The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Abstract

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson's disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.

Introduction

Parkinson's disease (PD) is a progressive neurological disease, which is marked by the extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Both experimental and epidemiological studies revealed a strong correlation between the disease onset and previous exposure to endogenous or exogenous mitochondrial toxins [1–5]. A leading model to study idiopathic PD in animals involves the use of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a precursor to the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), which targets nigrostriatal dopaminergic neurons in the substantia nigra in a pattern precipitating pathogenesis analogous to human PD. MPP+ mediates dopaminergic degeneration by inhibiting electron transport chain activity at complex I [6], where a loss of oxidative metabolism and production of ATP through the electron transport chain can bring about rapid neuronal depolarization and a calcium mediated cascade of cell death [7, 8].

There is little doubt that COX-2 plays a critical role in exacerbating the pathogenesis of PD, as SNc COX-2 protein expression is heightened in both animal models susceptible to MPTP and humans, respectively [9, 10]. Likewise, COX-2 knockout mice are resistant to MPTP toxicity [11] and administration of COX-2 inhibitors such as parecoxib and celecoxib effectively ameliorate MPTP toxicity in rodents [12] a result of down-regulating proinflammatory prostaglandins [13] activated by the JNK/c-Jun signaling pathway [14]. In the current study, we investigate several COX inhibitors for neuroprotection against direct MPP+ toxicity in vitro to evaluate if COX is involved with the direct action of the toxin, and further investigate potential protective effects by piroxicam in vivo against MPTP.

Materials and Methods

Materials

Neuro-2A (N-2A) brain neuroblastoma cells were obtained from American Type Culture Collection (Manassas, VA). Dulbecco's Modified Eagle Medium (DMEM), l-glutamine, fetal bovine serum—heat inactivated (FBS), phosphate buffered saline (PBS) and penicillin/streptomycin were supplied by Fischer Scientific, Mediatech (Pittsburgh, PA, USA). All other chemicals and supplies were purchased from Sigma Chemical (St. Louis, MO, USA).

Cell Culture—N-2A Cells

N-2A cells were chosen for this study since they are originally derived from the brain of an albino mouse, an animal susceptible to MPTP toxicity [15]. Furthermore, N-2A cells exhibit true neuronal morphology, unlike commonly used immortal neuroblastoma cells that exhibit epithelial or fibroblastic characters [16]. N-2A cells also have a high affinity for MPP+ uptake and exhibit a vulnerability to the toxin that exceeds that of other cell lines [19]. Previous data obtained in our lab indicate that N-2A cells are more vulnerable to MPP+ than both rat PC-12 and human SH-SY5Y cell models, when glucose in the medium is reduced [17]. The N-2A cells were grown in DMEM with phenol red, 10% FBS, 4 mM l-glutamine and penicillin/streptomycin (100 Units/0.1 mg/ml). Cells were grown at 37°C in 5% CO₂/atmosphere. Cells were scraped and sub-cultured every 2-5 days. Experimental plating media consisted of DMEM minus phenol red, 1.8% FBS, penicillin/streptomycin (100 Units/0.1 mg/ml) and 4 mM l-glutamine. For experiments, cells were plated in 96 well plates at a density of approximately 1 × 10⁶ cells/ml. A stock solution for each experimental compound was prepared in PBS+ 10 mM (N-[2-hydroxyethylpiperazine-N′-[2-ethanesulfonic acid]) (HEPES) and adjusted to a pH of 7.4. Six dilutions of each experimental compound were prepared to span a 1000-fold experimental dilution range. The solution of MPP+ was prepared fresh daily.

Somatic Cell ATP

The levels of adenosine—5′-triphosphate (ATP) was measured using the firefly bioluminescence technique. Briefly, a lyses buffer containing 4 mM dl-dithiothreitol (DTT), 2 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris-HCL, 5 μg/ml apoprotin, pepstatin A, and 200 μM phenylmethylsulfonyl fluoride (PMSF) was prepared in sterile PBS (pH = 7.4). The lysis buffer was added directly to the cell suspension and samples were frozen immediately and stored at −80°C. A luciferase-luciferin solution in glycine buffer (Sigma Chemical, MO, USA) was reconstituted as instructed with sterile deionized water. The final concentration of ATP reagent contained 0.04 mg/ml luciferase, 0.26 mM luciferin, 19.5 mM MgSO₄, 1.95 mM EDTA, 195 mM glycine and 324 mM Tris (hydroxymethyl) aminomethane hydrochloride (pH = 7.4). The cell lysate was thawed slowly, and diluted in serum free plating medium (v/v) 1:2. The ATP reagent was added at 1.28% of the final sample volume. Light was quantified on a Beckman LS 6500 Scintillation Counter (Beckman, Schaumburg, IL, USA). Settings were set at: Isotope = Manual, Count = CPM, Background = None, Time = 20 s, Quench = off with no chemiluminescence adjustment. A standard curve was generated by dilutions of ATP in serum free plating medium and Light Units were plotted as CPM versus ATP concentration. The data were compared to untreated cells exposed to phosphate buffer solution (% live control).

O₂ Consumption—Clark Electrode

The Oxygraph respirometer (Hansatech Instruments Ltd, Norfolk, England) was used to measure oxygen uptake as an indicator of cellular and mitochondrial respiration. Data from the electrode are plotted in a real time display chart-recorder. The electrode was calibrated with both air saturated deionized water and deionized water with sodium dithionite. Briefly, 400 μl of each sample was loaded into the chamber. After rate equilibration, a 50 s reading was taken for each sample. Data are plotted as nM of O₂ consumed/ml and is converted to % control.

Cell Viability

Cell viability was assessed by resazurin oxidoreduction indicator dye [17]. A working solution of resazurin was prepared in PBS minus phenol red (0.5 mg/ml). Reduction of the dye by viable cells reduces the amount of oxidized form and increases the amount of its bright red fluorescent intermediate. The dye solution was added to an amount equal to 15% of the original culture medium volume. Cultures were returned to the incubator for 6-8 h. Quantitative analysis of dye conversion was measured on a microplate fluorometer—Model 7620-version 5.02 (Cambridge Technologies Inc, Watertown, MA, USA) set at 550/580 (excitation/emission). There was no washout period before cell viability was measured. The data were expressed as % live control.

Animals

C57/B6 male mice (8 weeks old) were acclimated to the housing facility for 5 days, where they were given free access to water and standard laboratory mouse chow and exposed to a 12-h light/dark standard cycle. The study commenced on the 6th day in which piroxicam (20 mg/kg) or vehicle (0.9% saline) was administered to the mice for three consecutive days prior to administration of MPTP/or saline and for the duration of the study. On the 10th day, MPTP was administered intraperitoneal (i.p.) (40 mg/kg) or an equivalent volume of saline (control). Seven days after the last MPTP injection, the mice were anesthesized with sodium pentobarbital 50 mg/kg and sacrificed by decapitation.

Western Blot

Western blot was preformed as to the protocol by Yan et al. with minor modifications [18]. The substantia nigra was dissected, washed in PBS and immediately placed at −80°C in lysis buffer. The lysis buffer consisted of 5% glycerol, 1 mM sucrose, 200 μM phenylmethylsulfonyl fluoride, 10 mM [Tris (hydroxymethyl) aminomethane hydrochloride] (Tris), 5 μg/ml pepstatin A, 1 mM EDTA, 10 μg/ml apoprotin, 10 μg/ml leupeptin, 2 mM-dithiothreitol, 3 mM urea prepared in 18 MΩ water. The samples were stored at −80°C for 24 h and lysed by freeze-thaw and cell membranes were destroyed using a sonic Dismembrator (Fischer Scientific, Model 550). Samples were placed in Laemmli sample buffer containing 3% mercaptoethanol and boiled for 5 min and re-centrifuged at 13,000 × g for 5 min. The supernatant was removed for Western blot and protein analysis. Proteins were separated on a 4–15% SDS-polyacrylamide gel using the buffer system of Laemmli and transferred to nitrocellulose at 100 V for 1 h in Towbin-SDS transfer buffer containing 25 mM Tris, 192 mM glycine and 20% methanol. After transfer, the blot was washed once with PBS containing 0.05% Tween 20 (TTBS). Briefly, the blot was thoroughly dried and placed in blocking buffer 1% BSA in TTBS for 1 h at room temperature. The primary antibody used was a monoclonal antibody, which “recognizes an epitope present in the N-terminal region between 40 and 152 of both rodent and human tyrosine hydroxylase. TH-16 detects both the intact subunits and the 59/57 kDa doublet and an array of decreasing molecular weight TH forms resulting from severe proteolysis” (Sigma Scientific). The primary antibody was diluted 1:4000 in a diluent buffer containing 1% BSA (Fraction V) in TTBS and 0.2% sodium azide along with mouse anti-β-actin monoclonal antibody (1:5,000) and incubated at 4°C for 24 h. After washing, the samples were placed in 1% BSA (Fraction V) in TTBS containing anti-mouse IgG (Fc specific) peroxidase conjugate (1:4000) and incubated for 1 h at room temperature. After a final wash, peroxidase was detected with Sigma FAST^TM DAB (3,3′-diaminobenzidine tetrahydrochloride) with a metal enhancer cobalt chloride. Gels were scanned and analyzed with Image J, 1.34S, National Institute of Health, USA for band intensity.

HPLC Analysis of Catecholamines

Briefly, 7 days after the last MPTP injections, animals were sacrificed and the SNc tissues were placed immediately at −80°C. Each tissue was sonicated in 0.1 M perchloric acid, centrifuged/filtered at 10,000 × g for 5 min through a 0.45-lm membrane. Quantification of DA was determined by HPLC using EZChrom Analytical Software (version 6.5) (Scientific Software, San Ramon CA, USA) and an ESA Coulochem analytical ECD detector [settings: E400 mV, CH1: −40 mV 100 μA CH₂: 400 mv 5 μA] using a Waters 117 Autosampler Plus and 510 HPLC pump (Waters Corporation, Milford, MA USA). The mobile phase consisted of 90 mM sodium phosphate dibasic, 50 mM citric acid, 0.05 mM EDTA, 1.7 mM of octanesulfonic acid, 10% methanol and adjusted to a pH of 3.0. The column was a C18 reverse phase ESA HR-80 with 3 μm particle size and dimensions of 8 cm × 4.6 mm, the flow rate was set at 1.5 ml/min and standards and samples were prepared in 0.1 M perchloric acid where the injection volume was set at 15 μl per sample.

Evaluation of Locomotor Activity

Locomotive function was analyzed using a Mini-Counter at 24 after MPTP administration (Columbus Instruments, Columbus, OH, USA). The apparatus quantifies count information from beam interruptions detecting ambulatory activity as horizontal movement and vertical motion (raised head motions or rearing). Locomotor activity was monitored 24 h after drug treatments every 10 s over 90 min testing duration.

Data Analyses

Statistical analysis was performed using Graphpad Prism—version 3.0, (Graphpad Software Inc.), San Diego, CA, USA. Data were expressed as the mean ± SEM for each group. Significance of difference between the groups was assessed using a one-way ANOVA followed by a Tukey post-hoc means comparison test.

Results

N-2A cells were incubated for 24 h in the presence of several experimental COX inhibitors + MPP+ (500 μM) and there was no washout period before cell death was measured. As it appears from the toxicity data tabulated in Table 1, there were no protective effects observed in the presence of curcumin (from tumeric), ibuprofen or acetylsalicylic acid. In contrast, piroxicam was effective in providing protection against MPP+ toxicity, with the highest level of protection at 5 mM (from 15 ± 2% to 89 ± 4.3%, P < 0.001). To confirm that the protective effects of piroxicam were related to overcoming complex I inhibition, toxicity experiments were preformed in the presence of 50 μM rotenone where piroxicam was also found effective in attenuating toxicity (Fig. 1). The results indicate that piroxicam treatment partly restored ATP, with no reversible effects to the insult of the mitochondria as measured by oxygen respiration (Table 2). Treatment with MPP+ (500 μM) resulted in a dramatic loss of ATP (1.1% of live controls, P < 0.001) where piroxicam was effective in restoring only ATP concentrations during MPP+ toxicity, with the highest level of protection at 5 mM (20.6 ± 1.3% of live controls, P < 0.001).

Table 1.

Effects of COX inhibitors on MPP+ (500 μM) Toxicity in Neuroblastoma Cells at 24 h at 37°C

COX inhibitors	Dose response(concentration range/% viability)
(From Tumeric) Curcumin (mM)	Live-Ctrl	MPP+ -Ctrl	0.005	0.01	0.05	0.5	2.5	5
	100.0 ± 1.1	16 ± 1.4	17 ± 1.9	14 ± 1.5	13 ± 1.7	13 ± 1.0	17 ± 1.2	15 ± 2.1
Ibuprofin (mM)	Live-Ctrl	MPP+ -Ctrl	0.005	0.01	0.05	0.5	2.5	5
	100.0 ± 1.6	8 ± 1.8	7 ± 2.7	8. ± 2.4	6 ± 2.5	8 ± 2.2	10 ± 1.4	3 ± 1.4
Acetylsalicylic acid (mM)	Live-Ctrl	MPP+ -Ctrl	0.001	0.002	0.01	0.1	0.5	1
	100.0 ± 1.2	15 ± 3.2	16 ± 4.1	19 ± 2.5	17 ± 1.3	16 ± 2.4	16 ± 3.4	18 ± 4.9
*Piroxicam (mM)	Live-Ctrl	MPP+-Ctrl	0.005	0.01	0.05	0.5	2.5	5
	100.0 ± 1.9	15 ± 2.0	13 ± 1.0	12 ± 1.3	14 ± 0.3	16 ± 1.9	60 ± 6.5*	89 ± 3.4*

Cell viability was quantified using resazurin dye conversion. N-2A cells were plated with MPP+ (500 μM) plus a variation in experimental compounds. The controls viability data were monitored with almar blue, which gave a reading of 449 ± 10.4 fluorescence intensity at excitation/emission—550/580. This was equivalent to 100 ± 2.34%, which was set as the untreated controls. Data are expressed as the mean ± SEM (n = 4). Significance of difference between treatment and MPP+ treated control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test.

^*P < 0.001

Fig. 1.

Protective effects of piroxicam against MPP+ and rotenone toxicity in neuroblastoma cells at 24 h at 37°C. Cell viability was determined using resazurin dye conversion. Cells were plated ± MPP+ (500 μM) or rotenone (50 μm) plus a variation in piroxicam concentration. Data are expressed as the mean ± SEM, (n = 4). Significance of difference between treatment and MPP treated control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test. *P < 0.001 for MPP+ toxicity trial, ⁺P < 0.001 for rotenone toxicity trial

Table 2.

O₂ consumption and ATP levels during cytoprotection by piroxicam on MPP+ (500 μM) toxicity in neuroblastoma

Piroxicam (mM)	Dose response (Concentration range/% live control)
	Live-Ctrl	MPP+-Ctrl	0.005 mM	0.01 mM	0.05 mM	0.5 mM	2.5 mM	5 mM
ATP	100 ± 2.9	1.1 ± 0.3	1.5 ± 0.2	2.6 ± 0.5	2.4 ± 0.6	3.0 ± 0.7	14.2 ± 4.1*	20.6 ± 1.3*
Oxygen Consumption	100 ± 0.1	4.5 ± 0.1	4.6 ± 0.1	4.4 ± 0.2	5.6 ± 0.2	4.6 ± 0.1	4.2 ± 0.1	4.4 ± 0.2

Oxygen consumption and ATP was determined. Cells were plated with MPP+ (500 μM) plus a variation in experimental compounds. The oxygen used by the control group was equivalent to 74.6 ± 0.09 nM/ml of dissolved oxygen compared to the media blank control. MPP+ blocked oxygen consumption The ATP was detected using a scintillation counter with the methods described for light intensity, where the control group (untreated cells) gave a reading of 647.5 ± 16.01, which was equivalent to 100 ± 1.4%. Data are expressed as the mean ± SEM (% of MPP+ treated control), (n = 4). Significance of difference between treatment and control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test.

^*P < 0.001

In vivo, the protective effects of piroxicam against MPTP administration (40 mg/kg) in mice were evidenced by reversal of attenuated locomotive function (Fig. 2), SNc dopamine concentration (Fig. 3) and SNc tyrosine hydroxylase protein expression (Fig. 4). These findings corroborate other findings in that COX-2 inhibitors play a protective role against the degenerative pathogenesis involving mitochondrial insult to the substantia nigra and striatal tract.

Fig. 2.

The protective effects of Piroxicam on locomotive activity in MPTP treated mice. The data represent locomotive activity-combined vertical and horizontal tested over 90 min. The data are expressed as the mean ± SEM (n = 4). Significance of difference between treatment and control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test. *P < 0.001

Fig. 3.

The effects of Piroxicam on SNc DA concentration in MPTP treated mice. SNc dopamine concentration in MPTP treated mice in the absence or presence of piroxicam. The data represent DA (ng/mg tissue) and are expressed as the mean ± SEM (n = 4). Significance of difference between treatment and control was determined by an one-way ANOVA, followed by a Tukey mean comparison post hoc test. **P < 0.001, *P < 0.05

Fig. 4.

The effects of piroxicam on SNc tyrosine hydroxylase immunoreactivity in MPTP treated mice. SNc tyrosine hydroxylase protein expression in MPTP treated mice in the absence or presence of piroxicam. The data represent band intensity relative to β-actin controls and are expressed as the mean ± SEM (n = 4). Significance of difference between treatment and control was determined by a oneway ANOVA, followed by a Tukey mean comparison post hoc test. *P < 0.001

Discussion

The obtained data indicate that N2-A, the cells used in this study, are more resistant to the toxic effects of MPP+ with the addition of piroxicam. MPP+ blocked mitochondrial O₂ consumption, which was not reversed in the presence of piroxicam. The data indicate that, piroxicam partially reversed the loss of cell viability and the loss of ATP. While somatic ATP levels were not fully restored to normal level in the presence of piroxicam, there appeared to be sufficient cellular energy to partially maintain cell viability. Determining a role for piroxicam in MPP+ toxicity is complex and there is very little research on the effect of cyclooxygenase inhibitors and MPTP toxicity. It appears from the current study that maintaining ATP through anaerobic conditions is critical event in preventing cellular death with MPP+ [17]. Meanwhile, it is known that exposure to the mitochondrial toxins MPP+ can lead to rapid depletion of ATP [19].

The findings in this study show a significant neuroprotective effect by piroxicam against MPP+ toxicity in vitro, with no observed restorative changes in mitochondrial O₂ consumption parallel to a rise in ATP and viability. These data suggest that neuroprotective effects of this particular compound may involve sustaining anaerobic cell function after shut down of oxidative metabolism, an effect unique to piroxicam. Recently, research in our lab has delineated a very critical role for upregulating glycolysis, in providing resistance to MPP+ mediated injury [17]. The upregulation of ATP production under mitochondrial shutdown in the presence of MPP+ would aid in preventing energy loss, which could provide explanation to the observed protection against MPP+ by piroxicam. Piroxicam is known to possess potentiating influence on carbohydrate metabolism [20] where it can stimulate glycolysis and glycogenolysis and enhance carbohydrate utilization. Upregulation of glycolysis under mitochondrial shutdown in the presence of MPP+ would aid in preventing ATP loss and could provide protection against ergogenic insult [17]. This could provide explanation to the observed protection against MPP+ by piroxicam in neuroblastoma in which glial cells are absent. Moreover, in the absence of glial cells, neuronal COX-2 would not likely to cause cell death since neurons releasing PGE2, activate glial cell inflammation which then initiates reciprocal damage to surrounding neurons [14].

In the current work, we investigated the neuroprotective properties of several COX inhibitors against MPP+ in N-2A cells in vitro The data obtained demonstrate a lack of protective effects observed by COX 1–2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, whereas, piroxicam was protective in a dose dependent manner. This effective neuroprotection of piroxicam in vitro, lead us to set an experiment to examine the action of piroxicam in vivo. The presence of piroxicam protection in mice treated with MPTP might help in establishing a great therapeutic potential for PD.

The in vivo data obtained demonstrate that piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the SNc and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. Piroxicam has previous relevant use as a general anti-inflammatory agent used for treatment of arthritis, fever and general pain [20]. It is known that COX-2 plays some role in PD pathology through invoking heightened production of pro-inflammatory prostaglandins. Both COX-1 (acetylsalicylic acid) and COX-2 (meroxicam) inhibitors are effective in attenuating MPTP-induced striatal dopamine depletion, locomotor activity, cell loss, and tyrosine hydroxylase immunoreactivity (TH-IR) in the SNc [20]. As noted earlier, some studies show no protection afforded by paracetamol, diclofenac, ibuprofen, indomethacin or dexamethasone, questioning a specific role for cyclooxygenase [21] both COX-1 (acetylsalicylic acid) and COX-2 (meroxicam) inhibitors can attenuate MPTP-induced striatal dopamine depletion, locomotor activity, cell loss, and TH-IR in the SNc [21]. Interestingly, another research study corroborates that systemic administration of salicylate and aspirin were protective against MPTP toxicity in vivo. However, these authors found no protection offered by paracetamol, diclofenac, ibuprofen, or indomethacin, questioning a specific role for cyclooxygenase [22].

It was concluded from this study that piroxicam appear to offer protection against MPP+ through upregulating anaerobic energy survival mechanisms, and this effect is independent of COX activity. The findings in this study demonstrate that piroxicam can be used as an effective agent to prevent MPTP-induced dopaminergic neuronal damage in experimental models of PD.