Ellen Niederberger

COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib

The regular use of various nonsteroidal anti‐inflammatory drugs (NSAIDs) was shown to decrease the incidence of colorectal cancer. This effect is thought to be caused predominantly by inhibition of cyclooxygenase‐2 (COX‐2) and, subsequently, prostaglandin synthesis. However, recent studies have suggested that COX‐independent pathways may contribute considerably to these antiproliferative effects. To evaluate the involvement of COX‐dependent and COX‐independent mechanisms further, we assessed the effects of celecoxib (selective COX‐2 inhibitor) and SC560 (selective COX‐1 inhibitor) on cell survival, cell cycle distribution, and apoptosis in three colon cancer cell lines, which differ in their expression of COX‐2. Both drugs induced a G0/G1 phase block and reduced cell survival independent of whether or not the cells expressed COX‐2. Celecoxib was more potent than SC560. The G0/G1 block caused by celecoxib could be attributed to a decreased expression of cyclin A, cyclin B1, and cyclin‐dependent kinase‐1 and an increased expression of the cell cycle inhibitory proteins p21Waf1 and p27Kip1. In addition, celecoxib, but not SC560, induced apoptosis, which was also independent of the COX‐2 expression of the cells. In vivo, celecoxib as well as SC560 reduced the proliferation of HCT‐15 (COX‐2 deficient) colon cancer xenografts in nude mice, but both substances had no significant effect on HT‐29 tumors, which express COX‐2 constitutively. Thus, our in vitro and in vivo data indicate that the antitumor effects of celecoxib probably are mediated through COX‐2 independent mechanisms and are not restricted to COX‐2 over‐expressing tumors.

Celecoxib loses its anti-inflammatory efficacy at high doses through activation of NF-κB

Celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, has recently been approved for the symptomatic treatment of arthritis. In some clinical studies, doses of 400 and 800 mg/day provided somewhat less efficacy compared with 200 mg/day, which suggests an early ceiling effect. Using the zymosan‐induced inflammation model in rats, we show that celecoxib significantly reduces paw swelling at 50 mg/kg but completely loses its anti‐inflammatory efficacy at doses ≥100 mg/kg. To evaluate the underlying mechanisms, we used rat renal mesangial cells as a cell culture model. In these cells, celecoxib (50 μM) increased the interleukin Iβ stimulated nuclear translocation and DNA binding of NF‐κB and facilitated the degradation of I‐κB. Consequently, COX‐2 and tumor necrosis factor α (TNF‐α) expression were increased. The up‐regulation of COX‐2 and TNF‐α also occurred in the spinal cord of rats treated with celecoxib (≥100 mg/kg), indicating that in vitro mechanisms were relevant in vivo. Clinically, the overexpression of COX‐2 might be less important because celecoxib inhibits COX‐2 enzymatically. However, the up‐regulation of TNF‐α and possibly other NF‐κB regulated proinflammatory genes might worsen the pathophysiological processes underlying chronic arthritis.

The IKK-NF-κB pathway: a source for novel molecular drug targets in pain therapy?

Several studies indicate that the nuclear factor‐kappa B (NF‐κB) ‐activation cascade plays a crucial role not only in immune responses, inflammation, and apoptosis but also in the development and processing of pathological pain. Accordingly, a pharmacological intervention into this pathway may have antinociceptive effects and could provide novel treatment strategies for pain and inflammation. In this review we summarize the role of NF‐κB in the nervous system, its impact on nociception, and several approaches that investigated the effects of various modulators of the classical I‐κB‐kinase‐NF‐κB signal transduction pathway in inflammatory nociception and neuropathic pain. The results indicate that NF‐κB has an impact on nociceptive transmission and processing and that a number of substances that inhibit the NF‐κB‐activating cascade are capable of reducing the nociceptive response in different animal models. Therefore, a modulation of specific participants in the NF‐κB signal transduction might exert a useful approach for the development of new painkillers.—Niederberger, E., Geisslinger, G. The IKK‐NF‐κB pathway: a source for novel molecular drug targets in pain therapy? FASEB J. 22, 3432–3442 (2008)

Inhibition of NF-κB and AP-1 activation by R- and S-flurbiprofen

R-flurbiprofen is considered the ‘inactive’ isomer of the nonsteroidal anti-inflammatory drug (NSAID), flurbiprofen, because it does not inhibit cyclooxygenase (COX) activity. However, previous studies have revealed that it has antinociceptive and antitumor effects not due to epimerization to the cyclooxygenase-inhibiting S-isomer. Here, we show that R-flurbiprofen has additional anti-inflammatory activity comparable with that of dexamethasone in the zymosaninduced paw inflammation model in rats. Different criteria suggest that the observed effects are !

Effects of selective COX‐1 and ‐2 inhibition on formalin‐evoked nociceptive behaviour and prostaglandin E2 release in the spinal cord

Nociception evoked prostaglandin (PG) release in the spinal cord considerably contributes to the induction of hyperalgesia and allodynia. To evaluate the relative contribution of cyclooxygenase‐1 (COX‐1) and COX‐2 in this process we assessed the effects of the selective COX‐1 inhibitor SC560 and the selective COX‐2 inhibitor celecoxib on formalin‐evoked nociceptive behaviour and spinal PGE2 release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin‐evoked PGE2 raise. In contrast, celecoxib (10 and 20 mg/kg) was ineffective in both regards, i.e. the flinching behaviour was largely unaltered and the formalin‐induced PGE2 raise as assessed using microdialysis was only slightly, not significantly reduced. This suggests that the formalin‐evoked rapid PG release was primarily caused by COX‐1 and was independent of COX‐2. Mean free spinal cord concentrations of celecoxib during the formalin assay were 32.0 ± 4.5 nm, thus considerably higher than the reported IC50 for COX‐2 (3–7 nm). Therefore, the lack of efficacy of celecoxib is most likely not to be a result of poor tissue distribution. COX‐2 mRNA and protein expression in the spinal cord were not affected by microdialysis alone but the mRNA rapidly increased following formalin injection and reached a maximum at 2 h. COX‐2 protein was unaltered up to 4 h after formalin injection. The time course of COX‐2 up‐regulation suggests that the formalin‐induced nociceptive response precedes COX‐2 protein de novo synthesis and may therefore be unresponsive to COX‐2 inhibition. Considering the results obtained with the formalin model it may be hypothesized that the efficacy of celecoxib in early injury evoked pain may be less than that of unselective NSAIDs.

cGMP Produced by NO-Sensitive Guanylyl Cyclase Essentially Contributes to Inflammatory and Neuropathic Pain by Using Targets Different from cGMP-Dependent Protein Kinase I

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoprotein

Morphine‐6‐glucuronide (M6G) is a substrate of P‐glycoprotein (P‐gp), which forms an outward transporter at the blood–brain barrier. Inhibition of P‐gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P‐gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8‐h infusion), five of them together with PSC833 to inhibit P‐gp (32‐h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P‐gp increased the M6G concentrations in the spinal cord approximately three‐fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 ± 0.03 for M6G alone to 0.17 ± 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P‐gp. Inhibition of P‐gp alters the transport of M6G across the blood–brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception.

NADPH Oxidase-4 Maintains Neuropathic Pain after Peripheral Nerve Injury

Reactive oxygen species (ROS) contribute to sensitization of pain pathways during neuropathic pain, but little is known about the primary sources of ROS production and how ROS mediate pain sensitization. Here, we show that the NADPH oxidase isoform Nox4, a major ROS source in somatic cells, is expressed in a subset of nonpeptidergic nociceptors and myelinated dorsal root ganglia neurons. Mice lacking Nox4 demonstrated a substantially reduced late-phase neuropathic pain behavior after peripheral nerve injury. The loss of Nox4 markedly attenuated injury-induced ROS production and dysmyelination processes of peripheral nerves. Moreover, persisting neuropathic pain behavior was inhibited after tamoxifen-induced deletion of Nox4 in adult transgenic mice. Our results suggest that Nox4 essentially contributes to nociceptive processing in neuropathic pain states. Accordingly, inhibition of Nox4 may provide a novel therapeutic modality for the treatment of neuropathic pain.

Modulation of spinal nociceptive processing through the glutamate transporter GLT-1

GLT-1 is the predominant glutamate transporter in most brain regions and therefore plays a major role in terminating synaptic transmission and protecting neurons from glutamate neurotoxicity. In the present study we assessed (i) the regulation of GLT-1 expression in the spinal cord after peripheral nociceptive stimulation and (ii) the nociceptive behavior of rats following inhibition or transient knockdown of spinal GLT-1. Formalin injection into one hindpaw caused a rapid transient upregulation of GLT-1 protein expression in the spinal cord which did not occur when rats were pretreated with morphine (10 mg/kg, i.p.) suggesting that the nociceptive input specifically caused the increase of GLT-1 transcription. Inhibition of GLT-1 by the transportable inhibitor trans-pyrrolidine-2,4-dicarboxylic acid resulted in a significant reduction of nociceptive behavior in the rat formalin assay. Similar results were obtained with a transient reduction of GLT-1 protein expression by antisense oligonucleotides. These data suggest that inhibition of GLT-1 activity or expression reduces excitatory synaptic efficacy and thereby nociception. Mechanisms that might explain this phenomenon may include activation of inhibitory metabotropic glutamate receptors, postsynaptic desensitization or disturbance of glutamate recycling.

Modulation of central nervous system-specific microRNA-124a alters the inflammatory response in the formalin test in mice.

Summary miRNA‐124a is expressed in “pain‐relevant” regions of the spinal cord and is regulated during inflammatory nociception. Modulation of miRNA‐124a has an impact on the nociceptive response. ABSTRACT microRNAs (miRNAs) are small noncoding RNAs that have been linked to a number of disease‐related signal transduction pathways. Several studies indicate that they are also involved in nociception. It is not clear, however, which miRNAs are important and which genes are modulated by miRNA‐associated mechanisms. This study focuses on the regulation and function of the central nervous system (CNS)–specific miRNA‐124a in the spinal cord of mice in a formalin model of inflammatory nociception. miRNA‐124a is constitutively expressed in the spinal cord of mice, particularly in neurons of the dorsal horn. Peripheral noxious stimulation with formalin led to significant down‐regulation of its expression. Knock‐down of miRNA‐124a by intravenous administration of a specific miRNA‐124a inhibitor further increased the nociceptive behavior associated with an upregulation of the pain‐relevant miRNA‐124a target MeCP2 and proinflammatory marker genes. In contrast, administration of a miRNA‐124a mimic counteracted these effects and decreased nociception by down‐regulation of the target gene. In conclusion, our results indicate that miRNA‐124a is involved in inflammatory nociception by regulation of relevant target proteins and might therefore constitute a novel target for anti‐inflammatory therapy.