Cleavage of SNAP-25 ameliorates cancer pain in a mouse model of melanoma.

BACKGROUND: Cancer pain is associated with increased pain sensitivity to noxious (hyperalgesia) and normally innocuous (allodynia) stimuli due to activation of nociceptors by tumour-derived mediators or tumour infiltration of nerves. The pain sensitization is accompanied by modifications in gene expression, but specifically regulated genes are largely unknown. The 25 kDa synaptosomal-associated protein (SNAP-25) is involved in chemical neurotransmission at the synaptic cleft. Its inhibition by Botulinum neurotoxin A (BoNT/A) has been associated with antinociceptive effects in migraine, inflammatory and neuropathic pain. However, its potential to reduce tumour-associated pain remains to be clarified. METHODS: We applied a melanoma model of tumour pain in C57BL/6 mice and investigated SNAP-25 expression and regulation by qRT-PCR, Western Blot and immunofluorescence as well as tumour-associated mechanical allodynia with and without BoNT/A treatment. RESULTS: We found increased SNAP-25 expression in the dorsal root ganglia and the sciatic nerve. Intraplantar injection of BoNT/A induced the cleavage of SNAP-25 in these tissues and was associated with decreased mechanical allodynia after therapeutic treatment at early and late stages of tumour pain while the tumour size was not affected. CONCLUSIONS: Our data indicate that SNAP-25 plays a role in tumour pain but has no influence on the initiation and progression of skin cancer. Its cleavage inhibits the development of allodynia in the mouse melanoma model and might be useful as new therapeutic approach for the treatment of cancer pain. WHAT DOES THIS STUDY ADD?: SNAP-25 is differentially regulated during melanoma-induced tumour pain. Its cleavage by BoNT/A might be a suitable therapeutic option for tumour pain patients since tumour-associated pain can be strongly and significantly reduced after preventive and therapeutic BoNT/A treatment, respectively.

[Pharmacological aspects of pain research in Germany]. / Pharmakologische Aspekte der Schmerzforschung in Deutschland.

In spite of several approved analgesics, the therapy of pain still constitutes a challenge due to the fact that the drugs do not exert sufficient efficacy or are associated with severe side effects. Therefore, the development of new and improved painkillers is still of great importance. A number of highly qualified scientists in Germany are investigating signal transduction pathways in pain, effectivity of new drugs and the so far incompletely investigated mechanisms of well-known analgesics in preclinical and clinical studies. The highlights of pharmacological pain research in Germany are summarized in this article.

[Epigenetics and pain]. / Epigenetik und Schmerz.

Chronic pain affects approximately 20 % of adults worldwide and is often associated with a decrease in the quality of life and various comorbidities. Conventional analgesic therapies are frequently insufficient and sometimes lead to severe side effects. Therefore, great efforts are still being made to elucidate the signalling pathways in pain and to develop new, safe and effective therapies. Epigenetic mechanisms which interfere with the regulation of gene expression are involved in the pathogenesis of several diseases and are gaining increasing impetus in medical research. As they are also involved in pain processing, a modulation of these mechanisms might represent a novel option for the therapy of pain patients.

Current role of CT and whole body MRI in multiple myeloma.

Radiology of bone lacunae can help differentiate between smouldering and symptomatic myeloma. CT seems to be more apt for this purpose than a standard X-ray but appropriate principles must be applied when performing and reading it. Lesions visible in an MRI above all allow myelomas to be monitored during treatment. Because of the radiation dose, whole body CT must be performed with a slice thickness of 2mm, increments of 1.5 and intensity of 40mAs. It should be read associating the reading of the axial slices with reading the mean coronal and sagittal projections of a thickness of 2cm. Whole body MRI must associate T1-weighted sagittal, STIR coronal and b-800 diffusion-weighted axial sequences. Changes in the disease correlate with changes in the diffusion, STIR and T1-weighted images interpreted together. While whole body CT has a place in clinical routine, the indication for whole body MRI still needs to be clarified and has yet to take its place in research protocols.

The non-canonical IκB kinases IKKε and TBK1 as potential targets for the development of novel therapeutic drugs.

IκB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are two non-canonical IKKs which are involved in interferon regulatory factor (IRF) as well as nuclear factor kappaB (NF-κB) signaling cascades. Both kinases have already been linked to the pathophysiology of several diseases and therefore been suggested as potential promising targets for the development of therapeutic drugs. In this review, we summarize the roles of IKKε and TBK1 in different diseases and outline therapeutic options for modulation of these kinases.

Imaging of snapping phenomena.

Snapping phenomena result from the sudden impingement between anatomical and/or heterotopical structures with subsequent abrupt movement and noise. Snaps are variously perceived by patients, from mild discomfort to significant pain requiring surgical management. Identifying the precise cause of snaps may be challenging when no abnormality is encountered on routinely performed static examinations. In this regard, dynamic imaging techniques have been developed over time, with various degrees of success. This review encompasses the main features of each imaging technique and proposes an overview of the main snapping phenomena in the musculoskeletal system.

Anatomic variants of the anterior part of the cerebral arterial circle at multidetector computed tomography angiography.

Imaging of the cerebral arterial circle (CAC) is essential in neurovascular diseases such as ischemic stroke for detecting arterial occlusions and evaluating arterial supply, and in subarachnoid or intralobar hemorrhage for detecting intracranial malformations. Multidetector computed tomography angiography (MD-CTA) is increasingly being used for the detection and treatment planning of intracranial aneurysm. For optimal interpretation and treatment planning, this method requires suitable post-processing equipment, and extensive knowledge of the relevant anatomy and anatomical variants. Anatomical variants of the CAC are common, particularly in the anterior CAC, the most common site of intracranial aneurysm. The aim of this review is to illustrate the normal anatomy and most common anatomical variants of the anterior CAC detected by MD-CTA, and to discuss the relevant embryological and technical considerations.

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.

A rapid screening method for a single nucleotide polymorphism (SNP) in the human MOR gene.

AIMS: Genetic association studies have suggested that the single nucleotide polymorphism (SNP) at position 118 of the human mu-opioid receptor (MOR) gene could be a potential risk factor for drug treatment variability in patients. Therefore, we wanted to develop a fast and reliable detection method for this SNP which is applicable in a clinical setting. METHODS: To detect the polymorphism at position A118-->G in the human MOR gene we used the fluorescence resonance energy transfer (FRET)-PCR technique with subsequent melting curve analysis. RESULTS: The polymorphism at position A118-->G in the human MOR gene could be clearly discriminated with melting peak temperatures of 69.8 degrees C and 63.8 degrees C, corresponding to the wild type and mutated MOR allele, respectively. The results from FRET-PCR were validated by sequencing and restriction-fragment length polymorphism (RFLP). Screening of blood samples from 100 subjects showed an allelic distribution for the human MOR alleles of 79% (homozygous wild type), 20% (heterozygous) and 0.9% (homozygous mutated). CONCLUSIONS: The FRET-PCR protocol for detection of the human MOR gene polymorphism at position 118 offers a rapid and reliable method which could be used for population screening of this and other genes.

Effects of selective COX-1 and -2 inhibition on formalin-evoked nociceptive behaviour and prostaglandin E(2) 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 PGE(2) release. SC560 (10 and 20 mg/kg) significantly reduced the nociceptive response and completely abolished the formalin-evoked PGE(2) 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 PGE(2) 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.

Lycobetaine acts as a selective topoisomerase II beta poison and inhibits the growth of human tumour cells.

The phenanthridine alkaloid lycobetaine is a minor constituent of Amaryllidaceae. Inhibition of cell growth was studied in the clonogenic assay on 21 human tumour xenografts (mean IC(50) = 0.8 microM). The growth of human leukaemia cell lines was also potently inhibited (mean IC(50) = 1.3 microM). Athymic nude mice, carrying s.c. implanted human gastric tumour xenograft GXF251, were treated i.p. with lycobetaine for 4 weeks, resulting in a marked tumour growth delay. Lycobetaine was found to act as a specific topoisomerase II beta poison. In the presence of calf thymus DNA, pure recombinant human topoisomerase II beta protein was selectively depleted from SDS-gels, whereas no depletion of topoisomerase II alpha protein was observed. In A431 cells immunoband-depletion of topoisomerase II beta was induced, suggesting stabilization of the covalent catalytic DNA-intermediate in living cells. It is reasonable to assume that this mechanism will cause or at least contribute significantly to the antitumour activity.

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.

Inhibition of NF-kappaB 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 antitum or 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 zymosan-induced paw inflammation model in rats. Different criteria suggest that the observed effects are mediated at least in part through inhibition of NF-kB activation: R-flurbiprofen inhibited i) LPS-induced NF-kB DNA binding activity in RAW 264.7 macrophages, ii) translocation of the p65 subunit of NF-kB into the nucleus of these cells, and iii) zymosan-induced NF-kB-dependent gene transcription in the inflamed paw and spinal cord of rats. S-flurbiprofen produced similar effects but was less potent. In addition, R-flurbiprofen inhibited DNA binding activity of AP-1, another key regulatory transcription factor in inflammatory processes. Because R-flurbiprofen does not cause gastrointestinal mucosal damage or other side effects associated with long-term NSAID or glucocorticoid use, it might be a useful drug in inflammatory or other diseases in which increased or constitutive NF-kB and AP-1 activation are involved in the pathophysiological processes.

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.