y cells were detached, fixed for 30 min in 2% paraformaldehyde and permeabilized with 0.1% Tryton-X100. After blocking with BSA for 30 min, the cells were incubated with a mouse monoclonal specific anti p21 antibody (Santa Cruz Biotechnology Inc, v Santa Cruz, CA) at 4 C overnight. After washing, a FITC-conjugated secondary antibody (Santa Cruz) was added for 1 h at room temperature and samples were analysed with an Elite flow cytometer. 2.5. Western blotting v
2. Materials and methods 2.1. Cell culture SH-SY5Y human neuroblastoma cells (ACTT, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum and a mixture of streptomycin/penicillin, v and maintained at 37 C in a humidified atmosphere of 5% CO2. Only cells of passages 22–36 were used in the present study. SH-SY5Y cells were detached once a week and seeded into dishes or multiwell plates, depending on their experimental use, and exposed to the drug after reaching 60–70% confluency except for long term studies (7 days) in which treatments were started in lower density cultures. 2.2. MTT cell viability assay After treatment with donepezil, medium was removed, and SH-SY5Y cells were incubated with fresh medium containing MTT (Sigma, St. Louis, MO; v 0.9 mg/ml final concentration) for 2 h at 37 C. The solubilization solution, containing acidified isopropanol and 20% SDS, was added and left for 20 min in order to extract the produced formazan which was then evaluated in a plate reader (absorbance ¼ 560 nm). 2.3. Assessment of cell cycle and apoptotic cell death Apoptosis was evaluated by cytofluorometric analysis. SH-SY5Y cells were detached with the aid of a cell
SH-SY5Y cells were harvested at 4 C with 10 mM Tris lysis buffer containing 5 mM EDTA, 1% TrytonX100, 1 mM phenylmethylsulfonylfluoride, 25 lg/ml leupeptin and 0.5% aprotinin (all from Sigma). After sonication, an aliquot of the samples was processed for protein concentrations according to the method of Bradford. Samples were diluted in sample buffer and boiled for 5 min. Electrophoresis was performed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (40 mA/h) using 80–120 lg of cell proteins per lane. After separation, proteins were transferred onto a nitrocellulose membrane for 2 h at room temperature using a transblot semidry transfer cell. Membranes were maintained in blocking serum prior to incubation with v specific antibodies at 4 C overnight. Following repeated washings, membranes were exposed to horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 1 h at room temperature. Antibodies used were rabbit polyclonals (anti-cyclin E and anti-cyclin B from Santa Cruz) and monoclonal (anti-actin from Sigma) and were used at concentrations of 1–2 lg/ml. Proteins were visualized using the enhancing chemiluminescence detection system (ECL, Amersham Italia). 2.6. Immunocytochemistry Cells were fixed in 4% paraformaldehyde and stained with the monoclonal anti-MAP-2 antibody (Chemicon, Temecula, CA; 1:750) prior to incubation with a biotinylated secondary anti-mouse and the Vectastain ABC kit (all from Vector, Burlingame, CA).
1200
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
2.7. Drugs and reagents Donepezil was kindly provided by Pfizer Italia, galantamine hydrochloride was from Calbiochem (San Diego, USA) and mecamylamine was from Sigma. All common laboratory reagents, unless otherwise specified, were from Sigma.
3. Results A long term exposure (7 days) of the human SHSY5Y neuroblastoma cells to increasing concentrations (100 nM–10 lM) of donepezil resulted in inhibition of cell proliferation that was significant at a concentration of 1 lM (Fig. 1A). A similar effect was observed when
Fig. 1. Inhibitory effect of donepezil on SH-SY5Y cell proliferation. Undifferentiated neuroblastoma cells were exposed to increasing concentrations of donepezil for 7 days (A) or to 10 lM donepezil (DPZ) for 48 h, (B) Cell viability was evaluated by the MTT proliferation assay. Blockade of muscarinic and nicotinic receptors by homatropine (hmt; 10 lM) and mecamylamine (mec; 10 lM), respectively, did not modify the inhibitory effect of donepezil. p< 0:05 by ANOVA followed by Bonferroni t-test for significance.
cells were counted by Trypan blue exclusion with the aid of a hemocytometer (ð8:3 0:52Þ 105 and ð5:9 0:31Þ 105 for control and 10 lM donepezil, respectively). The effect was time-dependent as a decrease of cell number, although of lower intensity (20% of control), was also present when cells were incubated for 48 h with the drug (Fig. 1B). This effect was not secondary to increased acetylcholine levels as it was not modified by receptor blockade with homatropine (10 lM) or mecamylamine (10 lM), antagonists for muscarinic and nicotinic receptors, respectively (Fig. 1B). The inhibitory effect of donepezil on cell proliferation was not accompanied by induction of apoptosis. In contrast, donepezil exhibited rather a protective activity against basal, unstimulated apoptosis that occurred in SH-SY5Y neuroblastoma cells in culture (Fig. 2), as shown by staining with the nuclear probe bisbenzimide (not shown), but more clearly, by cytofluorometric analysis from which a quantification of the apoptotic cell population was achieved (Fig. 2). Again this effect was not modified by treatment with the nicotinic receptor antagonist mecamylamine (10 lM), being the apoptotic population 10:2 þ 1:0%, 5:15 þ 0:3% and 5:4 þ 0:2% in control, donepeziland donepezil þ mecamylamine-treated cells, respectively. This effect was not mimicked by galantamine
Fig. 2. Donepezil reduces SH-SY5Y apoptotic cell death. Following treatment with donepezil (DPZ; 10 lM) for 48 h, cells were harvested, fixed with 70% ethanol and processed for ethidium iodide staining prior to analysis of cell cycle by the use of flow cytometry. The percent of the prediploid cell population representing the apoptotic cells is indicated. Data are from one representative experiment; similar results were obtained from three independent experiments each run in triplicate. Single values were comprised between 7.4–13.8% and 1.6–4.9% for control and donepezil-treated cells, respectively.
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
1201
Table 1 Effect of a 48 h-exposure to donepezil (10 lM) on the viability of SHSY5Y neuroblastoma cells differentiated by a 7 day-treatment with retinoic acid Treatment
Cells/ml (105)
Control Donepezil
4:9 0:2 5:8 0:3
After completion of differentiation by exposure to 10 lM retinoic acid, cells were treated with donepezil for additional 48 h. Cell number was evaluated by cell counting at the light microscope after labelling with a 0.04% solution of trypan blue. Values are mean SE of four independent studies each run in triplicate. p< 0:05 by Student’s t-test.
(100–300 nM) that did not modify the apoptotic population under basal conditions and did not affect SH-SY5Y cell number (not shown). In addition, in retinoic acid-differentiated SH-SY5Y neuroblastoma cells in which the occurrence of apoptotic death was markedly increased, exposure for 48 h to 10 lM donepezil significantly enhanced neuronal survival (Table 1). To examine more closely, the effect of donepezil on cellular dynamics, the cell cycle distribution profiles were analysed in SH-SY5Y cells exposed to the drug. As shown in Fig. 3A, treatment with 10 lM donepezil significantly modified the distribution of cells within the cell cycle by causing an increase of the cells in the G0/G1 phases and a corresponding decrease in the ‘‘proliferative’’ components of the cycle (S-G2/M). In particular, the greatest change was evident in the G2/ M population where approximately a 40% reduction was observed in cells exposed to donepezil treatment (Fig. 3A). Concomitant to changes in cell cycle progression, the expression of cyclin E, a key regulator of the G1/S transition was slightly reduced in donepeziltreated SH-SY5Y neuroblastoma cells, but more importantly, the levels of cyclin B1 appeared markedly diminished (Fig. 3B). This was accompanied by an enhanced expression of the cell cycle inhibitor p21 the levels of which slightly increased 6 and 24 h following exposure to donepezil (Fig. 3C). Fig. 4 shows immucytochemical analysis of the neuronal marker microtubule associated protein-2 (MAP2) in control (left panel) and donepezil-treated (right panel) SH-SY5Y cells. Selected cells exhibited increased staining for MAP-2; however, this differentiative activity was not supported by cytofluorometric analysis that revealed a similar immunostaining, as expressed by mean fluorescence intensity, for control and donepezil-treated cultures (3.35 and 3.86, respectively).
4. Discussion Acetylcholinesterase inhibitors, including donepezil, exert multiple actions at the cholinergic synapse.
Fig. 3. Modifications of different cell cycle-related parameters by donepezil in SH-SY5Y neuroblastoma cells. Treatment with donepezil for 48 h markedly reduced the percent of cells in the S and G2/M phases of the cycle while increasing the population in G0/G1 (A) The expression of the cell cycle proteins cyclin B and cyclin E was reduced, (B) as shown by western blot analysis whereas the expression of the cell cycle inhibitor p21 was rapidly enhanced, as shown by cytofluorometric analysis, (C) following exposure to the drug. Data reported in (B) are mean SE of three independent experiments. Data in (C) are from one representative determination. Consistent results were obtained in three to four similar experiments. MFI ¼ mean fluorescence intensity. p< 0:05 by Student’s t-test; x p< 0:05 by one-way ANOVA followed by Bonferroni t-test for significance.
Besides elevating acetylcholine levels, they are in fact known to interact with muscarinic and nicotinic receptors (Snape et al., 1999; Svensson and Nordberg, 1997; Hellstrom-Lindahl et al., 2000), to increase the levels of
1202
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Fig. 4. Immunostaining of control (left panel) and donepezil-treated (right panel) SH-SY5Y cells with the neuronal marker MAP-2. Cells were repeatedly (7 days) exposed to donepezil (10 lM) prior to incubation with a mouse anti-MAP-2 antibody and processing for immunocytochemistry using the ABC avidin/biotin system.
nicotinic receptors (Barnes et al., 2000; Schrattenholz et al., 1996) and to stimulate choline acetyltransferase activity (Kato et al., 1999). The pharmacodynamic profile of donepezil, in particular, includes an antagonistic activity on muscarinic cholinergic (Snape et al., 1999) and NMDA (Wang et al., 1999) receptors and a supposed interaction with an allosteric site on nicotinic receptors (Takada et al., 2003) that however appears rather controversial (Santos et al., 2002). The clinical efficacy of donepezil in the treatment of AD resides also in other effects of the drug on neuronal function. Evidence in fact exists that donepezil, as well as other acetylcholinesterase inhibitors, are neuroprotective (Takada et al., 2003; Zhang and Tang, 2000; Zhou et al., 2001; Svensson and Nordberg, 1998) and increase the release of the soluble form of amyloid precursor protein in vitro (Racchi et al., 2001). In the present study, we have investigated additional cellular targets for the action of donepezil by analysing the ability of the drug to modify the proliferation and viability of a neuroblastoma cell line. This cellular model appears particularly suitable for the purpose of this study giving the opportunity, in contrast to primary neuronal cultures, to analyse specifically cell cycle kinetics as modified by the drug. The inhibitory effect of donepezil on SH-SY5Y cell proliferation does not seem to be due to enhanced acetylcholine levels or to a direct effect on muscarinic or nicotinic receptors as demonstrated by the lack of effect of cholinergic receptor antagonists. The use of mecamylamine, however, cannot completely rule out an interaction of donepezil with the nicotinic receptor, as the interaction of both drugs is thought to occur allosterically (Woodruff-Pak et al., 2003). The direct effects of donepezil at the nicotinic receptor sites are however rather controversial. In fact, in PC12 cells, the neuroprotective action of donepezil on b-amyloid-induced toxicity, in contrast to that of tacrine, was not prevented by any nicotinic antagonist (Svensson and Nordberg, 1998), whereas, in rat cortical neurons, neuroprotection by donepezil against glutamate neurotoxicity was sensitive to nicotinic antagonists (Takada
et al., 2003); on the other hand, single channel activity elicited by donepezil could be blocked by mecamylamine (Woodruff-Pak et al., 2003; Maelicke et al., 2000). Thus, at least some of the effects of donepezil are independent of its interaction with the nicotinic receptor site. Although the above reported interactions of donepezil might not be relevant for the action of the drug in vivo, they had to be taken into account in SH-SY5Y cells due to the marked presence of cholinergic receptors in this in vitro system (Lukas et al., 1993; Steel and Buckley, 1993). Moreover, the increased availability of acetylcholine at the cell membrane is unlikely to mediate inhibition of cell proliferation as stimulation of both muscarinic and nicotinic cholinergic receptors, in most cases and in different cellular systems, is responsible for increased proliferation (Song et al., 2003; Metzen et al., 2003; Fucile et al., 1997); however, in rare cases, inhibition of proliferation mediated by muscarinic receptors has been reported (Williams, 2003; dos Santos et al., 2003). The inhibitory action of donepezil on cell proliferation is not accompanied by apoptosis, but rather by an increased cell viability as suggested by the slight reduction of the apoptotic population under basal conditions and by the increased cell number following donepezil treatment in retinoic acid-differentiated cells. This appears in line with results previously reported in other cellular systems in which donepezil exerted neuroprotective activity under conditions of different toxic insults including oxygen-glucose deprivation (Zhou et al., 2001), and oxidative stress (Zhang and Tang, 2000) and in the presence of b-amyloid peptide (Svensson and Nordberg, 1998), or glutamate (Takada et al., 2003). In most of these studies, the neuroprotective effect of donepezil in basal conditions has not been analysed or was not present, but, in all of them, indirect indexes that could not be extremely sensitive and indicative of apoptotic death were applied. The protective effect we have observed seems specific for donepezil as it was not mimicked by galantamine which is endowed with both acetylcholinesterase inhibiting activity and allosteric modulating properties on
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
nicotinic receptors. This appears rather surprising as galantamine in SH-SY5Y cells has recently been shown to increase the basal levels of the anti-apoptotic protein bcl-2 (Arias et al., 2004). However, according to our results, the neuroprotective effect of galantamine was not observed in basal conditions, but only following the apoptotic challenge of thapsigargin or b-amyloid (Arias et al., 2004). To elucidate further the mechanisms involved in donepezil effects, the expression of cell cycle regulatory proteins was analysed. The reduced expression of specific proteins that are critical for cell cycle advance suggests that donepezil may inhibit cell proliferation by reducing or slowing down the progression of cell cycle. Due to the different roles of the two cyclins analysed (Grana and Reddy, 1995), the observed distribution during distinct phases of the cycle is well in agreement with the expression of cyclin proteins. Cyclin E, which is involved in the early phases of cell cycle activation, was in fact slightly reduced in donepezil-treated cells in accordance with a moderate reduction of the S phase population. In contrast, the expression of cyclin B, which drives the transition into G2/M, was markedly inhibited, supporting the pronounced reduction of cell subpopulation in the G2/M phases of the cycle. However, the expression of the cell cycle inhibitor p21 (Roninson, 2002), which is known to affect the interaction between various cyclins and CDKs, mainly G1 specific cyclin/CDKs, but also cyclin B/CDC2 (Roninson, 2002), involved in G1/S as well as S/G2 transition, was slightly increased following donepezil treatment. This suggests that, as indicated by the lowered cyclin B expression, the action of donepezil on cell cycle was more pronounced in the late phases, but was also significant in the progression of cells through G1/S transition of which p21 is a negative modulator (Harper et al., 1993). The apparent discrepancy between the concomitant induction of p21 and inhibition of cyclin E expression could be due to the heterogeneity of SH-SY5Y cells that include different phenotypes probably displaying different sensitivity to donepezil action. Proliferation, differentiation and survival are strictly related phenomena in the cell (Blagosklonny, 2003). Hence, the decreased cell growth observed following donepezil treatment could be coupled to neuronal differentiation. This was investigated by analysing the expression of the specific neuronal marker, MAP-2 following exposure of SH-SY5Y cells to donepezil for 7 days. Although selected cells exhibited an increased staining with the anti-MAP-2 antibody, indicative of an enhanced expression of the neuronal marker, a clear differentiating activity of donepezil was not supported by quantitative cytofluorometric analysis. This is not surprising due to the different subpopulations that characterize SH-SY5Y neuroblastoma cells (Ross et al., 1983) and to the peculiar differentiation induced by
1203
various morphogens that does not necessarily coincide with the appearance of an intense neuritic network (Lombet et al., 2001). The differentiating activity of donepezil and other acetylcholinesterase inhibitors was suggested by the increased expression of tau protein, a neuronal-specific differentiation marker, observed following exposure of SH-SY5Y neuroblastoma cells to this class of compounds (Hellstrom-Lindahl et al., 2000). Interestingly, differentiation in neuronal cells may strictly correlate with neuroprotective activity as suggested by the modified expression of the bcl-2 protein family following treatment with various morphogens (Lombet et al., 2001; La Sorella et al., 1995). In conclusion, changes in SH-SY5Y cell growth and cycle-related dynamics induced by donepezil disclose novel cellular effects that may contribute to the action of donepezil in delaying and/or preventing the loss of neuronal function.
Acknowledgements Supported in part by a grant from Pfizer Italia Medical Department.
References Arias, E., Ale´s, E., Gabilan, N.H., Cano-Abad, N.F., Villaroya, M., Garcia, A.G., Lopez, M.G., 2004. Galantamine prevents apoptosis induced by b-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors. Neuropharmacology 46, 103–114. Barnes, C.A., Meltzer, J., Houston, F., Orr, G., McGann, K., Wenk, G.L., 2000. Chronic treatment of old rats with donepezil or galantamine: effects on memory, hippocampal plasticity and nicotinic receptors. Neuroscience 99, 17–23. Blagosklonny, M.V., 2003. Apoptosis, proliferation, differentiation: in search of the order. Semin. Cancer Biol. 13, 97–105. Busser, J., Geldmacher, D.S., Herrup, K., 1998. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer’s disease brain. J. Neurosci. 18, 2801–2807. Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Canonico, P.L., Nicoletti, F., Sortino, M.A., 1999. Mitotic signaling by bamyloid peptide causes neuronal death. FASEB J. 13, 2225–2234. Copani, A., Caricasole, A., Caruso, A., Caraci, F., Iacovelli, L., Sortino, M.A., Terstappen, G.C., Nicoletti, F., 2003. The Wnt pathway, cell-cycle activation and beta-amyloid: novel therapeutic strategies in Alzheimer’s disease? Trends Pharmacol. Sci. 24, 233–238. dos Santos, A.A., Medina, S.V., Sholl-Franco, A., de Araujo, E.G., 2003. PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF. Neurochem. Int. 42, 73–80. Fucile, S., Napolitano, M., Mattei, E., 1997. Cholinergic stimulation of human microcytoma cell line H69. Biochem. Biophys. Res. Commun. 230, 501–504. Giacobini, E., 2000. Cholinesterase inhibitors stabilize Alzheimer’s disease. Ann. N. Y. Acad. Sci. 920, 321–327. Grana, X., Reddy, E.P., 1995. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11, 211–219.
1204
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., Elledge, S.J., 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816. Hellstrom-Lindahl, E., Moore, H., Nordberg, A., 2000. Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists. J. Neurochem. 74, 777–784. Husseman, J.W., Nochlin, D., Vincent, I., 2000. Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases. Neurobiol. Aging 21, 815–828. Kato, K., Hayako, H., Ishihara, Y., Marui, S., Iwane, M., Miyamoto, M., 1999. TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons. Neurosci. Lett. 260, 5–8. La Sorella, A., Iavarone, A., Israel, M.A., 1995. Differentiation of neuroblastoma enhances bcl-2 expression and induces alterations of apoptosis and drug resistance. Cancer Res. 55, 4711–4716. Lombet, A., Zujovic, V., Kandouz, M., Billardon, C., CarvajalGonzales, S., Gompel, A., Roste`ne, W., 2001. Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of bcl-2 protein family. Eur. J. Biochem. 268, 1352–1362. Lukas, R.J., Norman, S.A., Lucero, L., 1993. Characterization of nicotinic acetylcholine receptors expressed by cells of the SHSY5Y human neuroblastoma clonal line. Mol. Cell. Neurosci. 4, 1–12. Maelicke, A., Schrattenholz, A., Samochocki, M., Radina, M., Albuquerque, E.X., 2000. Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer’s disease. Behav. Brain Res. 113, 199–206. McShea, A., Harris, P.L., Webster, K.R., Wahl, A.F., Smith, M.A., 1997. Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer’s disease. Am. J. Pathol. 150, 1933–1939. McShea, A., Wahl, A.F., Smith, M.A., 1999. Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med. Hypotheses 52, 525–527. Metzen, J., Bittinger, F., Kirkpatrick, C.J., Kilbinger, H., Wessler, I., 2003. Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype. Life Sci. 72, 2075–2080. Nagy, Z., Esiri, M.M., Cato, A.M., Smith, A.D., 1997. Cell cycle markers in the hippocampus in Alzheimer’s disease. Acta Neuropathol. (Berl.) 94, 6–15. Nordberg, A., Svensson, A.-L., 1998. Cholinesterase inhibitors in the treatment of Alzheimer’s disease. A comparison of tolerability and pharmacology. Drug Saf. 19, 465–480. Racchi, M., Sironi, M., Caprera, A., Konig, G., Govoni, S., 2001. Short and long-term effects of acetylcholinesterase inhibition on the expression and metabolism of the amyloid precursor protein. Mol. Psychiatry 6, 520–528. Roninson, I.B., 2002. Oncogenic functions of tumour suppressor p21Waf1/Cip1/Sdi1: association with cell senescence and tumour-promoting activities of stromal fibroblasts. Cancer Lett. 179, 1–14. Ross, R.A., Spengler, B.A., Biedler, J.L., 1983. Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J. Natl. Cancer Inst. 71, 741–747. Santos, M.D., Alkondon, M., Pereira, E.F., Aracava, Y., Eisenberg, H.M., Maelicke, A., Albuquerque, E.X., 2002. The nicotinic
allosteric potentiating ligand galantamine facilitates synaptic transmission in the mammalian central nervous system. Mol. Pharmacol. 61, 1222–1234. Schrattenholz, A., Pereira, E.F., Roth, U., Weber, K.H., Albuquerque, E.X., Maelicke, A., 1996. Agonist responses of neuronal nicotinic acetylcholine receptors are potentiated by a novel class of allosterically acting ligands. Mol. Pharmacol. 49, 1–6. Snape, M.F., Misra, A., Murray, T.K., De Souza, R.J., Williams, J.L., Cross, A.J., Green, A.R., 1999. A comparative study in rats of the in vitro and in vivo pharmacology of acetylcholinesterase inhibitors tacrine, donepezil and NXX-066. Neuropharmacology 38, 181–193. Song, P., Sekhon, H.S., Jia, Y., Keller, J.A., Blusztajn, J.K., Mark, G.P., Spindel, E.R., 2003. Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res. 63, 214–221. Steel, M.C., Buckley, N.J., 1993. Differential regulation of muscarinic receptor mRNA levels in neuroblastoma cells by chronic agonist exposure: a comparative polymerase chain reaction study. Mol. Pharmacol. 43, 694–701. Svensson, A-L., Nordberg, A., 1997. Interaction of tacrine, galanthamine, NXX-066 and E2020 with neuronal a4b2 nicotinic receptors expressed in fibroblast cells. In: Iqbal, K., Winblad, B., Nishimura, T., Takeda, M., Wisniewski, H.M. (Eds.), Alzheimer’s Disease: Biology, Diagnosis and Therapeutics. John Wiley & Sons Ltd, New York, pp. 753–758. Svensson, A.L., Nordberg, A., 1998. Tacrine and donepezil attenuate the neurotoxic effect of Ab(25-35) in rat PC12 cells. Neuroreport 9, 1519–1522. Takada, Y., Yonezawa, A., Kume, T., Katsuki, H., Keneko, S., Sugimoto, H., Akaike, A., 2003. Nicotinic acetylcholine receptormediated neuroprotection by donepezil against glutamate neurotoxicity in rat cotical neurons. J. Pharm. Exp. Ther. 306, 772–777. Vincent, I., Rosado, M., Davies, P., 1996. Mitotic mechanisms in Alzheimer’s disease? J. Cell Biol. 132, 413–425. Vincent, I., Jicha, G., Rosado, M., Dickson, D., 1997. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer’s disease brain. J. Neurosci. 17, 3588–3598. Wang, X.D., Zhang, J.M., Yang, H.H., Hu, G.Y., 1999. Modulation of NMDA receptor by huperzine A in rat cerebral cortex. Acta Pharmacol. Sin. 20, 21–35. Williams, C.L., 2003. Muscarinic signaling in carcinoma cells. Life Sci. 72, 2173–2182. Woodruff-Pak, D.S., Vogel, R.W., Wenk, G.L., 2003. Mecamylamine interactions with galantamine and donepezil: effects on learning, acetylcholinesterase, and nicotinic acetylcholine receptors. Neuroscience 117, 439–447. Yang, Y., Mufson, E.J., Herrup, K., 2003. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer’s disease. J. Neurosci. 23, 2557–2563. Zhang, H.Y., Tang, X.C., 2000. Huperzine B, a novel acetylcholinesterase inhibitor, attenuates hydrogen peroxide induced injury in PC12 cells. Neurosci. Lett. 292, 41–44. Zhou, J., Fu, Y., Tang, X.C., 2001. Huperazine A and donepezil protect rat pheochromocytoma cells against oxygen-glucose deprivation. Neurosci. Lett. 306, 53–56.
Novel neuronal targets for the acetylcholinesterase inhibitor donepezil M.A. Sortino a,, G. Frasca a, M. Chisari a, P. Platania a, S. Chiechio b, C. Vancheri c, A. Copani b, P.L. Canonico d a
Department of Experimental and Clinical Pharmacology, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy b Department of Pharmaceutical Sciences, University of Catania, Catania, Italy c Department of Internal and Specialistic Medicine, University of Catania, Catania, Italy d DISCAFF, University of Piemonte Orientale, Novara, Italy Received 24 October 2003; received in revised form 16 July 2004; accepted 17 August 2004
Abstract The effects of the acetylcholinesterase inhibitor donepezil on cell viability and proliferation events have been analysed in SH-SY5Y human neuroblastoma cells. Short- (48 h) or long-term (7 days) exposure of SH-SY5Y cells to donepezil (100 nM–10 lM) induced a concentration-dependent inhibition of cell proliferation that was not modified by muscarinic and nicotinic receptor antagonists, or mimicked by galantamine, and was not related to induction of apoptosis. By analysing the distribution profile of cell populations within the cell cycle following treatment with 10 lM donepezil, a reduction of cells in the S-G2/M phases of the cycle and a parallel increase of the G0/G1 population were observed. In addition, the expression of two cyclins of the G1/S and G2/M transitions, cyclin E and cyclin B, was significantly reduced in donepezil-treated cells. In contrast, the expression of the cell cycle inhibitor p21 rapidly (6 h) increased following exposure to the drug. Finally, donepezil increased the expression of the neuronal marker MAP-2 in selected subpopulations of SH-SY5Y cells, suggesting that the effect on cell proliferation by donepezil may correlate to a trend to neuronal differentiation. # 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cell cycle; Differentiation; Alzheimer’s disease
1. Introduction Re-expression of the complex machinery of cell cycle activation in neurons has been recently related to several neuropathologies including Alzheimer’s disease (AD) (Husseman et al., 2000; McShea et al., 1999; Yang et al., 2003). An attempt to re-enter a mitotic cycle precedes neuronal death as demonstrated by the expression of several cell cycle-related proteins in neurons at risk for death (Busser et al., 1998; McShea et al., 1997; Nagy et al., 1997; Vincent et al., 1996, 1997). Thus, cyclins D, E and B as well as the regulators of cell cycle progression, cyclin dependent kinases (CDK) 1 and 4 are expressed in neurons from Corresponding author. Tel.: +39-095-7384086; fax: +39-0957384228. E-mail address: [email protected] (M.A. Sortino).
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.08.011
AD brain (Busser et al., 1998; McShea et al., 1997; Nagy et al., 1997; Vincent et al., 1996, 1997). In addition, in post-mitotic cortical neurons in vitro, b-amyloid induces expression of specific markers of the G1-S phases such as cyclins D, E and A, whereas blockade of CDK4/6 and CDK2 protects against bAPinduced neuronal death (Copani et al., 1999). Interestingly, in these neurons, an attempt to duplicate DNA precedes death as demonstrated by the appearance of an unexpected S phase by cell cycle analysis (Copani et al., 1999). All these findings have contributed to provide a novel view of neuronal death by highlighting a link between cell division and neurodegeneration. Although the precise sequence of intracellular events driving neurons to death has not been identified, cell cycle regulators have been suggested as possible, novel therapeutic targets for the treatment of neurodegenerative disorders (Copani et al., 2003).
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Donepezil belongs to the class of acetylcholinesterase inhibitors and represents at present one of the most used drug for the treatment of early and/or mild stages of AD (Giacobini, 2000; Nordberg and Svensson, 1998). Its clinical efficacy has been related to the ability of the drug to inhibit acetylcholinesterase activity (Snape et al., 1999), thus increasing acetylcholine extracellular levels. However, it still remains uncertain whether the therapeutic benefits of this class of compounds can be entirely explained by the increased availability of acetylcholine at the synapse. Certainly this action will contribute to the amelioration of memory and cognition, but other mechanisms may underlie the efficacy of these drugs. With all this in mind, we have asked whether these alternative mechanisms for donepezil may include modifications of cell cycle profiles. In the present study, we have used the SH-SY5Y human neuroblastoma cell line that is sensitive to changes in cell replication rates and differentiation (Ross et al., 1983). We here demonstrate that donepezil modifies proliferation and cell cycle profiles of SH-SY5Y neuroblastoma cells suggesting potential, additional molecular targets for the clinical efficacy of this drug.
1199
scraper, washed with phosphate buffer (PBS), and fixed v in 70% ethanol at 20 C overnight. Cells were repeatedly washed, incubated with 100 lg/ml RNase in PBS v for 1 h at 37 C, and stained with the nuclear dye propidium iodide (final concentration, 50 lg/ml PBS). Analysis was carried out on a Coulter Elite flow cytometer. Cell cycle distribution was analysed by the Multicycle AV software program (Phoenix Flow System, San Diego, CA, USA). 2.4. Flow cytometry SH-SY5Y cells were detached, fixed for 30 min in 2% paraformaldehyde and permeabilized with 0.1% Tryton-X100. After blocking with BSA for 30 min, the cells were incubated with a mouse monoclonal specific anti p21 antibody (Santa Cruz Biotechnology Inc, v Santa Cruz, CA) at 4 C overnight. After washing, a FITC-conjugated secondary antibody (Santa Cruz) was added for 1 h at room temperature and samples were analysed with an Elite flow cytometer. 2.5. Western blotting v
2. Materials and methods 2.1. Cell culture SH-SY5Y human neuroblastoma cells (ACTT, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum and a mixture of streptomycin/penicillin, v and maintained at 37 C in a humidified atmosphere of 5% CO2. Only cells of passages 22–36 were used in the present study. SH-SY5Y cells were detached once a week and seeded into dishes or multiwell plates, depending on their experimental use, and exposed to the drug after reaching 60–70% confluency except for long term studies (7 days) in which treatments were started in lower density cultures. 2.2. MTT cell viability assay After treatment with donepezil, medium was removed, and SH-SY5Y cells were incubated with fresh medium containing MTT (Sigma, St. Louis, MO; v 0.9 mg/ml final concentration) for 2 h at 37 C. The solubilization solution, containing acidified isopropanol and 20% SDS, was added and left for 20 min in order to extract the produced formazan which was then evaluated in a plate reader (absorbance ¼ 560 nm). 2.3. Assessment of cell cycle and apoptotic cell death Apoptosis was evaluated by cytofluorometric analysis. SH-SY5Y cells were detached with the aid of a cell
SH-SY5Y cells were harvested at 4 C with 10 mM Tris lysis buffer containing 5 mM EDTA, 1% TrytonX100, 1 mM phenylmethylsulfonylfluoride, 25 lg/ml leupeptin and 0.5% aprotinin (all from Sigma). After sonication, an aliquot of the samples was processed for protein concentrations according to the method of Bradford. Samples were diluted in sample buffer and boiled for 5 min. Electrophoresis was performed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (40 mA/h) using 80–120 lg of cell proteins per lane. After separation, proteins were transferred onto a nitrocellulose membrane for 2 h at room temperature using a transblot semidry transfer cell. Membranes were maintained in blocking serum prior to incubation with v specific antibodies at 4 C overnight. Following repeated washings, membranes were exposed to horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 1 h at room temperature. Antibodies used were rabbit polyclonals (anti-cyclin E and anti-cyclin B from Santa Cruz) and monoclonal (anti-actin from Sigma) and were used at concentrations of 1–2 lg/ml. Proteins were visualized using the enhancing chemiluminescence detection system (ECL, Amersham Italia). 2.6. Immunocytochemistry Cells were fixed in 4% paraformaldehyde and stained with the monoclonal anti-MAP-2 antibody (Chemicon, Temecula, CA; 1:750) prior to incubation with a biotinylated secondary anti-mouse and the Vectastain ABC kit (all from Vector, Burlingame, CA).
1200
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
2.7. Drugs and reagents Donepezil was kindly provided by Pfizer Italia, galantamine hydrochloride was from Calbiochem (San Diego, USA) and mecamylamine was from Sigma. All common laboratory reagents, unless otherwise specified, were from Sigma.
3. Results A long term exposure (7 days) of the human SHSY5Y neuroblastoma cells to increasing concentrations (100 nM–10 lM) of donepezil resulted in inhibition of cell proliferation that was significant at a concentration of 1 lM (Fig. 1A). A similar effect was observed when
Fig. 1. Inhibitory effect of donepezil on SH-SY5Y cell proliferation. Undifferentiated neuroblastoma cells were exposed to increasing concentrations of donepezil for 7 days (A) or to 10 lM donepezil (DPZ) for 48 h, (B) Cell viability was evaluated by the MTT proliferation assay. Blockade of muscarinic and nicotinic receptors by homatropine (hmt; 10 lM) and mecamylamine (mec; 10 lM), respectively, did not modify the inhibitory effect of donepezil. p< 0:05 by ANOVA followed by Bonferroni t-test for significance.
cells were counted by Trypan blue exclusion with the aid of a hemocytometer (ð8:3 0:52Þ 105 and ð5:9 0:31Þ 105 for control and 10 lM donepezil, respectively). The effect was time-dependent as a decrease of cell number, although of lower intensity (20% of control), was also present when cells were incubated for 48 h with the drug (Fig. 1B). This effect was not secondary to increased acetylcholine levels as it was not modified by receptor blockade with homatropine (10 lM) or mecamylamine (10 lM), antagonists for muscarinic and nicotinic receptors, respectively (Fig. 1B). The inhibitory effect of donepezil on cell proliferation was not accompanied by induction of apoptosis. In contrast, donepezil exhibited rather a protective activity against basal, unstimulated apoptosis that occurred in SH-SY5Y neuroblastoma cells in culture (Fig. 2), as shown by staining with the nuclear probe bisbenzimide (not shown), but more clearly, by cytofluorometric analysis from which a quantification of the apoptotic cell population was achieved (Fig. 2). Again this effect was not modified by treatment with the nicotinic receptor antagonist mecamylamine (10 lM), being the apoptotic population 10:2 þ 1:0%, 5:15 þ 0:3% and 5:4 þ 0:2% in control, donepeziland donepezil þ mecamylamine-treated cells, respectively. This effect was not mimicked by galantamine
Fig. 2. Donepezil reduces SH-SY5Y apoptotic cell death. Following treatment with donepezil (DPZ; 10 lM) for 48 h, cells were harvested, fixed with 70% ethanol and processed for ethidium iodide staining prior to analysis of cell cycle by the use of flow cytometry. The percent of the prediploid cell population representing the apoptotic cells is indicated. Data are from one representative experiment; similar results were obtained from three independent experiments each run in triplicate. Single values were comprised between 7.4–13.8% and 1.6–4.9% for control and donepezil-treated cells, respectively.
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
1201
Table 1 Effect of a 48 h-exposure to donepezil (10 lM) on the viability of SHSY5Y neuroblastoma cells differentiated by a 7 day-treatment with retinoic acid Treatment
Cells/ml (105)
Control Donepezil
4:9 0:2 5:8 0:3
After completion of differentiation by exposure to 10 lM retinoic acid, cells were treated with donepezil for additional 48 h. Cell number was evaluated by cell counting at the light microscope after labelling with a 0.04% solution of trypan blue. Values are mean SE of four independent studies each run in triplicate. p< 0:05 by Student’s t-test.
(100–300 nM) that did not modify the apoptotic population under basal conditions and did not affect SH-SY5Y cell number (not shown). In addition, in retinoic acid-differentiated SH-SY5Y neuroblastoma cells in which the occurrence of apoptotic death was markedly increased, exposure for 48 h to 10 lM donepezil significantly enhanced neuronal survival (Table 1). To examine more closely, the effect of donepezil on cellular dynamics, the cell cycle distribution profiles were analysed in SH-SY5Y cells exposed to the drug. As shown in Fig. 3A, treatment with 10 lM donepezil significantly modified the distribution of cells within the cell cycle by causing an increase of the cells in the G0/G1 phases and a corresponding decrease in the ‘‘proliferative’’ components of the cycle (S-G2/M). In particular, the greatest change was evident in the G2/ M population where approximately a 40% reduction was observed in cells exposed to donepezil treatment (Fig. 3A). Concomitant to changes in cell cycle progression, the expression of cyclin E, a key regulator of the G1/S transition was slightly reduced in donepeziltreated SH-SY5Y neuroblastoma cells, but more importantly, the levels of cyclin B1 appeared markedly diminished (Fig. 3B). This was accompanied by an enhanced expression of the cell cycle inhibitor p21 the levels of which slightly increased 6 and 24 h following exposure to donepezil (Fig. 3C). Fig. 4 shows immucytochemical analysis of the neuronal marker microtubule associated protein-2 (MAP2) in control (left panel) and donepezil-treated (right panel) SH-SY5Y cells. Selected cells exhibited increased staining for MAP-2; however, this differentiative activity was not supported by cytofluorometric analysis that revealed a similar immunostaining, as expressed by mean fluorescence intensity, for control and donepezil-treated cultures (3.35 and 3.86, respectively).
4. Discussion Acetylcholinesterase inhibitors, including donepezil, exert multiple actions at the cholinergic synapse.
Fig. 3. Modifications of different cell cycle-related parameters by donepezil in SH-SY5Y neuroblastoma cells. Treatment with donepezil for 48 h markedly reduced the percent of cells in the S and G2/M phases of the cycle while increasing the population in G0/G1 (A) The expression of the cell cycle proteins cyclin B and cyclin E was reduced, (B) as shown by western blot analysis whereas the expression of the cell cycle inhibitor p21 was rapidly enhanced, as shown by cytofluorometric analysis, (C) following exposure to the drug. Data reported in (B) are mean SE of three independent experiments. Data in (C) are from one representative determination. Consistent results were obtained in three to four similar experiments. MFI ¼ mean fluorescence intensity. p< 0:05 by Student’s t-test; x p< 0:05 by one-way ANOVA followed by Bonferroni t-test for significance.
Besides elevating acetylcholine levels, they are in fact known to interact with muscarinic and nicotinic receptors (Snape et al., 1999; Svensson and Nordberg, 1997; Hellstrom-Lindahl et al., 2000), to increase the levels of
1202
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Fig. 4. Immunostaining of control (left panel) and donepezil-treated (right panel) SH-SY5Y cells with the neuronal marker MAP-2. Cells were repeatedly (7 days) exposed to donepezil (10 lM) prior to incubation with a mouse anti-MAP-2 antibody and processing for immunocytochemistry using the ABC avidin/biotin system.
nicotinic receptors (Barnes et al., 2000; Schrattenholz et al., 1996) and to stimulate choline acetyltransferase activity (Kato et al., 1999). The pharmacodynamic profile of donepezil, in particular, includes an antagonistic activity on muscarinic cholinergic (Snape et al., 1999) and NMDA (Wang et al., 1999) receptors and a supposed interaction with an allosteric site on nicotinic receptors (Takada et al., 2003) that however appears rather controversial (Santos et al., 2002). The clinical efficacy of donepezil in the treatment of AD resides also in other effects of the drug on neuronal function. Evidence in fact exists that donepezil, as well as other acetylcholinesterase inhibitors, are neuroprotective (Takada et al., 2003; Zhang and Tang, 2000; Zhou et al., 2001; Svensson and Nordberg, 1998) and increase the release of the soluble form of amyloid precursor protein in vitro (Racchi et al., 2001). In the present study, we have investigated additional cellular targets for the action of donepezil by analysing the ability of the drug to modify the proliferation and viability of a neuroblastoma cell line. This cellular model appears particularly suitable for the purpose of this study giving the opportunity, in contrast to primary neuronal cultures, to analyse specifically cell cycle kinetics as modified by the drug. The inhibitory effect of donepezil on SH-SY5Y cell proliferation does not seem to be due to enhanced acetylcholine levels or to a direct effect on muscarinic or nicotinic receptors as demonstrated by the lack of effect of cholinergic receptor antagonists. The use of mecamylamine, however, cannot completely rule out an interaction of donepezil with the nicotinic receptor, as the interaction of both drugs is thought to occur allosterically (Woodruff-Pak et al., 2003). The direct effects of donepezil at the nicotinic receptor sites are however rather controversial. In fact, in PC12 cells, the neuroprotective action of donepezil on b-amyloid-induced toxicity, in contrast to that of tacrine, was not prevented by any nicotinic antagonist (Svensson and Nordberg, 1998), whereas, in rat cortical neurons, neuroprotection by donepezil against glutamate neurotoxicity was sensitive to nicotinic antagonists (Takada
et al., 2003); on the other hand, single channel activity elicited by donepezil could be blocked by mecamylamine (Woodruff-Pak et al., 2003; Maelicke et al., 2000). Thus, at least some of the effects of donepezil are independent of its interaction with the nicotinic receptor site. Although the above reported interactions of donepezil might not be relevant for the action of the drug in vivo, they had to be taken into account in SH-SY5Y cells due to the marked presence of cholinergic receptors in this in vitro system (Lukas et al., 1993; Steel and Buckley, 1993). Moreover, the increased availability of acetylcholine at the cell membrane is unlikely to mediate inhibition of cell proliferation as stimulation of both muscarinic and nicotinic cholinergic receptors, in most cases and in different cellular systems, is responsible for increased proliferation (Song et al., 2003; Metzen et al., 2003; Fucile et al., 1997); however, in rare cases, inhibition of proliferation mediated by muscarinic receptors has been reported (Williams, 2003; dos Santos et al., 2003). The inhibitory action of donepezil on cell proliferation is not accompanied by apoptosis, but rather by an increased cell viability as suggested by the slight reduction of the apoptotic population under basal conditions and by the increased cell number following donepezil treatment in retinoic acid-differentiated cells. This appears in line with results previously reported in other cellular systems in which donepezil exerted neuroprotective activity under conditions of different toxic insults including oxygen-glucose deprivation (Zhou et al., 2001), and oxidative stress (Zhang and Tang, 2000) and in the presence of b-amyloid peptide (Svensson and Nordberg, 1998), or glutamate (Takada et al., 2003). In most of these studies, the neuroprotective effect of donepezil in basal conditions has not been analysed or was not present, but, in all of them, indirect indexes that could not be extremely sensitive and indicative of apoptotic death were applied. The protective effect we have observed seems specific for donepezil as it was not mimicked by galantamine which is endowed with both acetylcholinesterase inhibiting activity and allosteric modulating properties on
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
nicotinic receptors. This appears rather surprising as galantamine in SH-SY5Y cells has recently been shown to increase the basal levels of the anti-apoptotic protein bcl-2 (Arias et al., 2004). However, according to our results, the neuroprotective effect of galantamine was not observed in basal conditions, but only following the apoptotic challenge of thapsigargin or b-amyloid (Arias et al., 2004). To elucidate further the mechanisms involved in donepezil effects, the expression of cell cycle regulatory proteins was analysed. The reduced expression of specific proteins that are critical for cell cycle advance suggests that donepezil may inhibit cell proliferation by reducing or slowing down the progression of cell cycle. Due to the different roles of the two cyclins analysed (Grana and Reddy, 1995), the observed distribution during distinct phases of the cycle is well in agreement with the expression of cyclin proteins. Cyclin E, which is involved in the early phases of cell cycle activation, was in fact slightly reduced in donepezil-treated cells in accordance with a moderate reduction of the S phase population. In contrast, the expression of cyclin B, which drives the transition into G2/M, was markedly inhibited, supporting the pronounced reduction of cell subpopulation in the G2/M phases of the cycle. However, the expression of the cell cycle inhibitor p21 (Roninson, 2002), which is known to affect the interaction between various cyclins and CDKs, mainly G1 specific cyclin/CDKs, but also cyclin B/CDC2 (Roninson, 2002), involved in G1/S as well as S/G2 transition, was slightly increased following donepezil treatment. This suggests that, as indicated by the lowered cyclin B expression, the action of donepezil on cell cycle was more pronounced in the late phases, but was also significant in the progression of cells through G1/S transition of which p21 is a negative modulator (Harper et al., 1993). The apparent discrepancy between the concomitant induction of p21 and inhibition of cyclin E expression could be due to the heterogeneity of SH-SY5Y cells that include different phenotypes probably displaying different sensitivity to donepezil action. Proliferation, differentiation and survival are strictly related phenomena in the cell (Blagosklonny, 2003). Hence, the decreased cell growth observed following donepezil treatment could be coupled to neuronal differentiation. This was investigated by analysing the expression of the specific neuronal marker, MAP-2 following exposure of SH-SY5Y cells to donepezil for 7 days. Although selected cells exhibited an increased staining with the anti-MAP-2 antibody, indicative of an enhanced expression of the neuronal marker, a clear differentiating activity of donepezil was not supported by quantitative cytofluorometric analysis. This is not surprising due to the different subpopulations that characterize SH-SY5Y neuroblastoma cells (Ross et al., 1983) and to the peculiar differentiation induced by
1203
various morphogens that does not necessarily coincide with the appearance of an intense neuritic network (Lombet et al., 2001). The differentiating activity of donepezil and other acetylcholinesterase inhibitors was suggested by the increased expression of tau protein, a neuronal-specific differentiation marker, observed following exposure of SH-SY5Y neuroblastoma cells to this class of compounds (Hellstrom-Lindahl et al., 2000). Interestingly, differentiation in neuronal cells may strictly correlate with neuroprotective activity as suggested by the modified expression of the bcl-2 protein family following treatment with various morphogens (Lombet et al., 2001; La Sorella et al., 1995). In conclusion, changes in SH-SY5Y cell growth and cycle-related dynamics induced by donepezil disclose novel cellular effects that may contribute to the action of donepezil in delaying and/or preventing the loss of neuronal function.
Acknowledgements Supported in part by a grant from Pfizer Italia Medical Department.
References Arias, E., Ale´s, E., Gabilan, N.H., Cano-Abad, N.F., Villaroya, M., Garcia, A.G., Lopez, M.G., 2004. Galantamine prevents apoptosis induced by b-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors. Neuropharmacology 46, 103–114. Barnes, C.A., Meltzer, J., Houston, F., Orr, G., McGann, K., Wenk, G.L., 2000. Chronic treatment of old rats with donepezil or galantamine: effects on memory, hippocampal plasticity and nicotinic receptors. Neuroscience 99, 17–23. Blagosklonny, M.V., 2003. Apoptosis, proliferation, differentiation: in search of the order. Semin. Cancer Biol. 13, 97–105. Busser, J., Geldmacher, D.S., Herrup, K., 1998. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer’s disease brain. J. Neurosci. 18, 2801–2807. Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Canonico, P.L., Nicoletti, F., Sortino, M.A., 1999. Mitotic signaling by bamyloid peptide causes neuronal death. FASEB J. 13, 2225–2234. Copani, A., Caricasole, A., Caruso, A., Caraci, F., Iacovelli, L., Sortino, M.A., Terstappen, G.C., Nicoletti, F., 2003. The Wnt pathway, cell-cycle activation and beta-amyloid: novel therapeutic strategies in Alzheimer’s disease? Trends Pharmacol. Sci. 24, 233–238. dos Santos, A.A., Medina, S.V., Sholl-Franco, A., de Araujo, E.G., 2003. PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF. Neurochem. Int. 42, 73–80. Fucile, S., Napolitano, M., Mattei, E., 1997. Cholinergic stimulation of human microcytoma cell line H69. Biochem. Biophys. Res. Commun. 230, 501–504. Giacobini, E., 2000. Cholinesterase inhibitors stabilize Alzheimer’s disease. Ann. N. Y. Acad. Sci. 920, 321–327. Grana, X., Reddy, E.P., 1995. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11, 211–219.
1204
M.A. Sortino et al. / Neuropharmacology 47 (2004) 1198–1204
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., Elledge, S.J., 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816. Hellstrom-Lindahl, E., Moore, H., Nordberg, A., 2000. Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists. J. Neurochem. 74, 777–784. Husseman, J.W., Nochlin, D., Vincent, I., 2000. Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases. Neurobiol. Aging 21, 815–828. Kato, K., Hayako, H., Ishihara, Y., Marui, S., Iwane, M., Miyamoto, M., 1999. TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons. Neurosci. Lett. 260, 5–8. La Sorella, A., Iavarone, A., Israel, M.A., 1995. Differentiation of neuroblastoma enhances bcl-2 expression and induces alterations of apoptosis and drug resistance. Cancer Res. 55, 4711–4716. Lombet, A., Zujovic, V., Kandouz, M., Billardon, C., CarvajalGonzales, S., Gompel, A., Roste`ne, W., 2001. Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of bcl-2 protein family. Eur. J. Biochem. 268, 1352–1362. Lukas, R.J., Norman, S.A., Lucero, L., 1993. Characterization of nicotinic acetylcholine receptors expressed by cells of the SHSY5Y human neuroblastoma clonal line. Mol. Cell. Neurosci. 4, 1–12. Maelicke, A., Schrattenholz, A., Samochocki, M., Radina, M., Albuquerque, E.X., 2000. Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer’s disease. Behav. Brain Res. 113, 199–206. McShea, A., Harris, P.L., Webster, K.R., Wahl, A.F., Smith, M.A., 1997. Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer’s disease. Am. J. Pathol. 150, 1933–1939. McShea, A., Wahl, A.F., Smith, M.A., 1999. Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med. Hypotheses 52, 525–527. Metzen, J., Bittinger, F., Kirkpatrick, C.J., Kilbinger, H., Wessler, I., 2003. Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype. Life Sci. 72, 2075–2080. Nagy, Z., Esiri, M.M., Cato, A.M., Smith, A.D., 1997. Cell cycle markers in the hippocampus in Alzheimer’s disease. Acta Neuropathol. (Berl.) 94, 6–15. Nordberg, A., Svensson, A.-L., 1998. Cholinesterase inhibitors in the treatment of Alzheimer’s disease. A comparison of tolerability and pharmacology. Drug Saf. 19, 465–480. Racchi, M., Sironi, M., Caprera, A., Konig, G., Govoni, S., 2001. Short and long-term effects of acetylcholinesterase inhibition on the expression and metabolism of the amyloid precursor protein. Mol. Psychiatry 6, 520–528. Roninson, I.B., 2002. Oncogenic functions of tumour suppressor p21Waf1/Cip1/Sdi1: association with cell senescence and tumour-promoting activities of stromal fibroblasts. Cancer Lett. 179, 1–14. Ross, R.A., Spengler, B.A., Biedler, J.L., 1983. Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J. Natl. Cancer Inst. 71, 741–747. Santos, M.D., Alkondon, M., Pereira, E.F., Aracava, Y., Eisenberg, H.M., Maelicke, A., Albuquerque, E.X., 2002. The nicotinic
allosteric potentiating ligand galantamine facilitates synaptic transmission in the mammalian central nervous system. Mol. Pharmacol. 61, 1222–1234. Schrattenholz, A., Pereira, E.F., Roth, U., Weber, K.H., Albuquerque, E.X., Maelicke, A., 1996. Agonist responses of neuronal nicotinic acetylcholine receptors are potentiated by a novel class of allosterically acting ligands. Mol. Pharmacol. 49, 1–6. Snape, M.F., Misra, A., Murray, T.K., De Souza, R.J., Williams, J.L., Cross, A.J., Green, A.R., 1999. A comparative study in rats of the in vitro and in vivo pharmacology of acetylcholinesterase inhibitors tacrine, donepezil and NXX-066. Neuropharmacology 38, 181–193. Song, P., Sekhon, H.S., Jia, Y., Keller, J.A., Blusztajn, J.K., Mark, G.P., Spindel, E.R., 2003. Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res. 63, 214–221. Steel, M.C., Buckley, N.J., 1993. Differential regulation of muscarinic receptor mRNA levels in neuroblastoma cells by chronic agonist exposure: a comparative polymerase chain reaction study. Mol. Pharmacol. 43, 694–701. Svensson, A-L., Nordberg, A., 1997. Interaction of tacrine, galanthamine, NXX-066 and E2020 with neuronal a4b2 nicotinic receptors expressed in fibroblast cells. In: Iqbal, K., Winblad, B., Nishimura, T., Takeda, M., Wisniewski, H.M. (Eds.), Alzheimer’s Disease: Biology, Diagnosis and Therapeutics. John Wiley & Sons Ltd, New York, pp. 753–758. Svensson, A.L., Nordberg, A., 1998. Tacrine and donepezil attenuate the neurotoxic effect of Ab(25-35) in rat PC12 cells. Neuroreport 9, 1519–1522. Takada, Y., Yonezawa, A., Kume, T., Katsuki, H., Keneko, S., Sugimoto, H., Akaike, A., 2003. Nicotinic acetylcholine receptormediated neuroprotection by donepezil against glutamate neurotoxicity in rat cotical neurons. J. Pharm. Exp. Ther. 306, 772–777. Vincent, I., Rosado, M., Davies, P., 1996. Mitotic mechanisms in Alzheimer’s disease? J. Cell Biol. 132, 413–425. Vincent, I., Jicha, G., Rosado, M., Dickson, D., 1997. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer’s disease brain. J. Neurosci. 17, 3588–3598. Wang, X.D., Zhang, J.M., Yang, H.H., Hu, G.Y., 1999. Modulation of NMDA receptor by huperzine A in rat cerebral cortex. Acta Pharmacol. Sin. 20, 21–35. Williams, C.L., 2003. Muscarinic signaling in carcinoma cells. Life Sci. 72, 2173–2182. Woodruff-Pak, D.S., Vogel, R.W., Wenk, G.L., 2003. Mecamylamine interactions with galantamine and donepezil: effects on learning, acetylcholinesterase, and nicotinic acetylcholine receptors. Neuroscience 117, 439–447. Yang, Y., Mufson, E.J., Herrup, K., 2003. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer’s disease. J. Neurosci. 23, 2557–2563. Zhang, H.Y., Tang, X.C., 2000. Huperzine B, a novel acetylcholinesterase inhibitor, attenuates hydrogen peroxide induced injury in PC12 cells. Neurosci. Lett. 292, 41–44. Zhou, J., Fu, Y., Tang, X.C., 2001. Huperazine A and donepezil protect rat pheochromocytoma cells against oxygen-glucose deprivation. Neurosci. Lett. 306, 53–56.