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Differential effects of COX inhibitors against β-amyloid-induced neurotoxicity in human neuroblastoma cells
Neurochemistry International 47 (2005) 589–596 www.elsevier.com/locate/neuint
Differential effects of COX inhibitors against b-amyloid-induced neurotoxicity in human neuroblastoma cells P. Ferrera, C. Arias * Departamento de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, AP 70-228, 04510 Me´xico, DF, Me´xico Received 19 January 2005; received in revised form 9 June 2005; accepted 13 June 2005 Available online 16 September 2005
Abstract Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer’s disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-b protein (Ab, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Ab and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Ab toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Ab neurotoxicity. # 2005 Elsevier Ltd. All rights reserved. Keywords: Amyloid-b protein; Neurotoxicity; Indomethacin; Human neuroblastoma; COX-2; Arachidonic acid; Neuroprotection
1. Introduction Alzheimer’s disease (AD) has a complex pathogenesis and may involve multiple etiological factors but a central role for b-amyloid protein (Ab) has been supported by genetic, neuropathological and biochemical evidence (Cotman et al., 1992; Hsiao et al., 1996; Holcomb et al., 1998; Walsh et al., 2002). There are several indicators suggesting that inflammatory processes may play a role in AD and it has been hypothesized that anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), may delay the onset or progression of AD. The pharmacological activity of NSAIDs is generally attributed to inhibition of cyclooxygenase (COX), the rate-limiting enzyme in the * Corresponding author. Tel.: +52 55 56223896; fax: +52 55 56223897. E-mail address: [email protected] (C. Arias). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.06.012
production of prostanoids from arachidonic acid (AA). The two isoforms of COX (COX-1 and COX-2) have been found in neurons (Yamagata et al., 1993). In some clinical trials the use of different NSAIDs on AD have provided conflicting data. While the treatment with the non-selective COX inhibitor indomethacin improved cognitive test in AD patients (Rogers et al., 1993) the selective COX-2 rofecoxib and the non-selective COX inhibitor naproxen failed to slow the progression of the disease (Aisen et al., 2003; Reines et al., 2004) suggesting that different NSAIDs have differential properties modulating metabolic pathways involved in AD pathology. Besides the well-known effects of NSAIDs on COX inhibition it has been found that some NSAIDs can also reduce nitric oxide (NO) production (Du and Li, 1999) and exert protective effects on neuronal apoptosis acting as NO scavengers (Asanuma et al., 2001). Moreover, new results
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indicate that some NSAIDs, like indomethacin might control the activity of g-secretase and, thus reduce the generation of Ab (Weggen et al., 2001). In AD, Ab aggregates have been associated with increased generation of oxygen reactive species (ROS), which leads to lipid, protein and DNA peroxidation, and to activation of transcription factors that are central to an array of neuroinflamatory gene signaling events in the AD brain (Lukiw and Bazan, 2000). It has been suggested that Ab, including its active fragment 25–35, may stimulate ROS production through an extracellular signal-regulated phospholipase A2 cascade (Lehtonen et al., 1996; Paris et al., 2000; Andersen et al., 2003), which leads to an increase in AA released from membrane phospholipids. In turn, the AA released is metabolized via the COX pathways leading to the generation of prostaglandins and by the lipo-oxygenase (LOX) pathways giving rise to leukotrienes. Although much of the mentioned evidence suggests the potential protective role of NSAIDs directly on Ab toxicity in neurons, the differential effects of selective or non-selective NSAIDs acting on COX or LOX enzymes have not been clarified yet. To study these effects, we compared the actions of indomethacin with selective COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxy-phenyl)-3-trifluoromethyl pyrazole (SC-560); the selective COX-2 blocker N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS-398) and with the 5-LOX inhibitor nordihydroguairetic acid (NDGA) on Ab-induced neurotoxicity alone or in combination with AA in differentiated and non-differentiated human neuroblastoma cells.
2. Experimental procedures 2.1. Cell culture Human neuroblastoma cell line MSN (Reynolds et al., 1986) was maintained in RPMI 1640 medium containing nonessential amino acids plus 15% fetal calf serum in 24 multidishes in an atmosphere of 5% CO2/95% O2, at 37 8C and were plated at a density of 1 106 cells per well. After 5 days in culture, neurons were incubated by 24 h with Ab fragment 25–35, 20 mM. When Ab 1–42 (10 mM) was added to cultures, we previously pre-incubated the peptide at 37 8C during 7 days and then cells were exposed to the peptide by 48 h. Different doses of compounds were tested: AA 10, 20 and 100 mM; NS-398 1, 5, 10 and 20 mM indomethacin 10, 50, 100 and 200 mM; SC-560 0.01, 0.1 and 1 mm and the 5-LOX inhibitor NDGA, 10 mM. Higher doses of indomethacin (50, 100 and 200 mM) were still neuroprotective to neuroblastoma cells and 1 mm of SC-560 became toxic to the cells. All drugs were purchased from Sigma Chemical (St. Louis, MO). To avoid the antiproliferative effects of NSAIDs (Eli et al., 2001), in some experiments MSN cells were differentiated into neurons by adding retinoic acid 10 mM plus NGF 50 ng/ml (both from Sigma Chemical, St. Louis, MO) (Encinas et al., 2000) during 7 days. The differentiated
neurons were immunocytochemically stained with the neuron-specific marker MAP2. 2.2. Mitochondrial reducing capacity As a measure of cell viability, the mitochondrial reducing capacity was evaluated 24 h after the onset of drug exposure. The method employed in the present study is commonly used in cultured cell studies to evaluate mitochondrial redox activity through the conversion of MTT tetrazolium salt to formazan crystals by mitochondrial respiratory chain reactions (Mossman, 1983). In brief, MTT was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/ ml and was added to MSN cells 1/10 (v/v) after 24 h of incubation with the different treatments and allowed to incubate for one more hour. Then, the incubation medium was removed and MSN cells were solubilized with isopropyl alcohol (0.8 ml). The absorbance of each sample was quantified at 570 nm using a spectrophotometer (Pharmacia Biotech, Gaithersburg, MD). Results are expressed as percentages of control values. Values are mean + S.E.M. of five to seven independent experiments made in triplicate. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by a Student’s t-test. 2.3. Cell viability In some experiments, cell survival was also assessed by using the membrane permeable dye calcein-AM (C-AM) and the DNA-binding fluorophere ethidium homodimer (EthD-1), according to the Live/Dead kit (Molecular Probes, Eugene, OR, USA). Because calcein-AM is converted to green fluorescence by an intracellular esterase, green staining indicates metabolically intact cells. EthD-1 is an indicator of membrane damage and reveals dead cells in red. The assay was performed following to the manufacturer’s instructions. In brief, MSN cells were grown on coverslips, and after 24 h to drugs exposure they were washed twice, 5 min each, in PBS and exposed to C-AM (2 mM) and HE (4 mM) in PBS for an additional 5 min. Then the cells were washed in PBS and fixed for 30 min with cold paraformaldehyde. Immediately after, cells were observed under an epifluorescent microscope and the number of live and dead cells was counted and results were expressed as a percentage of dead cells relative to the total number of cells counted in five different fields of four independent experiments. 2.4. Western blotting Western immunoblots were used to assess inducible COX-2 expression after stimulation with interleukine-1b (IL-1b). For stimulation experiments, cultured MSN cells were treated with Ab, AA, indomethacin and NS-398 in the same doses as described before. 1 h after exposure to these drugs, 100 U/ml IL-1b (Chemicon International, Temecula, CA) were added and cells were incubated for 24 h. Duplicate
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experiments were performed for each condition and they were repeated three times. At the end of incubation, cells were harvested and processed by immunoblotting. Briefly, 30 mg protein from the lysed MSN cells were loaded in a 5– 10% gradient SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane. They were incubated for 2 h in PBS solution containing 5% albumin. Blots were incubated with a polyclonal antibody against COX-2 (5 ug/mL Chemicon International, Temecula, CA) overnight at 4 8C, then washed three times with PBS (5 min each), incubated with an anti-rabbit IgG conjugated to horseradish peroxidase (1:1000 Vector, Laboratories, Burlingame, CA) and detected by chemiluminescence (ECL kit from Amersham, Arlington Heights, IL, USA) on Kodak X-Omat film. Negative controls consisted in eliminating the primary antibody from the procedure. The intensity of COX-2 bands in the immunoblots was quantified by densitometry using the Scion Image program for Windows 2000 (Scion Corporation, NIH, USA). COX-2 densitometric values were normalized with respect to those obtained with an antibody against a-tubulin (1 mg/mL; Clone DM 1A, Sigma Chemical, St. Louis, MO) used as an internal control.
3. Results 3.1. Neuroprotection by indomethacin from Ab 25–35and AA-induced toxicity Cultured MSN were treated with retinoic acid plus NGF in order to induce neuronal differentiation. The neuron-like
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phenotype was quite obvious 72 h after incubation when the cells produced cytoplasmic extensions. After 6 days, most of the population of the treated MSN neuroblastoma cells presented some neurite extensions (Fig. 1D) that were positive to the microtubule-associated protein 2 (MAP-2) (data not shown). Once this stage of differentiation was achieved, we proceeded to study the effects of Ab 25–35, AA or both, in the absence or presence of indomethacin on mitochondrial reducing capacity and the results were compared with those obtained in non-differentiated naive MSN cells. In naive MSN cells (Fig. 1C), after 24 h incubation with Ab the mitochondrial reducing capacity was inhibited by approximately 15%. To determine whether neuron survival was affected by incubation with the main substrate of COX enzymes, MSN cells were exposed to different AA concentrations. There was a significant difference between the survival of untreated MSN cells and cells exposed to 10 mM AA (25% less of MTT reduction). Higher concentrations of AA (20 or 100 mM) reduced the number of surviving neurons by more than 50% (data not shown). Interestingly, the combined incubation with Ab plus 10 mM AA did not increase the number of cell deaths produced by the treatment with Ab alone (18%, compared to control). The addition of indomethacin to the incubation media reverted the effects of Ab, AA and Ab plus AA on MTT reduction almost to control values (Fig. 1A). Interestingly, in differentiated MSN neuroblastoma cells Ab induced similar toxicity as in naive cells (15%), but the inhibition of MTT reduction induced by AA 10 mM alone or in combination with Abwas more evident (35 and 40%, respectively) and the protective effect of indomethacin incubation was remarkable (Fig. 1B).
Fig. 1. Effects of Ab 25–35 and AA on mitochondrial redox activity in the absence or presence of indomethacin in naive (A) and in differentiated (B) MSN cells. Cells were incubated for 24 h in the presence of different drugs (Ab, 20 mM; AA, 10 mM and indomethacin, 10 mM) and MTT reduction was assessed at the end of incubation. The morphological shape of naive neurons (C) or differentiated neurons with retinoic acid and NGF (D). Data expressed as the percentage of control values are the mean S.E.M. of duplicate determinations of five to seven different experiments: *P < 0.05; **P < 0.005.
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Fig. 2. Viability of MSN cells assessed by calcein AM (green) and ethidium homodimer (red). Living cells were stained green and dead cells were stained red. The amount of surviving cells exposed for 24 h to Ab 25–35 20 mM (B) or AA 10 mM (D) was lower compared with untreated cultures (A). After incubation with indomethacin the amount of surviving cells was significantly higher compared with the amount of surviving cells after Ab (C) or AA (E). The right panel shows the percentage of living neurons from each experimental condition compared with controls. Each bar represents the mean S.E.M. of total number of cells counted in five different fields from four independent experiments: *P < 0.05; **P < 0.005.
To confirm that the inhibition of MTT reduction reflected an increase in neuronal death, cultures were processed with the membrane-permeable dye calcein and with the DNAbinding fluorophere ethidium homodimer, 24 h after the onset of drug exposure. In each case, neuronal cultures were observed through a fluorescence microscope and the percentage of dead cells (red nuclei) in differentiated MSN cells was calculated. After 24 h, Ab 25–35 incubation induced 18% of cell death compared with 40% in the case of AA exposure. Pharmacological treatment with indomethacin prevented the loss of neurons in Ab- or AA-treated cultures, a finding consistent with the results obtained in the MTT assay (Fig. 2). Because inhibition of COX enzymes occurs at nanomolar and micromolar indomethacin concentrations, we explored the dose-response effects of indomethacin on Ab- and AA-induced toxicity. Fig. 3 shows that in cultured MSN cells indomethacin promotes cell viability in the range of 10 nM–10 mM with similar potency, which supports a role of COX inhibition as the main mechanism involved in its protective properties.
Ab 25–35 and totally blocked the AA-induced neuronal death (Fig. 4). To study the role of 5-LOX activity on the toxicity elicited by Ab 25–35, AA or the combination of Ab 25–35 plus AA, MSN cell were treated with NDGA for 24 h. Analysis of cell viability with the MTT assay showed that 5-LOX inhibition did not attenuate the toxicity induced by the mentioned compounds and, in addition, significantly inhibited cell viability by 15% in control neurons by itself. 3.3. Differential effects of COX inhibitors on Ab1–42-induced toxicity In view that the relevant peptide in AD is the 1–42 fragment we studied in parallel the protective effects of
3.2. Effects of NS-398, SC-560 and NDGA on Ab 25–35-induced neurotoxicity To expand the above results we compared the effects of indomethacin with the selective inhibitor of COX-2, NS-398 and the selective COX-1 inhibitor SC-560. In differentiated cultures, NS-398 at a dose of 10 mM protected neurons from the toxicity induced by AA or by the combination of Ab 25– 35 plus AA (Fig. 4). However, contrary to the results obtained with indomethacin, NS-398 did not show a significant effect on the inhibition of MTT reduction induced by the Ab 25–35. Similar to above results SC560 (0.1 mM) slightly reduce the neurotoxicity induced by
Fig. 3. Survival of MSN cells exposed to 20 mM Ab 25–35 (filled squares) or 10 mM AA (filled circles) in the presence of different concentration of indomethacin. Cells treated with indomethacin show an increase in MTT reduction that was clear since the lowest dose of 10 nM. Each point represents the mean S.E.M. of triplicate determinations from three independent experiments.
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Fig. 4. Effects of Ab 25–35 and AA on mitochondrial redox activity in the absence or presence of NS-398, SC-560 and NDGA in MSN cells. Cells were exposed to Ab 25–35 or AA in the presence or absence of COX/LOX inhibitors. NS-398 protected cells from AA toxicity but not from Ab toxicity while SC-560 partially inhibited cell death induced by Ab and totally protected cells from AA-induced cell death. NDGA inhibited the mitochondrial reducing capacity by itself and did not show any protection of Ab or AA effects. The data expressed as the percentage of control values are the mean S.E.M. of duplicate determinations of five to seven different experiments: *P < 0.05; **P < 0.005.
indomethacin, SC-560 and NS-398 on the neurotoxicity induced by this peptide. Differentiated MSN cells exposed by 48 h to Ab 1–42 showed a 75% of viability. Indomethacin was the most potent NSAID to protect MSN cells (100% of protection) while the selective COX-1 SC-560, slightly but statistically significant reverted cell death (90% viability). However co-treatment with the selective COX-2 inhibitor, NS-398 did not restore MTT reduction (Fig. 5).
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Fig. 6. Representative Western blot and densitometric analysis of IL-1binduced COX-2 expression (72 kDa) from MSN cells exposed for 24 h to Ab 25–35, AA and COX-2 inhibitors indomethacin and NS-398. Lane 1: control; lanes 2, 3, 4 and 5: Ab 25–35, 20 mM, NS-398, 10 mM, AA, 10 mm and Ab 25–35 plus AA, respectively; and lanes 6, 7 and 8: Ab 25–35, AA, and Ab 25–35 plus AA, respectively, in the presence of NS-398. Lane 9: indomethacin, 10 mM and lanes 10, 11 and 12: Ab 25–35, AA, and Ab 25– 35 plus AA, respectively, in the presence of indomethacin. Each bar represents the mean S.E.M. of COX-2 expression normalized with tubulin expression (55 kDa) of three independent experiments done in duplicate: *P < 0.05; **P < 0.005.
3.4. Changes in COX-2 expression
Fig. 5. Differential effects of COX inhibitors on Ab 1–42-induced toxicity in differentiated MSN cells. Cell were incubated in the presence of 20 mM Ab 1–42 by 48 h in the absence or presence of 10 mM indomethacin, 0.1 mM SC-560 or 10 mM NS-398. Data expressed as the percentage of control values are the mean S.E.M. of triplicate determinations of three different experiments: *P < 0.05.
To determine whether the different treatments induce changes directly of COX-2 expression, Western blot analyses were performed for each condition. We did not detect COX-2 immunoreactivity under basal conditions, neither in the presence of Ab nor of AA (data not shown). When cultured in the presence of 100 U/ml IL-1b, a band of approximately 70 kDa clearly appeared (Fig. 6, lane 1) consistent with previous reports of the appearance of COX2 immunoreactivity in neuroblastoma cells (Fiebich et al., 2000). IL-1b-induced COX-2 was slightly reduced in the presence of Ab 25–35 but not affected by AA or by the combined treatment with Ab plus AA (Fig. 6, compare lanes 1 with 2 and 4). Interestingly, NS-398, the specific inhibitor of COX-2, reduced the amount of COX-2 expression by about 70% while the reduction of COX-2 expression in the presence of indomethacin was approximately 20% (Fig. 6, lanes 3 and 9, respectively). Coincubation of NS-398 with Ab, AA or Ab plus AA did not have significant effects when compared with control COX-2 expression (Fig. 6, lanes 6, 7 and 8, respectively). On the other hand, co-incubation of indomethacin with Ab, AA or Ab plus AA slightly but significantly reduced the IL-1binduced COX-2 expression (Fig. 6, lanes 10, 11 and 12, respectively).
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4. Discussion There is growing interest in understanding the mechanisms mediating the protective effects of NSAIDs on Alzheimer’s disease. In this report we present data indicating that indomethacin completely protects from Ab toxicity, in naive and differentiated human neuroblastoma cells at a concentration range from 0.01 to 10 mM and more efficiently than the specific COX-1 inhibitor SC-560 or COX-2 inhibitor NS-398. Indomethacin is a conventional NSAID affecting both the constitutive COX-1 and the inducible COX-2 enzymes with similar potency in human (Cryer and Feldman, 1998). However indomethacin actions may include mechanisms both dependent and independent of COX pathways. Although from the results of our experiments we can not exclude whether some protective actions of indomethacin occurring via COX/independent inhibition, the doses of indomethacin tested here were in the range at which COX enzymes are inhibited, suggesting that their main neuroprotective effect was probably due to modulation of COXdependent signaling pathways (Turini and DuBois, 2002). Supporting this conclusion, a neuroprotective effect of indomethacin has been found in a rat neuroblastoma cell line by direct scavenging of nitric oxide radicals at much higher doses (Asanuma et al., 2001) and the concentration of indomethacin required to activates peroxisome proliferator activated receptor g was one to two orders of magnitude greater than the concentrations needed to block protagladin synthesis (Lehmann et al., 1997). xAlthough it has been reported that certain NSAIDs are capable of inhibiting cell proliferation of cultured cancer cells (Bayer and Beaven, 1979; Eli et al., 2001), in the present model we did not observe reduction of cell viability by indomethacin at the tested doses of either naive or differentiated MSN cells. Moreover, indomethacin promoted an increase in cell viability compared with control cells in the presence of Ab or AA. On the other hand, the selective COX-2 inhibitor, NS-398, protected MSN cells from AA toxicity but had no effect on cell death induced by Ab. Although COX-2 represents a potential target for NSAIDs action in neurodegenerative mechanisms involving inflammation, it does not seem to play a prominent role in the mechanism related with Ab neurotoxicity in the present model. Because indomethacin is capable of inhibiting both the constitutive COX-1 enzyme and the inducible COX-2 with almost same efficacy, our results may support also the involvement of the COX-1 enzyme in Ab-induced neurotoxicity as previously suggested (Bate et al., 2003). In view that the selective COX-1 inhibitor SC-560 partially protected differentiated MSN cells from Ab 25–35- or 1–42mediated neuronal damage the mechanism of action of indomethacin on cell viability may be considered as based on both COX-1 and COX-2 inhibition. There is substantial evidence indicating that Ab toxicity involves free radical production (Hensley et al., 1994; Mattson
and Goodman, 1995) and, interestingly, the activation of the AA cascade has been implicated (Fagarasan and Efthimiopoulos, 1996). In the present work, we found reduction in cell viability by incubation with AA but in no case did we find a potentiation of Ab toxicity by AA. These results could be due to the used dose of AA and Ab at which their neurotoxic effects rose to higher levels and did not allow observation of the additive effects. NSAIDs exert anti-inflammatory activity by inhibiting the AA metabolism of inflammatory mediators that are involved in causing cell death and are implicated in neurodegenerative diseases (Farooqui et al., 1997). Interestingly, both selective and non-selective COX inhibitors, but not the 5-LOX inhibitor NDGA, protected neurons from AA toxicity indicating that the COX/LOX metabolites may play dual roles regulating cell death and cell survival. Although previous studies indicate that LOX inhibitors reduced the toxicity of Ab (Goodman et al., 1994; Fagarasan and Efthimiopoulos, 1996) we found that NDGA diminished MSN cell survival and increased the toxicity of Ab. Supporting our findings, it has been reported that NDGA induces neuronal death in neuroblastoma cell line SH-SY5Y (Bate et al., 2003) and in carcinoma cells through a mechanism involving lipid peroxidation and depletion of GSH (Tang and Honn, 1997). NDGA inhibits 5-LOX over 12-LOX, 15-LOX and COX, with Ki values of 200 nM, 30 mM, 30 mM and 100 mM, respectively (Salari et al., 1984), and a variety of cellular effects of NDGA have been reported. For example, NDGA blocks the autophosphorylation of the platelet derived growth factor receptor (Domin et al., 1994) and inhibits TNFa receptor and cell respiration in tumoral cell lines (West et al., 2004; Pavani et al., 1994). Interestingly, NDGA also binds to AP-1 and thereby interfere with DNA binding by the transcription complex (Kwon et al., 2001). Thus, it is probably that the observed effects of NDGA on neuroblastoma cells survival results from its multiple actions on 5-LOX, receptor protein kinases and specific transcription factors. As mentioned, COX-2 is the inducible form of the COX enzyme, which is located in microglia as well as in neurons. In MSN cells under basal conditions COX-2 levels were undetectable. In order to compare the effects of indomethacin with the selective COX-2 inhibitor NS-398 on COX-2 levels, we induced its expression with IL-1b as has been previously done in the human neuroblastoma cell line SK-NSH (Fiebich et al., 2000). We showed that the IL-1b-induced COX-2 expression was remarkably reduced by NS-398 but only partially by indomethacin. Currently, this data provide evidence indicating that a specific activity inhibitor of COX2 can also modulate its expression. While AA had no significant effect on IL-1b-induced COX-2 expression, incubation with Ab slightly reduced it. On the other hand, it is interesting that in the presence of Ab, NS-398 did not decrease the overexpressed COX-2 contrary to indomethacin did. Present results support the notion that indomethacin has a poor effect on COX-2 levels in contrast with the
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specific inhibitor NS-398 but in the presence of Ab can potentiate the reduction of COX-2. Taken together, present results demonstrate that Ab toxicity can be reduced more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Ab neurotoxicity. Thus, it is possible that the therapeutic potential of indomethacin could be prominent during the early stages of AD retarding the neurodegenerative process induced by Ab.
Acknowledgments The authors thank O. Mercado-Go´mez and A. ReynaNeyra for assistance with statistical analysis and Isabel Pe´rez-Montfort for the revision of the English manuscript.
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Differential effects of COX inhibitors against b-amyloid-induced neurotoxicity in human neuroblastoma cells P. Ferrera, C. Arias * Departamento de Biologı´a Celular y Fisiologı´a, Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, AP 70-228, 04510 Me´xico, DF, Me´xico Received 19 January 2005; received in revised form 9 June 2005; accepted 13 June 2005 Available online 16 September 2005
Abstract Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer’s disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-b protein (Ab, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Ab and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Ab toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Ab neurotoxicity. # 2005 Elsevier Ltd. All rights reserved. Keywords: Amyloid-b protein; Neurotoxicity; Indomethacin; Human neuroblastoma; COX-2; Arachidonic acid; Neuroprotection
1. Introduction Alzheimer’s disease (AD) has a complex pathogenesis and may involve multiple etiological factors but a central role for b-amyloid protein (Ab) has been supported by genetic, neuropathological and biochemical evidence (Cotman et al., 1992; Hsiao et al., 1996; Holcomb et al., 1998; Walsh et al., 2002). There are several indicators suggesting that inflammatory processes may play a role in AD and it has been hypothesized that anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), may delay the onset or progression of AD. The pharmacological activity of NSAIDs is generally attributed to inhibition of cyclooxygenase (COX), the rate-limiting enzyme in the * Corresponding author. Tel.: +52 55 56223896; fax: +52 55 56223897. E-mail address: [email protected] (C. Arias). 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.06.012
production of prostanoids from arachidonic acid (AA). The two isoforms of COX (COX-1 and COX-2) have been found in neurons (Yamagata et al., 1993). In some clinical trials the use of different NSAIDs on AD have provided conflicting data. While the treatment with the non-selective COX inhibitor indomethacin improved cognitive test in AD patients (Rogers et al., 1993) the selective COX-2 rofecoxib and the non-selective COX inhibitor naproxen failed to slow the progression of the disease (Aisen et al., 2003; Reines et al., 2004) suggesting that different NSAIDs have differential properties modulating metabolic pathways involved in AD pathology. Besides the well-known effects of NSAIDs on COX inhibition it has been found that some NSAIDs can also reduce nitric oxide (NO) production (Du and Li, 1999) and exert protective effects on neuronal apoptosis acting as NO scavengers (Asanuma et al., 2001). Moreover, new results
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indicate that some NSAIDs, like indomethacin might control the activity of g-secretase and, thus reduce the generation of Ab (Weggen et al., 2001). In AD, Ab aggregates have been associated with increased generation of oxygen reactive species (ROS), which leads to lipid, protein and DNA peroxidation, and to activation of transcription factors that are central to an array of neuroinflamatory gene signaling events in the AD brain (Lukiw and Bazan, 2000). It has been suggested that Ab, including its active fragment 25–35, may stimulate ROS production through an extracellular signal-regulated phospholipase A2 cascade (Lehtonen et al., 1996; Paris et al., 2000; Andersen et al., 2003), which leads to an increase in AA released from membrane phospholipids. In turn, the AA released is metabolized via the COX pathways leading to the generation of prostaglandins and by the lipo-oxygenase (LOX) pathways giving rise to leukotrienes. Although much of the mentioned evidence suggests the potential protective role of NSAIDs directly on Ab toxicity in neurons, the differential effects of selective or non-selective NSAIDs acting on COX or LOX enzymes have not been clarified yet. To study these effects, we compared the actions of indomethacin with selective COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxy-phenyl)-3-trifluoromethyl pyrazole (SC-560); the selective COX-2 blocker N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide (NS-398) and with the 5-LOX inhibitor nordihydroguairetic acid (NDGA) on Ab-induced neurotoxicity alone or in combination with AA in differentiated and non-differentiated human neuroblastoma cells.
2. Experimental procedures 2.1. Cell culture Human neuroblastoma cell line MSN (Reynolds et al., 1986) was maintained in RPMI 1640 medium containing nonessential amino acids plus 15% fetal calf serum in 24 multidishes in an atmosphere of 5% CO2/95% O2, at 37 8C and were plated at a density of 1 106 cells per well. After 5 days in culture, neurons were incubated by 24 h with Ab fragment 25–35, 20 mM. When Ab 1–42 (10 mM) was added to cultures, we previously pre-incubated the peptide at 37 8C during 7 days and then cells were exposed to the peptide by 48 h. Different doses of compounds were tested: AA 10, 20 and 100 mM; NS-398 1, 5, 10 and 20 mM indomethacin 10, 50, 100 and 200 mM; SC-560 0.01, 0.1 and 1 mm and the 5-LOX inhibitor NDGA, 10 mM. Higher doses of indomethacin (50, 100 and 200 mM) were still neuroprotective to neuroblastoma cells and 1 mm of SC-560 became toxic to the cells. All drugs were purchased from Sigma Chemical (St. Louis, MO). To avoid the antiproliferative effects of NSAIDs (Eli et al., 2001), in some experiments MSN cells were differentiated into neurons by adding retinoic acid 10 mM plus NGF 50 ng/ml (both from Sigma Chemical, St. Louis, MO) (Encinas et al., 2000) during 7 days. The differentiated
neurons were immunocytochemically stained with the neuron-specific marker MAP2. 2.2. Mitochondrial reducing capacity As a measure of cell viability, the mitochondrial reducing capacity was evaluated 24 h after the onset of drug exposure. The method employed in the present study is commonly used in cultured cell studies to evaluate mitochondrial redox activity through the conversion of MTT tetrazolium salt to formazan crystals by mitochondrial respiratory chain reactions (Mossman, 1983). In brief, MTT was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/ ml and was added to MSN cells 1/10 (v/v) after 24 h of incubation with the different treatments and allowed to incubate for one more hour. Then, the incubation medium was removed and MSN cells were solubilized with isopropyl alcohol (0.8 ml). The absorbance of each sample was quantified at 570 nm using a spectrophotometer (Pharmacia Biotech, Gaithersburg, MD). Results are expressed as percentages of control values. Values are mean + S.E.M. of five to seven independent experiments made in triplicate. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by a Student’s t-test. 2.3. Cell viability In some experiments, cell survival was also assessed by using the membrane permeable dye calcein-AM (C-AM) and the DNA-binding fluorophere ethidium homodimer (EthD-1), according to the Live/Dead kit (Molecular Probes, Eugene, OR, USA). Because calcein-AM is converted to green fluorescence by an intracellular esterase, green staining indicates metabolically intact cells. EthD-1 is an indicator of membrane damage and reveals dead cells in red. The assay was performed following to the manufacturer’s instructions. In brief, MSN cells were grown on coverslips, and after 24 h to drugs exposure they were washed twice, 5 min each, in PBS and exposed to C-AM (2 mM) and HE (4 mM) in PBS for an additional 5 min. Then the cells were washed in PBS and fixed for 30 min with cold paraformaldehyde. Immediately after, cells were observed under an epifluorescent microscope and the number of live and dead cells was counted and results were expressed as a percentage of dead cells relative to the total number of cells counted in five different fields of four independent experiments. 2.4. Western blotting Western immunoblots were used to assess inducible COX-2 expression after stimulation with interleukine-1b (IL-1b). For stimulation experiments, cultured MSN cells were treated with Ab, AA, indomethacin and NS-398 in the same doses as described before. 1 h after exposure to these drugs, 100 U/ml IL-1b (Chemicon International, Temecula, CA) were added and cells were incubated for 24 h. Duplicate
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experiments were performed for each condition and they were repeated three times. At the end of incubation, cells were harvested and processed by immunoblotting. Briefly, 30 mg protein from the lysed MSN cells were loaded in a 5– 10% gradient SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane. They were incubated for 2 h in PBS solution containing 5% albumin. Blots were incubated with a polyclonal antibody against COX-2 (5 ug/mL Chemicon International, Temecula, CA) overnight at 4 8C, then washed three times with PBS (5 min each), incubated with an anti-rabbit IgG conjugated to horseradish peroxidase (1:1000 Vector, Laboratories, Burlingame, CA) and detected by chemiluminescence (ECL kit from Amersham, Arlington Heights, IL, USA) on Kodak X-Omat film. Negative controls consisted in eliminating the primary antibody from the procedure. The intensity of COX-2 bands in the immunoblots was quantified by densitometry using the Scion Image program for Windows 2000 (Scion Corporation, NIH, USA). COX-2 densitometric values were normalized with respect to those obtained with an antibody against a-tubulin (1 mg/mL; Clone DM 1A, Sigma Chemical, St. Louis, MO) used as an internal control.
3. Results 3.1. Neuroprotection by indomethacin from Ab 25–35and AA-induced toxicity Cultured MSN were treated with retinoic acid plus NGF in order to induce neuronal differentiation. The neuron-like
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phenotype was quite obvious 72 h after incubation when the cells produced cytoplasmic extensions. After 6 days, most of the population of the treated MSN neuroblastoma cells presented some neurite extensions (Fig. 1D) that were positive to the microtubule-associated protein 2 (MAP-2) (data not shown). Once this stage of differentiation was achieved, we proceeded to study the effects of Ab 25–35, AA or both, in the absence or presence of indomethacin on mitochondrial reducing capacity and the results were compared with those obtained in non-differentiated naive MSN cells. In naive MSN cells (Fig. 1C), after 24 h incubation with Ab the mitochondrial reducing capacity was inhibited by approximately 15%. To determine whether neuron survival was affected by incubation with the main substrate of COX enzymes, MSN cells were exposed to different AA concentrations. There was a significant difference between the survival of untreated MSN cells and cells exposed to 10 mM AA (25% less of MTT reduction). Higher concentrations of AA (20 or 100 mM) reduced the number of surviving neurons by more than 50% (data not shown). Interestingly, the combined incubation with Ab plus 10 mM AA did not increase the number of cell deaths produced by the treatment with Ab alone (18%, compared to control). The addition of indomethacin to the incubation media reverted the effects of Ab, AA and Ab plus AA on MTT reduction almost to control values (Fig. 1A). Interestingly, in differentiated MSN neuroblastoma cells Ab induced similar toxicity as in naive cells (15%), but the inhibition of MTT reduction induced by AA 10 mM alone or in combination with Abwas more evident (35 and 40%, respectively) and the protective effect of indomethacin incubation was remarkable (Fig. 1B).
Fig. 1. Effects of Ab 25–35 and AA on mitochondrial redox activity in the absence or presence of indomethacin in naive (A) and in differentiated (B) MSN cells. Cells were incubated for 24 h in the presence of different drugs (Ab, 20 mM; AA, 10 mM and indomethacin, 10 mM) and MTT reduction was assessed at the end of incubation. The morphological shape of naive neurons (C) or differentiated neurons with retinoic acid and NGF (D). Data expressed as the percentage of control values are the mean S.E.M. of duplicate determinations of five to seven different experiments: *P < 0.05; **P < 0.005.
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Fig. 2. Viability of MSN cells assessed by calcein AM (green) and ethidium homodimer (red). Living cells were stained green and dead cells were stained red. The amount of surviving cells exposed for 24 h to Ab 25–35 20 mM (B) or AA 10 mM (D) was lower compared with untreated cultures (A). After incubation with indomethacin the amount of surviving cells was significantly higher compared with the amount of surviving cells after Ab (C) or AA (E). The right panel shows the percentage of living neurons from each experimental condition compared with controls. Each bar represents the mean S.E.M. of total number of cells counted in five different fields from four independent experiments: *P < 0.05; **P < 0.005.
To confirm that the inhibition of MTT reduction reflected an increase in neuronal death, cultures were processed with the membrane-permeable dye calcein and with the DNAbinding fluorophere ethidium homodimer, 24 h after the onset of drug exposure. In each case, neuronal cultures were observed through a fluorescence microscope and the percentage of dead cells (red nuclei) in differentiated MSN cells was calculated. After 24 h, Ab 25–35 incubation induced 18% of cell death compared with 40% in the case of AA exposure. Pharmacological treatment with indomethacin prevented the loss of neurons in Ab- or AA-treated cultures, a finding consistent with the results obtained in the MTT assay (Fig. 2). Because inhibition of COX enzymes occurs at nanomolar and micromolar indomethacin concentrations, we explored the dose-response effects of indomethacin on Ab- and AA-induced toxicity. Fig. 3 shows that in cultured MSN cells indomethacin promotes cell viability in the range of 10 nM–10 mM with similar potency, which supports a role of COX inhibition as the main mechanism involved in its protective properties.
Ab 25–35 and totally blocked the AA-induced neuronal death (Fig. 4). To study the role of 5-LOX activity on the toxicity elicited by Ab 25–35, AA or the combination of Ab 25–35 plus AA, MSN cell were treated with NDGA for 24 h. Analysis of cell viability with the MTT assay showed that 5-LOX inhibition did not attenuate the toxicity induced by the mentioned compounds and, in addition, significantly inhibited cell viability by 15% in control neurons by itself. 3.3. Differential effects of COX inhibitors on Ab1–42-induced toxicity In view that the relevant peptide in AD is the 1–42 fragment we studied in parallel the protective effects of
3.2. Effects of NS-398, SC-560 and NDGA on Ab 25–35-induced neurotoxicity To expand the above results we compared the effects of indomethacin with the selective inhibitor of COX-2, NS-398 and the selective COX-1 inhibitor SC-560. In differentiated cultures, NS-398 at a dose of 10 mM protected neurons from the toxicity induced by AA or by the combination of Ab 25– 35 plus AA (Fig. 4). However, contrary to the results obtained with indomethacin, NS-398 did not show a significant effect on the inhibition of MTT reduction induced by the Ab 25–35. Similar to above results SC560 (0.1 mM) slightly reduce the neurotoxicity induced by
Fig. 3. Survival of MSN cells exposed to 20 mM Ab 25–35 (filled squares) or 10 mM AA (filled circles) in the presence of different concentration of indomethacin. Cells treated with indomethacin show an increase in MTT reduction that was clear since the lowest dose of 10 nM. Each point represents the mean S.E.M. of triplicate determinations from three independent experiments.
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Fig. 4. Effects of Ab 25–35 and AA on mitochondrial redox activity in the absence or presence of NS-398, SC-560 and NDGA in MSN cells. Cells were exposed to Ab 25–35 or AA in the presence or absence of COX/LOX inhibitors. NS-398 protected cells from AA toxicity but not from Ab toxicity while SC-560 partially inhibited cell death induced by Ab and totally protected cells from AA-induced cell death. NDGA inhibited the mitochondrial reducing capacity by itself and did not show any protection of Ab or AA effects. The data expressed as the percentage of control values are the mean S.E.M. of duplicate determinations of five to seven different experiments: *P < 0.05; **P < 0.005.
indomethacin, SC-560 and NS-398 on the neurotoxicity induced by this peptide. Differentiated MSN cells exposed by 48 h to Ab 1–42 showed a 75% of viability. Indomethacin was the most potent NSAID to protect MSN cells (100% of protection) while the selective COX-1 SC-560, slightly but statistically significant reverted cell death (90% viability). However co-treatment with the selective COX-2 inhibitor, NS-398 did not restore MTT reduction (Fig. 5).
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Fig. 6. Representative Western blot and densitometric analysis of IL-1binduced COX-2 expression (72 kDa) from MSN cells exposed for 24 h to Ab 25–35, AA and COX-2 inhibitors indomethacin and NS-398. Lane 1: control; lanes 2, 3, 4 and 5: Ab 25–35, 20 mM, NS-398, 10 mM, AA, 10 mm and Ab 25–35 plus AA, respectively; and lanes 6, 7 and 8: Ab 25–35, AA, and Ab 25–35 plus AA, respectively, in the presence of NS-398. Lane 9: indomethacin, 10 mM and lanes 10, 11 and 12: Ab 25–35, AA, and Ab 25– 35 plus AA, respectively, in the presence of indomethacin. Each bar represents the mean S.E.M. of COX-2 expression normalized with tubulin expression (55 kDa) of three independent experiments done in duplicate: *P < 0.05; **P < 0.005.
3.4. Changes in COX-2 expression
Fig. 5. Differential effects of COX inhibitors on Ab 1–42-induced toxicity in differentiated MSN cells. Cell were incubated in the presence of 20 mM Ab 1–42 by 48 h in the absence or presence of 10 mM indomethacin, 0.1 mM SC-560 or 10 mM NS-398. Data expressed as the percentage of control values are the mean S.E.M. of triplicate determinations of three different experiments: *P < 0.05.
To determine whether the different treatments induce changes directly of COX-2 expression, Western blot analyses were performed for each condition. We did not detect COX-2 immunoreactivity under basal conditions, neither in the presence of Ab nor of AA (data not shown). When cultured in the presence of 100 U/ml IL-1b, a band of approximately 70 kDa clearly appeared (Fig. 6, lane 1) consistent with previous reports of the appearance of COX2 immunoreactivity in neuroblastoma cells (Fiebich et al., 2000). IL-1b-induced COX-2 was slightly reduced in the presence of Ab 25–35 but not affected by AA or by the combined treatment with Ab plus AA (Fig. 6, compare lanes 1 with 2 and 4). Interestingly, NS-398, the specific inhibitor of COX-2, reduced the amount of COX-2 expression by about 70% while the reduction of COX-2 expression in the presence of indomethacin was approximately 20% (Fig. 6, lanes 3 and 9, respectively). Coincubation of NS-398 with Ab, AA or Ab plus AA did not have significant effects when compared with control COX-2 expression (Fig. 6, lanes 6, 7 and 8, respectively). On the other hand, co-incubation of indomethacin with Ab, AA or Ab plus AA slightly but significantly reduced the IL-1binduced COX-2 expression (Fig. 6, lanes 10, 11 and 12, respectively).
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4. Discussion There is growing interest in understanding the mechanisms mediating the protective effects of NSAIDs on Alzheimer’s disease. In this report we present data indicating that indomethacin completely protects from Ab toxicity, in naive and differentiated human neuroblastoma cells at a concentration range from 0.01 to 10 mM and more efficiently than the specific COX-1 inhibitor SC-560 or COX-2 inhibitor NS-398. Indomethacin is a conventional NSAID affecting both the constitutive COX-1 and the inducible COX-2 enzymes with similar potency in human (Cryer and Feldman, 1998). However indomethacin actions may include mechanisms both dependent and independent of COX pathways. Although from the results of our experiments we can not exclude whether some protective actions of indomethacin occurring via COX/independent inhibition, the doses of indomethacin tested here were in the range at which COX enzymes are inhibited, suggesting that their main neuroprotective effect was probably due to modulation of COXdependent signaling pathways (Turini and DuBois, 2002). Supporting this conclusion, a neuroprotective effect of indomethacin has been found in a rat neuroblastoma cell line by direct scavenging of nitric oxide radicals at much higher doses (Asanuma et al., 2001) and the concentration of indomethacin required to activates peroxisome proliferator activated receptor g was one to two orders of magnitude greater than the concentrations needed to block protagladin synthesis (Lehmann et al., 1997). xAlthough it has been reported that certain NSAIDs are capable of inhibiting cell proliferation of cultured cancer cells (Bayer and Beaven, 1979; Eli et al., 2001), in the present model we did not observe reduction of cell viability by indomethacin at the tested doses of either naive or differentiated MSN cells. Moreover, indomethacin promoted an increase in cell viability compared with control cells in the presence of Ab or AA. On the other hand, the selective COX-2 inhibitor, NS-398, protected MSN cells from AA toxicity but had no effect on cell death induced by Ab. Although COX-2 represents a potential target for NSAIDs action in neurodegenerative mechanisms involving inflammation, it does not seem to play a prominent role in the mechanism related with Ab neurotoxicity in the present model. Because indomethacin is capable of inhibiting both the constitutive COX-1 enzyme and the inducible COX-2 with almost same efficacy, our results may support also the involvement of the COX-1 enzyme in Ab-induced neurotoxicity as previously suggested (Bate et al., 2003). In view that the selective COX-1 inhibitor SC-560 partially protected differentiated MSN cells from Ab 25–35- or 1–42mediated neuronal damage the mechanism of action of indomethacin on cell viability may be considered as based on both COX-1 and COX-2 inhibition. There is substantial evidence indicating that Ab toxicity involves free radical production (Hensley et al., 1994; Mattson
and Goodman, 1995) and, interestingly, the activation of the AA cascade has been implicated (Fagarasan and Efthimiopoulos, 1996). In the present work, we found reduction in cell viability by incubation with AA but in no case did we find a potentiation of Ab toxicity by AA. These results could be due to the used dose of AA and Ab at which their neurotoxic effects rose to higher levels and did not allow observation of the additive effects. NSAIDs exert anti-inflammatory activity by inhibiting the AA metabolism of inflammatory mediators that are involved in causing cell death and are implicated in neurodegenerative diseases (Farooqui et al., 1997). Interestingly, both selective and non-selective COX inhibitors, but not the 5-LOX inhibitor NDGA, protected neurons from AA toxicity indicating that the COX/LOX metabolites may play dual roles regulating cell death and cell survival. Although previous studies indicate that LOX inhibitors reduced the toxicity of Ab (Goodman et al., 1994; Fagarasan and Efthimiopoulos, 1996) we found that NDGA diminished MSN cell survival and increased the toxicity of Ab. Supporting our findings, it has been reported that NDGA induces neuronal death in neuroblastoma cell line SH-SY5Y (Bate et al., 2003) and in carcinoma cells through a mechanism involving lipid peroxidation and depletion of GSH (Tang and Honn, 1997). NDGA inhibits 5-LOX over 12-LOX, 15-LOX and COX, with Ki values of 200 nM, 30 mM, 30 mM and 100 mM, respectively (Salari et al., 1984), and a variety of cellular effects of NDGA have been reported. For example, NDGA blocks the autophosphorylation of the platelet derived growth factor receptor (Domin et al., 1994) and inhibits TNFa receptor and cell respiration in tumoral cell lines (West et al., 2004; Pavani et al., 1994). Interestingly, NDGA also binds to AP-1 and thereby interfere with DNA binding by the transcription complex (Kwon et al., 2001). Thus, it is probably that the observed effects of NDGA on neuroblastoma cells survival results from its multiple actions on 5-LOX, receptor protein kinases and specific transcription factors. As mentioned, COX-2 is the inducible form of the COX enzyme, which is located in microglia as well as in neurons. In MSN cells under basal conditions COX-2 levels were undetectable. In order to compare the effects of indomethacin with the selective COX-2 inhibitor NS-398 on COX-2 levels, we induced its expression with IL-1b as has been previously done in the human neuroblastoma cell line SK-NSH (Fiebich et al., 2000). We showed that the IL-1b-induced COX-2 expression was remarkably reduced by NS-398 but only partially by indomethacin. Currently, this data provide evidence indicating that a specific activity inhibitor of COX2 can also modulate its expression. While AA had no significant effect on IL-1b-induced COX-2 expression, incubation with Ab slightly reduced it. On the other hand, it is interesting that in the presence of Ab, NS-398 did not decrease the overexpressed COX-2 contrary to indomethacin did. Present results support the notion that indomethacin has a poor effect on COX-2 levels in contrast with the
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specific inhibitor NS-398 but in the presence of Ab can potentiate the reduction of COX-2. Taken together, present results demonstrate that Ab toxicity can be reduced more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Ab neurotoxicity. Thus, it is possible that the therapeutic potential of indomethacin could be prominent during the early stages of AD retarding the neurodegenerative process induced by Ab.
Acknowledgments The authors thank O. Mercado-Go´mez and A. ReynaNeyra for assistance with statistical analysis and Isabel Pe´rez-Montfort for the revision of the English manuscript.
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