BackgroundBone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model.ResultsIn a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aβ-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain.ConclusionsThis work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

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The cystine/glutamate antiporter has been implicated in a variety of cancers as a major mediator of redox homeostasis. The excess glutamate secreted by this transporter in aggressive cancer cells has been associated with cancer-induced bone pain (CIBP) from distal breast cancer metastases. High-throughput screening of small molecule inhibitors of glutamate release from breast cancer cells identified several potential compounds. One such compound, capsazepine (CPZ), was confirmed to inhibit the functional unit of system xc− (xCT) through its ability to block uptake of its radiolabeled substrate, cystine. Blockade of this antiporter induced production of reactive oxygen species (ROS) within 4 hours and induced cell death within 48 hours at concentrations exceeding 25 μM. Furthermore, cell death and ROS production were significantly reduced by co-treatment with N-acetylcysteine, suggesting that CPZ toxicity is associated with ROS-induced cell death. These data suggest that CPZ can modulate system xc− activity in vitro and this translates into antinociception in an in vivo model of CIBP where systemic administration of CPZ successfully delayed the onset and reversed CIBP-induced nociceptive behaviors resulting from intrafemoral MDA-MB-231 tumors.

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO ؊ ) to CO 2 and NH 3 through the action of a cytosolic cyanase which required HCO 3 ؊ as a second substrate. The ability to metabolize NCO ؊ relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO 2 -concentrating mechanism (CCM), and light. (3,28,49). Spontaneous decarboxylation of the carbamate subsequently yields a second CO 2 and NH 3 . Assimilation of cyanate-derived NH 3 and CO 2 then proceeds through conventional metabolic pathways providing a unique source of nitrogen (N) for growth in a variety of bacteria and a source of carbon (C) for autotrophic metabolism (7,15,30,41,50,53).In E. coli, the coexpression of carbonic anhydrase (CA) is vital to maintain ongoing cyanate metabolism (17, 23). Mutants lacking CA activity do not readily catalyze cyanate decomposition, are unable to grow with NCO Ϫ as the sole N source, and are far more susceptible than wild-type cells to the toxic effects of cyanate itself on growth (18,(22)(23)(24). CA involvement is related to the absolute requirement by cyanase for HCO 3 Ϫ (rather than CO 2 ) as a substrate in the reaction. Physiological studies (17,18,22,23) indicate that in the absence of CA, the CO 2 generated from cyanate decomposition diffuses out of cells faster than it can be hydrated nonenzymatically to HCO 3 Ϫ . This leads to a cellular depletion of HCO 3 Ϫ and cessation of cyanate metabolism through substrate deprivation. CA prevents this cellular depletion by trapping CO 2 and catalytically regenerating HCO 3 Ϫ within cells at a rate that is not limiting for cyanase.Carbonic anhydrase, cyanase, and a hydrophobic protein designated as CynX are encoded by cynT, cynS, and cynX (4, 44), respectively, which are arranged in an operon in E. coli, ensuring the coordinated expression of the two enzymes required for cyanate decomposition. Expression of the cynTSX operon is induced by exogenous cyanate and positively regulated by CynR (45), a member of the LysR family of regulatory proteins. The cynR gene is located immediately upstream of the cynTSX operon but is transcribed in the opposite direction.The photoautotrophic cyanobacterium Synechococcus sp. strain UTEX 625 also converts exogenous cyanate to CO 2 and NH 3 as described in the reaction above (30). Inhibitor studies (30) have shown that cyanate-derived NH 3 is rapidly incorporated by this cyanobacterium via the central nitrogen assimilation pathway, and it has recently been suggested that NCO Ϫ can serve as the sole source of N for growth of the globally important marine cyanobacterium Synechococcus sp. strain WH8102 (35,43). CO 2 arising from cyanate decomposition is also rapidly assimilated by Synechococcus sp. strain UTEX 625

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Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.

Despite the lack of robust evidence of effectiveness, current treatment options for cancer-induced depression (CID) are limited to those developed for non-cancer related depression. Here, anhedonia-like and coping behaviours were assessed in female BALB/c mice inoculated with 4T1 mammary carcinoma cells. The behavioural effects of orally administered sulfasalazine (SSZ), a system xc− inhibitor, were compared with fluoxetine (FLX). FLX and SSZ prevented the development of anhedonia-like behaviour on the sucrose preference test (SPT) and passive coping behaviour on the forced swim test (FST). The SSZ metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) exerted an effect on the SPT but not on the FST. Although 5-ASA is a known anti-inflammatory agent, neither treatment with SSZ nor 5-ASA/SP prevented tumour-induced increases in serum levels of interleukin-1β (IL-1β) and IL-6, which are indicated in depressive disorders. Thus, the observed antidepressant-like effect of SSZ may primarily be attributable to the intact form of the drug, which inhibits system xc−. This study represents the first attempt at targeting cancer cells as a therapeutic strategy for CID, rather than targeting downstream effects of tumour burden on the central nervous system. In doing so, we have also begun to characterize the molecular pathways of CID.

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