A₃ adenosine receptor agonist prevents the development of paclitaxel-induced neuropathic pain by modulating spinal glial-restricted redox-dependent signaling pathways

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain is the major dose-limiting toxicity of several anticancer agents including the taxane paclitaxel (Taxol^®). A critical mechanism underlying paclitaxel-induced neuropathic pain is the increased production of peroxynitrite (PN) in spinal cord generated in response to activation of the superoxide-generating enzyme, NADPH oxidase. PN in turn contributes to the development of neuropathic pain by modulating several redox-dependent events in spinal cord. We recently reported that activation of the G_i/G_q-coupled A₃ adenosine receptor (A₃AR) with selective A₃AR agonists (i.e., IB-MECA) blocked the development of chemotherapy induced-neuropathic pain evoked by distinct agents, including paclitaxel, without interfering with anticancer effects. The mechanism(s) of action underlying these beneficial effects has yet to be explored. We now demonstrate that IB-MECA attenuates the development of paclitaxel-induced neuropathic pain by inhibiting the activation of spinal NADPH oxidase and two downstream redox-dependent systems. The first relies on inhibition of the redox-sensitive transcription factor (NFκB) and mitogen activated protein kinases (ERK and p38) resulting in a decreased production of neuroexcitatory/pro-inflammatory cytokines (TNF-α, IL-1β) and increased formation of the neuroprotective/anti-inflammatory IL-10. The second involves inhibition of redox-mediated posttranslational tyrosine nitration and modification (inactivation) of glia-restricted proteins known to play key roles in regulating synaptic glutamate homeostasis: the glutamate transporter GLT-1 and glutamine synthetase. Our results unravel a mechanistic link into biomolecular signaling pathways employed by A₃AR activation in neuropathic pain while providing the foundation to consider use of A₃AR agonists as therapeutic agents in CIPN patients.

Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain represents the most common dose-limiting complication associated with several first-line chemotherapeutics [12] including the taxane, paclitaxel (Taxol^®) used for breast, ovarian, non-small cell lung carcinomas, and Kaposi’s sarcoma. This chronic neuropathy can persist for years after treatment [55] diminishing quality-of-life [12] and restricting optimal chemotherapeutic dosages. Clinical management becomes problematic as the causative mechanisms are poorly understood and current pain drugs are only marginally effective with unacceptable side effects [12]. Identification of novel therapeutics as adjuncts to chemotherapeutics to minimize side-effects and maximize anticancer effects is urgently needed.

We recently identified that highly-specific A₃ adenosine receptor (A₃AR) agonism is a novel and viable therapeutic strategy for CIPN [7]. Adenosine exerts its effects via four G protein-coupled receptor subtypes: A₁AR and A₃AR couple to G_i/G_q and A_2AAR and A_2BAR to G_s/olf/o [17]. Selective A₃AR agonists, like IB-MECA or its 2-chloro analogue, Cl-IB-MECA, block neuropathic pain caused by diverse chemotherapeutics including paclitaxel, oxaliplatin, and bortezomib without interfering with anticancer effects [7]. Noteworthy, A₃AR agonists have advanced to clinical trials for cancer and autoimmune conditions displaying promising beneficial effects and a good safety profile [17]. The beneficial mechanism(s) underlying A₃AR agonism remain unexplored.

A₃AR is expressed in endothelial cells, inflammatory cells, glial cells, and neurons within the periphery and central nervous system (CNS) [23]. Pathways proposed to mediate A₃AR’s actions include inhibition of redox-sensitive NFκB, modulation of glycogen synthase kinase (GSK) 3β, attenuation of TNF-α/IL-1β, and increased formation of the anti-inflammatory IL-10 [21]. A₃AR agonists are also neuroprotective [17,23]. Neuroprotection may occur by activating pro-survival RhoA-phospholipase D (PLD) signaling pathways. In cardiomyocytes, PLD activity is decreased in response to prolonged reactive oxygen species production during apoptosis [2]; an A₃AR agonist can prevent this [30]. In breast cancer cells, increased PLD-mTOR activity and corresponding decreases in GSK3β generate pro-survival signaling [6]. Activating PLD can also increase production of choline, which activates α7 nicotinic acetylcholine receptors [29] known to be antinociceptive in chronic neuropathic pain [13]. A₃AR agonists additionally stimulate glial-production of neuroprotective substances like CCL2 [58] and inhibit glial-derived pro-inflammatory cytokines [33]. A₃AR activation protects against the neurotoxic P2X7-mediated [62] or the glutamate and NMDA-mediated rise in Ca²⁺ and thus neuronal excitability of neurons in vitro [61], suggesting that A₃AR impacts glutamatergic signaling. While the underlying mechanisms of CIPN are multifactorial and include changes in the periphery [5], prominent neuropathological CNS changes have been implicated in the dysregulation of spinal neuro-glia communication brought about by neuroinflammatory processes [10,18,25]. For example, activation of NFκB and MAPKs (ERK, p38) [18,25] and overt production of pro-inflammatory cytokines (TNF-α, IL-1β) [10,18,25] have been reported.

In a first attempt to define potential mechanisms underlying A₃AR’s protective actions in CIPN, we examined whether these effects are exerted via attenuation of spinal neuroinflammatory processes known to contribute to alterations in neuro-glia communication.

Methods

Experimental animals

Male Sprague Dawley rats (200–220 g starting weight) from Harlan Laboratories (Indianapolis, IN; Frederick, MD breeding colony) were housed 3–4 per cage in a controlled environment (12 h light/dark cycle) with food and water available ad libitum. All experiments were performed in accordance with the International Association for the Study of Pain and the National Institutes of Health guidelines on laboratory animal welfare and the recommendations by Saint Louis University Institutional Animal Care and Use Committee (IACUC). Animal use at the University of Messina likewise complied with Italian regulations for the protection of animals used for experimental and other scientific purpose (D.M. 116192) and with European Economic Community regulations. All experiments were conducted with the experimenters blinded to treatment conditions.

Test Compounds

In prophylactic experiments performed for biochemical analysis, all test compounds were given 15–20 min prior to chemotherapeutic (D0, D2, D4, and D6) and then daily thereafter up to D16. In the shortened prophylactic dosing regimen (concomitant), IB-MECA or its vehicle was given 15–20 minutes prior to paclitaxel only on the same days as paclitaxel (D0, 2, 4, 6). MRS1523 was given 15–20 minutes prior to IB-MECA. IB-MECA (1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide) was purchased from Tocris Bioscience (Bristol, United Kingdom). MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate) was obtained from Sigma-Aldrich (St. Louis, MO).

Paclitaxel-induced neuropathic pain

Rats were treated with paclitaxel (Parenta Pharmaceuticals; Yardley, PA) or its vehicle as previously described [10,25]. In brief, doses of pharmaceutical-grade paclitaxel were prepared from original stock (6 mg/ml in Cremophor EL and 95% dehydrated ethanol in 1:1 ratio, Sigma) by diluting in sterile saline. Paclitaxel (2 mg/kg in 0.2 ml) was delivered by intraperitoneal (i.p.) injection alternating days for four days (D): D0, D2, D4 and D6 with a final cumulative dose of 8 mg/kg [45]. Control animals received an equivalent volume of the vehicle with proportional amounts of Cremophor EL and 95% dehydrated ethanol diluted in saline. This paclitaxel dosing regimen produces a time-dependent development of mechano-allodynia and mechano-hyperalgesia (mechano-hypersensitivity) evidenced by a decrease in paw withdrawal thresholds (PWTs (g)) by D12 (onset) that peaked by D16, and plateaued throughout our observation period (D25; times relative to first injection) [10,25]. We saw no evidence for the development of thermal hypersensitivity. The delay between the last exposure to paclitaxel and onset of mechano-hypersensitivity mimics the clinical “coasting” phenomenon described in patients [12]. In this study, all measurements were done at time of peak pain behaviors, D16 or D25.

Behavioral testing

Behavioral measurements were always taken prior to the administration of test substances. Mechano-allodynia was measured as previously described [10,25]. Briefly, rats were allowed to acclimate to behavioral chambers for 15 min prior to measuring the mechanical paw withdrawal thresholds, grams [PWT, (g)] by manual von Frey filaments (Stoelting, Wood Dale, IL, ranging from 3.61 [0.407 g] to 5.46 [26 g] bending force) [9] or electronic Von Frey test (dynamic plantar aesthesiometer, model 37450; Ugo Basile, Italy) with a cut off set at 50 g. Mechano-allodynia was defined by a significant (P<0.05) reduction in mechanical mean absolute PWT (g) at forces that failed to elicit withdrawal responses before chemotherapy treatment (D0). Mechano-hyperalgesia was measured by stimulating the dorsum of the rat’s hind paw as previously described [10,25] by the Randall and Sellitto paw pressure test [47] using a Ugo-Basile analgesiometer (Italy. model 37215). The nociceptive paw withdrawal threshold [PWT, (g)] was defined as the force (g) which caused the rat withdrew its paw (cut off set at 250 g). Since, chemotherapy-induced neuropathy results in bilateral allodynia and hyperalgesia with no differences between left and right hind PWT (g), the values from both paws were averaged. Animals receiving chemotherapeutic agents in the presence or absence of the experimental test substances tested did not display signs of any toxicities, as previously described [10,25].

Tissue Collection

At a time point of peak mechano-hypersensitivity (D16 or D25), animals were terminally anesthetized and decapitated for tissue collection. Immediately afterwards, the lumbar enlargement portion of the spinal cord (L5–L6) was harvested, flash frozen in liquid nitrogen, and stored at -80 °C until the appropriate biochemical assay could be performed.

Measurement of NADPH oxidase activity

Spinal cord tissues (L4–L6) were homogenized in HEPES buffer [10 mM, pH 7.5, containing 250 mM sucrose, 1 mM EGTA, 25 mM potassium chloride, 10 μg/ml soybean trypsin inhibitor, 2 μg/ml aprotinin and 10 μg/ml leupeptin] and centrifuged at 1,000 × g to obtain the nuclear-free supernatants. The NADPH cytochrome c reductase activity in these supernatants was measured using Cytochrome c Reductase (NADPH) Assay Kit as previously described [10].

Western blot analysis for transcription factors

The levels of IκBα, phospho-NFκB p65 (serine 536), phospho-ERK1/2, ERK1/2, and phospho-p38 MAP kinase were quantified in cytosolic fractions from spinal cord tissue, while NFκB p65 levels were quantified in nuclear fractions prepared as previously described [43]. All lysates were stored at -80°C until further analysis. Western blot analysis was performed as previously described [43] using specific antibodies to IκBα, NFκB p65, ERK2, phospho-ERK1/2 (1:1000; Santa Cruz Biotechnology; Dallas, TX), phospho-p38 MAP kinase (threonine180/tyrosine182), phospho-NFκB p65 (serine 536) (1:1000; Cell Signaling Technology; Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (1:2000; Jackson ImmunoResearch Labratories; West Grove, PA) and enhanced chemiluminescence system (Amersham Biosciences; Amersham, UK). For loading controls, membranes were stripped and probed with antibodies against β-actin (1:10,000 Sigma-Aldrich) for whole lysates or cytosolic fractions or laminin-B1 protein (1:10,000, Sigma-Aldrich) for nuclear lysates. The relative expression of the protein bands were quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories; Milan, Italy) and Molecular Analyst software (IBM).

Cytokine Assay

The levels of cytokines in spinal cord (L4–6) cytosolic fraction isolated for Western Blot were assessed using by commercially available ELISA kits (R&D Systems; Milano, Italy) as previously described [10,25] or by custom-ordered magnetic multiplex cytokine kit (Bio-Rad Laboratories; Hercules, CA) according to manufacturer’s protocol.

Measurement of GLT-1 and GS

Cytosolic fractions and P2 membranes were obtained as previously described [38,39] and stored immediately at -80°C. Immunoprecipitation and Western blot analyses were performed as described previously [38,39]. Proteins were resolved with 7.5% (GLT-1), or 10% (GS) SDS-PAGE and electrophoretically transferred to PVDF membrane. The membranes were probed with mouse monoclonal anti-glutamine synthetase (1:2000) or polyclonal rabbit anti-GLT-1 (1:1000). Membranes were visualized with horseradish peroxidase-conjugated secondary antibodies (1 h, RT) and enhanced chemiluminescence. Rat brain lysate containing our proteins of interest was used as positive control. The blots were stripped and probed with a murine monoclonal anti-β-actin antibody (1:2000). The relative density of the protein bands of interest was determined from film using ImageQuant 5.2 software (Molecular Dynamics) and normalized to β-actin bands.

Statistical Analysis

Data are expressed as mean ± SD for n animals. Data from the time course studies were analyzed by two-way repeated measures ANOVA with Bonferroni comparisons. All other biochemical data collected were analyzed by one-way ANOVA with Dunnett’s-corrected comparisons. Significance was defined as P<0.05. All statistical analyses were performed using GraphPad Prism (v5.03, GraphPad Software, Inc.; San Diego, CA).

Results

Spinal activation of NADPH oxidase in response to paclitaxel is blocked by IB-MECA

We recently reported that the development of paclitaxel-induced neuropathic pain is dependent upon activation of NADPH oxidase which provides superoxide, the key precursor in the biosynthesis of peroxynitrite; blocking spinal NADPH oxidase blocked the formation of peroxynitrite and the development of paclitaxel-induced neuropathic pain [10]. We now demonstrate that on D16, a time of peak mechano-hypersensitivity (mechano-allodynia and –hyperalgesia; Fig. 1A,B; n=6), activation of NADPH oxidase and thus superoxide production in the spinal cord (Fig. 1C; n=6) was blocked by prophylactic treatment with IB-MECA (0.1 mg/kg/d; given i.p. from D0 to D15) (Fig. 1C; n=6); this was associated with significant attenuation of mechano-allodynia (Fig. 1A; n=6) and mechano-hyperalgesia (Fig. 1B; n=6). These beneficial events are dependent on selective A₃AR-mediated mechanisms since pretreatment with 2 mg/kg/d (n=6) [7] of the selective A₃AR antagonist, MRS1523, blocked the effects of IB-MECA on NADPH oxidase activity (Fig. 1C; n=6) and mechano-hypersensitivity (Fig. 1A,B; n=6). We also examined whether similar protection would be afforded by restricting IB-MECA treatment to when paclitaxel was given; a preferred adjunct regimen for treating patients only when they receive the chemotherapeutic agent. IB-MECA (0.1 mg/kg; n=6) given at the same time as paclitaxel (D0, 2, 4, and 6) blocked neuropathic pain through D25 (not shown); inhibitory effects at peak pain displayed a 98% and 98.7% inhibition of mechano-allodynia and mechano–hyperalgesia, respectively (n=6, P<0.001).

Fig. 1. IB-MECA prevents paclitaxel-induced mechano-hypersensitivity and spinal NADPH oxidase activity.

As compared to baseline on D16, administration of paclitaxel (P, black bars) but not vehicle (V, open bars) leads to the development of mechano-allodynia (A) and mechano–hyperalgesia (B). Spinal cords from paclitaxel-treated animals displayed higher levels of NADPH oxidase activity than their vehicle-treated counterparts (C). These paclitaxel-induced events were blocked by daily administration of the A₃AR agonist IB-MECA (D0–15, 0.1 mg/kg/d; dark gray bars, A–C). The selective A₃AR antagonist MRS1523 (2 mg/kg/d, D0–15, dark gray hatched bars) prevented IB-MECA’s beneficial actions (A–C). MRS1523 (white hatched bars) administered on its own to paclitaxel animals had no effect on PWTs (A,B). Results are expressed as mean ± SD for n=5–6 rats and analyzed by one-way ANOVA with Dunnett’s comparisons. *P<0.05 vs. Vehicle; †P<0.05 vs. Paclitaxel

IB-MECA prevents activation of NFκB and MAPK pathways and the production of TNF-α and IL-1β

The development of paclitaxel-induced neuropathic pain is dependent on increased production of peroxynitrite in the spinal cord that activates a series of redox-dependent signal transduction pathways, such as NFκB and MAPKs (ERK, p38) [25] and ultimately leading to the overt production of glia-dependent pro-inflammatory cytokines [10,25]. We now demonstrate that IB-MECA impinges upon these peroxynitrite-driven events. Consistent with our previous findings [25], administration of paclitaxel increased NFκB signaling in lumbar spinal cord indicated by IκBα degradation (Fig. 2A; n=5), p65 phosphorylation at serine 536 (Fig. 2B; n=5), and NFκB p65 nuclear translocation (Fig. 2C; n=5) as well as increased phosphorylation of MAPKs ERK1/2 (Fig. 2D; n=5) and p38 (Fig. 2E; n=5) and increased spinal formation of TNF-α and IL-1β (Fig. 3A; n=6). When compared to vehicle-treated rats, IB-MECA attenuated paclitaxel-induced activation of NFκB (Fig. 2A–C; n=5) and MAPKs (Fig. 2D,E; n=5) as well as the over-production of TNF-α and IL-1β (Fig. 3A; n=6) in the spinal cord measured by ELISA as previously reported [10,25]. Moreover, IB-MECA increased the level of the neuroprotective and anti-inflammatory cytokine, IL-10 (Fig. 3B; n=6). These events are A₃AR-specific as MRS1523 (2 mg/kg/d, n=6) prevented IB-MECA-mediated reductions in TNF-α and IL-1β and enhancement of IL-10 formation (Fig. 3A,B). Similar results were obtained when IB-MECA treatment was restricted to coincide with paclitaxel dosing (Table 1; n=6).

Fig. 2. IB-MECA prevents activation of NFκB and MAPKs in the spinal cord during paclitaxel-induced neuropathic pain.

When compared to vehicle (V, open bars), administration of paclitaxel (P, black bars) increased IκBα degradation (A), cytosolic phosphorylation of NFκB p65 (B), nuclear translocation of NFκB p65 (C), and phosphorylation of ERK1/2 (D) and p38 (E). These paclitaxel-induced events were blocked by daily administration of IB-MECA (D0–15, 0.1 mg/kg/d; gray bars, A–E). Representative blots are shown. Results are expressed as mean ± SD for n=5–6 rats and analyzed by one-way ANOVA with Dunnett’s comparisons. *P<0.05 vs. Vehicle; †P<0.05 vs. Paclitaxel

Fig. 3. IB-MECA decreases paclitaxel-induced spinal formation of pro-inflammatory TNF-α and IL-1β and increases the anti-inflammatory IL-10.

On D16, IB-MECA (0.1 mg/kg/d; D0–15; dark gray bars) attenuated paclitaxel-induced (P, black bars) elevations in TNF-α and IL-1β expression (A) and increased the levels of the anti-inflammatory cytokine, IL-10 (B) as compared to vehicle (V, open bars). The A₃AR antagonist MRS1523 blocked IB-MECA’s effects (dark gray hatched bars) but had no effect on vehicle-treated (light gray bars) animals (A,B). Results are expressed as mean ± SD for n=6 and analyzed by one-way ANOVA with Dunnett’s post hoc comparisons. *P<0.05 vs. Vehicle; †P<0.05 vs. Paclitaxel

Table 1. Cytokine levels for animals treated concomitantly with IB-MECA.

Concurrent IB-MECA retains efficacy in modulating spinal pro-/anti-inflammatory cytokines with paclitaxel-treatment.

IB-MECA (0.1 mg/kg/d; D0–4) given 15–20 minutes prior to paclitaxel (D0, 2, 4, and 6) effectively prevented the increase in pro-inflammatory cytokines (TNF-α, IL-1β) and increased the anti-inflammatory cytokine IL-10 as measured on D25. Results are expressed as mean ± SD for n=6 and analyzed by one-way ANOVA with Dunnett’s post hoc comparisons.

Treatment	TNF-α (pg/mg protein)		IL-1β (pg/mg protein)		IL-10 (pg/mg protein)
	Mean ± SD	95% CI	Mean ± SD	95% CI	Mean ± SD	95% CI
Vehicle	27.1 ± 5.2	21.6 – 32.5	40.0 ± 13.4	26.0 – 54.0	110.9 ± 19.5	90.4 – 131.3
Paclitaxel	40 ± 8.3*	31.5 – 49.0	73.1 ± 13.8*	58.6 – 87.6	94.6 ± 19.4	74.3 – 114.9
P+IB-MECA	26 ± 4.6**	22.1 – 31.8	40.5 ± 12.6**	27.3 – 53.7	146.9 ± 13.1	133.2 – 160.7**

^*P<0.05 vs. Vehicle;

^**P<0.05 vs. Paclitaxel

IB-MECA prevents peroxynitrite-mediated post-translational tyrosine nitration of the spinal glutamate transporter, GLT-1, and glutamine synthetase

A key property of peroxynitrite lies in its ability to post-translationally nitrate tyrosine and consequently modify protein function [22]. Protein nitration is increasingly recognized as an important occurrence during cell signaling and regulation of protein activity (resulting in no effect, loss or gain of function) [22]. The loss of function is best demonstrated in the development of central sensitization [49], including paclitaxel-induced neuropathic pain [10], with the in vivo nitration of glial glutamate transporters and glutamine synthetase; proteins known to be essential in regulating synaptic concentrations of glutamate and glutamate neurotransmission. As can be seen in figure 4, when compared to vehicle-treated rats, the development of paclitaxel-induced mechano-hypersensitivity was associated with increased nitration of GLT-1 (Fig. 4A; n=5) and GS (Fig. 4B; n=5) in spinal cords harvested on D16 from paclitaxel-treated rats, and this nitration was significantly (P<0.01) attenuated by IB-MECA (0.1 mg/kg/d, Fig. 4; n=5).

Fig. 4. IB-MECA prevents post-translational nitration of GLT-1 & GS in the spinal cord during paclitaxel-induced neuropathic pain.

Compared with vehicle (V, open bars), paclitaxel (P, black bars) led to significant nitration of the glutamate transporter GLT-1 (A) and GS (B) in spinal cord tissues, events attenuated in rats treated with IB-MECA (0.1 mg/kg/d, D0–15; gray bars). Representative blots are shown. Results are expressed as mean ± SD for n=5 rats and analyzed by one-way ANOVA with Dunnett’s comparisons. *P<0.05 vs. Vehicle; †P<0.05 vs. Paclitaxel

Discussion

A₃AR agonists are potent non-narcotic analgesics able to block and reverse neuropathic pain [7] by acting at peripheral, spinal, and supraspinal sites [32]. This study, the first to examine potential signaling pathways, identifies inhibition of NADPH oxidase with subsequent modulation of two well-described glia-restricted redox-dependent signaling pathways as an important spinal mechanism of action (Fig. 5).

Fig. 5. Proposed schematic representation of mechanisms underlying IB-MECA’s beneficial actions on CIPN.

Chemotherapy (paclitaxel)-induced neuropathic pain is associated with increased NADPH oxidase activity within the spinal cord contributing to enhanced peroxynitrite (PN) production. Due to its ability to post-translationally modify protein function, peroxynitrite could be a driver behind many of the spinal neuropathological changes underlying CIPN including 1) the nitration/inactivation of glutamate transporter 1 (GLT-1) and glutamine synthetase (GS) and 2) the activation of redox-dependent signaling pathways (NFκB and MAPK) leading to a surge in glial-associated pro-inflammatory cytokine production (TNF-α and IL-1β). Treatment with the selective A₃AR agonist, IB-MECA, not only inhibits paclitaxel-induced pain and the associated spinal events, it also increases the formation of the neuroprotective/anti-inflammatory cytokine, IL-10.

Accumulating evidence implicates neuroinflammatory processes in the alteration of spinal glia-neuronal communication during paclitaxel-induced neuropathic pain. For example, the hyperactivation of glial cells (astrocytes [18,60] and microglia [44]), the activation of redox-sensitive NFκB and MAPKs (ERK, p38) [18,25], and overt production of glia-derived pro-inflammatory cytokines (TNF-α and IL-1β) [10,18,25] have been documented. We recently reported that increased production of spinal superoxide-derived peroxynitrite after nerve injury is the linchpin in setting into motion well-defined processes essential to the initiation, propagation, and maintenance of central sensitization associated with paclitaxel-induced neuropathic pain [10]. Noteworthy, these findings are not unique to CIPN but are also reported in inflammatory pain, neuropathic pain, and opioid-induced antinociceptive tolerance and hyperalgesia underscoring the key contribution of peroxynitrite to the development of central sensitization underlying pain of several etiologies (reviewed in [49]). In spinal cord, two important enzymatic sources have been identified in providing sustained elevated levels of peroxynitrite: activation of NADPH oxidase (expressed in neurons, astrocytes and microglia [4]) and inactivation of mitochondrial manganese superoxide dismutase (MnSOD) following post-translational nitration of Tyr-34 by peroxynitrite [36]. This disruption in enzymatic activity provides a “feed-forward” mechanism sustaining elevated peroxynitrite through elevated superoxide (reviewed in [49]). Our results demonstrate that IB-MECA attenuated spinal activation of NADPH oxidase with subsequent inhibition of NFκB and MAPKs resulting in decreased production of TNF-α and IL-1β and increased formation of IL-10. In addition to its well-recognized anti-inflammatory role, IL-10 is also a powerful neuroinhibitory cytokine; therapeutic manipulations aimed at increasing its presence in spinal cord (i.e., with plasmid DNA encoding IL-10) [28] or by indirectly increasing its production through the removal of peroxynitrite [10] blocked paclitaxel-induced neuropathic pain. Therefore, increased spinal formation of IL-10 may represent a major component of A₃AR’s beneficial actions.

A recent study revealed that increased GSK3β activation in spinal cord contributes to paclitaxel-induced neuropathic pain by activating astrocytes and causing overt production of IL-1β; GSK3β inhibition with lithium was found to be beneficial [18]. Whether paclitaxel-induced activation of spinal GSK3β is also redox-modulated remains to be established, but is a clear possibility considering previous findings in non-pain related fields demonstrating a direct involvement of superoxide/peroxynitrite in Akt/GSK3β signaling [51] and since the pharmacological profile of lithium in the paclitaxel model [18] is identical to the one reported with peroxynitrite decomposition catalysts [10]. Once formed, nitroxidative species [40] and cytokines like IL-1β [59] contribute to excessive activation of synaptic glutamate receptors through several mechanisms including increasing the activities of AMPA and NMDA receptors in spinal dorsal horn neurons, and glutamate release from presynaptic terminals which has been reported to accompany paclitaxel-induced neuropathic pain [18]. It is currently unknown how A₃AR inhibits NADPH oxidase activation; however, a recent report revealed that IB-MECA inhibits NADPH oxidase activation in prostate cancer cells by inhibiting a cyclic AMP/PKA pathway [24]. Additionally, IB-MECA treatment correlated with a reduction in the expression of the Rac1 and p47^phox subunits of NADPH oxidase by inhibiting ERK1/2 activity [24]. Other mechanisms of A₃AR-mediated inhibition of NADPH oxidase activation may stem from the observed A₃AR-mediated shift from pro-inflammatory to anti-inflammatory environments. The provocation of NADPH oxidase activity by TNF-κ and toll-like receptors (TLRs) is well-established [4], and enhanced expression of endogenous IL-10 attenuates the production of pro-inflammatory cytokines and NADPH oxidase activity in LPS-stimulated cerebral microglia and prevents neuronal death [42]. The mitoprotective effects ascribed to A₃AR agonists [11] may also be attributed to attenuation of NADPH oxidase activity. Excessive glutamatergic signaling [50] sparks mitochondrial uptake of Ca²⁺ leading to increased superoxide production [56]. Elevations in superoxide from mitochondria can then trigger NADPH oxidase activity to further exacerbate mitochondrial dysfunction [8]. Moreover, the addition of pro-inflammatory mediators promotes increased metabotropic glutamate receptor (mGluR) 3 and reduced mGluR5 expression in cultured glia [1]. Such mGluR expression profiles favor increased NADPH oxidase activity in microglia [37] as well as promote the development of their neurotoxic phenotype [53]. In neurons, A₃AR agonists inhibit mGluR signaling [34]; thus, it is possible that A₃AR’s effects on glial NADPH oxidase activity occur through similar inhibition of mGluR signaling.

Alterations in glutamatergic neurotransmission and increased neuronal excitability are also present in paclitaxel-induced neuropathic pain [10,60]. Synaptic levels of glutamate are tightly regulated by GTs whose appropriate function is critical in ensuring optimal glutamatergic signaling [19]. Three GT subtypes are found in spinal cord: GLAST and GLT-1 in glia [48] and the excitatory amino acid carrier-1 (EACC1) in neurons [26]. Glia-restricted GTs account for >90% of glutamate reuptake and thus control the termination of glutamatergic signaling [19]. Compromising the glutamate reuptake efficiencies of GTs by either downregulating their expression and/or inactivating their transport activity ensures excessive activation of AMPA and NMDA receptors in the spinal dorsal horn and failure to terminate excitatory signaling [19]. Downregulation of spinal GTs is reported to accompany paclitaxel-induced neuropathic pain [60], but the mechanism(s) involved are unclear. However, inactivation of GTs is the consequence of specific tyrosine nitration and post-translational modifications, a process carried out uniquely by peroxynitrite [54]. In contradistinction to GT-regulation of extracellular glutamate homeostasis, GS plays a pivotal role in its intracellular metabolic fate [52]. In CNS, GS is located mainly in astrocytes and protects neurons against excitotoxicity by converting excess ammonia and glutamate into non-toxic glutamine [52] and returning it to neurons as a precursor for glutamate and GABA; its inactivation maintains neuronal excitability [52]. Spinal astrocyte hyperactivation plays a central role in paclitaxel-induced neuroapthic pain [60]; therefore, compromising the enzymatic activity of GS is expected to maintain neuronal excitation [52]. GS is exquisitively sensitive to peroxynitrite with nitration on Tyr-160 leading to significant loss of enzymatic activity [20]. Results of our study revealed that a second consequence of A₃AR activation is the inhibition of peroxynitrite-mediated posttranslational nitration and modification (inactivation) of GLT-1 and GS. It is therefore possible that A₃AR agonists, by lowering the production of spinal peroxynitrite and preventing GT and GS nitration, “reset” optimal glutamatergic neurotransmission by reducing glutamatergic post-synaptic excitability.

The mechanistic connections between paclitaxel and activation of NADPH oxidase resulting in peroxynitrite formation in spinal cord and downstream effects remain unknown. A growing body of data recently emerged to implicate activation of TLR4 on glial cells in the development of neuropathic pain [57]. More recently activation of TLR4 expressed on spinal astrocytes has also been linked to paclitaxel-induced neuropathic pain [31]. It is well established that redox-signaling following activation of NADPH oxidase is essential to the downstream effects (i.e., NFκB activation) engaged by TLR4 [41]. Noteworthy, peroxynitrite can sustain the activation of NADPH oxidase by nitrating and increasing PKC activity [3]. PKC phosphorylates the p47^phox subunit facilitating its translocation to the membrane and binding to the catalytic p67^phox subunit forming the active holoenzyme [27]. Moreover, PKC also phosphorylates the membrane-associated gp91^phox increasing its diaphorase activity and it’s binding of the Rac2, p67^phox, and p47^phox cytosolic subunits to form the active complex [46]. It is therefore tempting to speculate that TLR4 provides the link to peroxynitrite formation through NADPH oxidase activation; peroxynitrite then sustains a “nitroxidative” rich milieu in spinal cord by maintaining an activated NADPH oxidase and by inactivating mitochondrial MnSOD [35]. In this scenario, activation of A₃AR disrupts this interplay therefore breaking the vicious circle. These reciprocal interactions are expected to prolong and exacerbate CIPN by instituting feedback loops that then sustain local neuroinflammatory processes in spinal cord. A similar process is envisioned in dorsal root ganglia where TLR4 activation has been implicated in paclitaxel-induced neuropathic pain [31].

Collectively, our study provides the first mechanistic insight into beneficial effects exerted by A₃AR agonists. A₃AR are highly expressed in a broad spectrum of malignant cells [14–16]. Furthermore, A₃AR agonists possess direct anticancer effects in a variety of cancer cells; as shown in in vitro and in vivo studies [7,14–16], they do not interfere with the anticancer effects of widely used chemotherapeutics including paclitaxel [7] but can actually enhance their actions [7,14,15] and attenuate other undesireable side effects of chemotherapeutics including myelosuppression [14,16]. In addition, Cl-IB-MECA is already advancing in clinical trials as an anticancer agent, thus providing potential for fast-track proof-of-concept testing for CIPN. Noteworthy, in 2012 the FDA granted orphan drug status to Cl-IB-MECA for the treatment of hepatocellular carcinoma. Use of A₃AR agonists may thus provide a distinct advantage/opportunity in this therapeutic area; direct anticancer effects and mitigation of a major dose limiting toxicity, CIPN. Molecules endowed with such complimentary and potentially synergistic properties have yet to be identified, so this is unprecedented. Further understanding of the adenosine-to-A₃AR axis could provide the framework for clinical development of A₃AR agonists in CIPN and perhaps other chronic neuropathic pain states such as diabetic neuropathy whose underlying pathology shares commonalities with CIPN [5].

Summary.

A₃AR agonists prevent paclitaxel-induced neuropathic pain via modulating spinal redox-dependent signaling pathways (restoring glutamatergic homeostasis, attenuating pro-inflammatory pathways).