Potential Therapeutic Targets for Bone Cancer Pain-P2X Receptors

Cancer pain is difficult to treat as it appears to be driven simultaneously by inflammatory, neuropathic and tumorigenic mechanisms. I have reported on multiple occasions publication referencing use of our Pain and Inflammation Research Antibodies in studying bone cancer pain.

I would like to alert you to the latest reference:

Timothy K. Y. Kaan, Ping K. Yip, Sital Patel, Meirion Davies, Fabien Marchand, Debra A. Cockayne, Philip A. Nunn, Anthony H. Dickenson, Anthony P. D. W. Ford, Yu Zhong, Marzia Malcangio, and Stephen B. McMahon Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats. Brain, September 2010; 133: 2549 - 2564.

......Slides were then incubated with rabbit anti-P2X3 (1:2000, Neuromics) and sheep anti-calcitonin gene-related peptide (1:1000, Biomol...anti-beta-III-tubulin (1:4000, Promega) and guinea pig anti-P2X3 (1:100, Neuromics). The next day, after three washes with phosphate-buffered......

Summary: Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

Related Reagents:

All Purinergic Receptors
Neurotransmission Research Antibodies

Making Gains on Pain Research

Neuromics' Pain Research Customers continue to make gains using our Pain and Inflammation Research Antibodies and Transfection Kits. Here are the latest pubs:

Hua Zhang and A. S. Verkman. Aquaporin-1 Tunes Pain Perception by Interaction with Nav1.8 Na+ Channels in Dorsal Root Ganglion Neurons. February 19, 2010 The Journal of Biological Chemistry, 285, 5896-5906.

...chicken anti-calcitonin gene-related peptide (CGRP; 1:500, Neuromics, Edina, MN)...

Nathaniel A. Sowa, Bonnie Taylor-Blake, and Mark J. Zylka. Ecto-5'-Nucleotidase (CD73) Inhibits Nociception by Hydrolyzing AMP to Adenosine in Nociceptive Circuits. The Journal of Neuroscience, February 10, 2010, 30(6):2235-2244; doi:10.1523/JNEUROSCI.5324-09.2010.

...rabbit anti-P2X3-RA10109, Neuromics; 1:750), rabbit anti- VR1 C-Terminus (TRPV1) - mouse specific (RA14113, Neuromics; 1:750

Images: Images: Confocal images showing the effect of RTX on mu opioid receptor and TRPV1 immunoreactive DRG neurons and afferent terminals in the spinal cord. A: representative confocal images showing mu opioid receptor (green) and TRPV1 (red) immunoreactivities in DRG neurons of one vehicle- and one RTX-treated rat. Scale bar, 40 um. B: confocal images showing mu opioid receptor (green) and TRPV1 (red) immunoreactivities in afferent terminals in the spinal dorsal horn of 1 vehicle- and 1 RTX-treated rat. Scale bar, 80 um. Inset: high-magnification images (scale bar = 5 um) showing co-localization of mu opioid receptor and TRPV1 immunoreactivity in the lamina I. Co-localization of the mu opioid receptor and TRPV1 immunoreactivity is indicated in yellow when 2 images are digitally merged. All images are single confocal optical sections. Shao-Rui Chen and Hui-Lin Pan. Loss of TRPV1-Expressing Sensory Neurons Reduces Spinal mu-Opioid Receptors But Paradoxically Potentiates Opioid Analgesia. doi:10.1152/jn.01343.2005.

Neuromics' i-Fect ™ siRNA Transfection Reagent continues to be used a tool for studying expression of genes suspected to paly a role in pain . Expression studies include: DOR , The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Ca_V1.2 and more.

Here's a link to all transfection publications: Transfection Kit Pubs

Related Reagents:

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels, Biogenic Amines and more Opioid Neuropeptides

Purinergic Receptors Primary Neurons and Astrocytes- Primary human, rat and mouse neurons and astrocytes by Category

Opioid Receptors TRPV (Vanilloid); TRPM; TRPA and TRPCs

Purinergic Receptor Pubs

Our Purinergic Receptor Antibodies have recently been referenced in several publications.

In the first study 5 of our antibodies are used. It investigates the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization.

Sylvia Sarman; Jorge Mancini; Ingeborg van der Ploeg; J. Oscar Croxatto; Anders Kvanta; Juan E. Gallo. Involvement of Purinergic P2 Receptors in Experimental Retinal Neovascularization. Current Eye Research. 10.1080/02713680701885470 .

...Five eyes (5 animals) from either hyperoxa- or non-hyperoxia-treated mice were enucleated and fixed for 48 hr at 4°C in paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Afterward, eyes were immersed for cryoprotection in graded sucrose solution (5% overnight, 10%, 15%, and 20%) and interlocked with resin. Fourteen micron sections were obtained (Shandon AS325 Retraction Microtome, Thermo Scientific, Waltman, MA, USA) and fixed on polylysine-treated glass slides. After overnight incubation in primary antibody (P2X1 1:1000, P2X2 1:1000,P2X3 1:1000, and P2Y2 1:800) (Neuromics, Minneapolis, MN, USA) at 4°C, sections were treated with biotinylated secondary antibodies followed by an avidin peroxidase complex step (Vectastin Elite ABC, Vector Laboratories, Burlingame, CA, USA). Finally, a color reaction was obtained using 3,3'-diaminobenzidine (DAB)/nickel-enhanced solution for staining (Sigma-Aldrich, St Louis, MO, USA). At least 6 sections per retina were analyzed. For immunofluorescence, the sections were labeled with lissamine rhodamine-conjugated goat anti-rabbit Ig-G or with fluorescein-5-isothiocyanate-conjugated goat anti-guinea pig Ig-G (Jackson Immunoresearch Laboratories, West Grove, PA, USA). At least 6 sections per retina were analyzed. Visualization was done using a Nikon Fluorescence Eclipse Microscope (Tokyo, Japan), and photographs were taken with a Nikon DN 100 Digital Camera (Tokyo, Japan)...

Images: Expression of P2X and P2Y receptors in normal mice and in a model of ischemia-induced pathological retinal angiogenesis as assessed by immunohistochemistry. (A) P2X2 receptor expression was found in the outer plexiform layer of control mice. (B) In mice
treated with oxygen, the expression was similarly found in the outer plexiform layer. In addition, a strong signal was also found in the inner plexiform layer. This induction was noted in all slides analyzed. (C) Retina without primary antibody for P2X2. (D) In control mice,
weak P2Y2 receptor expression was seen in the ganglion cell and in the nerve fiber layer. (E) In mice treated with oxygen, P2Y2 expression
showed a similar overall distribution as control mice. The signal was, however, strongly up-regulated. This increase was noted in all slides analyzed. (F) Retina without primary antibody for P2Y2. Black bar = 20 μm.

The second studies P2Xs' role in neurotransmision.

Elsa Fabbretti*, Elena Sokolova*, Lara Masten, Marianna D’Arco, Alessandra Fabbro, Andrea Nistri and Rashid Giniatullin. Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+ sensing modulatory sites. JBC Papers in Press. Published on October 8, 2004 as Manuscript M409772200.

...Western immunoblots of transfected or untransfected HEK cells were performed as recently reported (19). In brief, these were lysed using a buffer containing 100 mM Tris HCl (pH 6.8), 200 mM dithiothreitol, 4 % SDS, 20 % glycerol and a cocktail of protease inhibitors (Sigma), and separated on 10 % polyacrylamide gel. After blocking with Tris saline buffer (TBS) containing milk, Tween 20 and preimmune serum, proteins were incubated overnight at 4°C with an anti-P2X3 antibody (Neuromics; 1:2000). Immunocomplexes were incubated for 1 h with a peroxidase-conjugated secondary antibody (Sigma; 1:4000) and detected with a chemiluminescence ECL kit (Amersham). Negative controls were obtained by mock transfection of HEK cells with the pEGFP-N1 plasmid (CLONTECH). Controls for efficient loading of different lysate materials were carried out by using an anti-β actin antibody (mouse monoclonal, 1:2000, Sigma)...

Related Reagents:

P2X1-Rabbit Antibody
P2X2-Guinea Pig Antibody
P2X2-Rabbit Antibody
P2X3-Rabbit Antibody
P2X3-Guinea Pig Antibody
Related Reagents:
All Purinergic Receptor Antibodies
All Pain and Inflammation Antibodies
Vision and Retina Research Antibodies