Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells.

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

The efficacy of Dynorphin fragments at the κ, µ and δ opioid receptor in transfected HEK cells and in an animal model of unilateral peripheral inflammation.

BACKGROUND: Dynorphin 1-17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1-17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, µ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1-17 and fragments (1-6, 1-7 and 1-9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1-7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund's Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1-7, 1-17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1-7 and 1-9 displayed the greatest activity across all receptor subtypes, while dynorphin 1-7, 1-9 and 1-17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1-7 and U50488H, but not dynorphin 1-17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1-7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1-7 mediates analgesia.

Cellular and molecular mechanisms of chronic rhinosinusitis and potential therapeutic strategies: review on cytokines, nuclear factor kappa B and transforming growth factor beta.

BACKGROUND: Chronic rhinosinusitis is characterised by persistent inflammation of the sinonasal mucosa. Multiple pathophysiological mechanisms are likely to exist. Previous research has focused predominantly on T-helper type cytokines to highlight the inflammatory mechanisms. However, proteins such as nuclear factor kappa B and transforming growth factor beta are increasingly recognised to have important roles in sinonasal inflammation and tissue remodelling. OBJECTIVE: This review article explores the roles of T-helper type cytokines, nuclear factor kappa B and transforming growth factor beta in the pathophysiological mechanisms of chronic rhinosinusitis. An understanding of these mechanisms will allow for better identification and classification of chronic rhinosinusitis endotypes, and, ultimately, improved therapeutic strategies.

Comparison and analysis of the animal models used to study the effect of morphine on tumour growth and metastasis.

UNLABELLED: The effect of opioids on tumour growth and metastasis has been debated for many years, with recent emphasis on the possibility that they might influence the rate of disease-free survival after tumour resection when used in the perioperative pain management of cancer surgery patients. The literature presents conflicting and inconclusive in vitro and in vivo data about the potential effect of opioids, especially morphine, on tumour growth and metastasis. To inform clinical practice, appropriate animal models are needed to test whether opioids alter the course of tumour growth and metastasis. Here, we review the literature on animal-based studies testing the effect of morphine on cancer so far, and analyse differences between the models used that may explain the discrepancies in published results. Such analysis should elucidate the role of opioids in cancer and help define ideal pre-clinical models to provide definitive answers. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Effect of lysine antifibrinolytics and cyclooxygenase inhibitors on the proteolytic profile of breast cancer cells interacting with macrophages or endothelial cells.

BACKGROUND: Extracellular matrix (ECM) proteases play a key role in the regulation of tumour invasion, growth, and transendothelial migration. The expression of ECM proteases and their endogenous inhibitors by cancer cells is regulated by stromal cells. We investigated the effect of commonly used perioperative medications on this regulation. METHODS: Breast cancer cells (4T1) were cultured alone or with endothelial cells (H5V) or macrophages (RAW264.7). Cell grown alone or in cocultures were treated with clinically relevant concentrations of cyclooxygenase (COX) inhibitors, aspirin (ASA), ketorolac, celecoxib, or lysine antifibrinolytics, ɛ-aminocaproic acid (EACA) and tranexamic acid (TXA). We determined the level of the ECM proteases urokinase-like plasminogen activator (uPA), matrix metalloproteinase (MMP)-2 and MMP-9, and endogenous MMP inhibitors, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in the conditioned media. RESULTS: Antifibrinolytics and COX inhibitors exerted a complex effect on cells grown alone and in cocultures. EACA increased the activity of MMP-9 and TIMP-1 in cocultures of 4T1 and RAW264.7. TXA increased TIMP-1 in the coculture without affecting MMP-9. EACA and TXA both attenuated MMP-2 detected in 4T1 and H5V cocultures. ASA and ketorolac both decreased the activity of MMP-2, MMP-9, and uPA. Celecoxib increased the activity of TIMP-1 in cocultures of 4T1 with both macrophages and endothelial cells. CONCLUSIONS: Antifibrinolytics and COX inhibitors can affect the proteolytic profile of the tumour microenvironment. Animal and clinical investigations are warranted to assess the effect of these proteolytic changes on the outcome of cancer surgery.

Dynorphin A 1-17 biotransformation in inflamed tissue, serum and trypsin solution analysed by liquid chromatography-tandem mass spectrometry.

Dynorphin A 1-17 (DYN A) is an endogenous neuropeptide that is of interest due to its diverse roles in analgesia, inflammation and addiction. Upon release, DYN A is subject to metabolism by a range of enzymes and its biotransformation is dependent on the site and environment into which it is released. In this study, we investigated the biotransformation of DYN A in rat inflamed tissue at pH 7.4 and 5.5, in rat serum and in trypsin solution. DYN A-porcine was incubated at 37 °C in each matrix over a range of incubation periods. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Incubation of DYN A in trypsin solution and in rat serum resulted in 6 and 14 fragments, respectively. Incubation in inflamed rat paw tissue occasioned 21 fragments at pH 7.4 and 31 fragments at pH 5.5. Secondary breakdown of some larger primary fragments was also observed in this study.

Study of beta endorphin metabolism in inflamed tissue, serum and trypsin solution by liquid chromatography-tandem mass spectrometric analysis.

Beta endorphin (ß-END) is recognised as one of the most significant endogenous neuropeptides, responsible for a wide range of biological activities in the body. However, within the body ß-END is exposed to hydrolysis by a variety of enzymes. In this study, we investigated the metabolism and fragmentation pattern of ß-END in rat inflamed tissue, in rat serum and in trypsin solution. ß-END (1-31)-rat was incubated at 37 °C in each matrix for different incubation times. The resultant fragments were separated using a C4 column and detected by mass spectrometry using total ion current mode. Structural information for the fragments was elucidated using tandem mass spectrometry. Incubation of ß-END (1-31)-rat in trypsin solution and in rat serum resulted in 8 and 13 fragments, respectively. Incubation in inflamed rat paw tissue resulted in 22 fragments at pH 7.4 and 26 fragments at pH 5.5. Some of these fragments were common to both pH values. The degradation of ß-END (1-31)-rat in inflamed tissue at pH 5.5 was faster than that at pH 7.4. Secondary fragmentation of some larger primary fragments was also observed in this study.

Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides--a case study using liquid chromatography/mass spectrometry method development for beta-endorphin assay.

Beta-endorphin was used as a model peptide to study the effect of solvent and electrospray mass spectrometer parameters in the optimisation of an assay method for multiply charged compounds using liquid chromatography/mass spectrometry (LC/MS). Unlike with singly charged compounds, the charge state distribution has a significant impact in the method development of multiply charged compounds such as peptides. Using a 50% acetonitrile/water solvent mixture, we found that the ion spray voltage had no influence on the charge state distribution. However, increasing declustering potential led to deprotonation of the higher charge states of the peptide thus causing a shift to lower charge states. The mechanism leading to the deprotonation was examined. It was concluded that the deprotonation is due to endoergic proton transfer from the peptide to solvent molecules clustered to the peptide that occurs in the declustering region. The extent of deprotonation increases with increasing proton affinity of the molecules of the non-aqueous solvent component used. Thus, if desired, deprotonation can be avoided by selecting a low proton affinity solvent such as methanol. The focusing potential was also found to have a great influence on the charge state distribution observed. The results of this study enabled us to select the optimum ion to be used in single ion/reaction monitoring mode. They also provided the most favourable parameter values to be used in the method to obtain the best sensitivity for the ion of choice. The results demonstrate the importance of considering the charge state distribution in the optimisation of electrospray LC/MS methods for multiply charged compounds.

Immune-derived opioids and peripheral antinociception.

1. Recent findings have suggested a significant involvement of the immune system in the control of pain. Immune cells contain opioid peptides that are released within inflamed tissue and act at opioid receptors on peripheral sensory nerve endings. It is also apparent that different types of lymphocytes contain beta-endorphin, memory T cells containing more beta-endorphin than naïve cells. 2. These findings highlight an integral link between immune cell migration and inflammatory pain. The present review highlights immune system involvement in the site-directed control of inflammatory pain. 3. Full-length mRNA transcripts for opioid precursor proteins are expressed in immune cells. Increased expression of pro-opiomelanocortin mRNA and beta-endorphin has been demonstrated in stimulated lymphocytes and lymphocytes from animals with inflammation. 4. Cytokines and corticotropin-releasing factor (CRF) release opioids from immune cells. Potent peripheral analgesia due to direct injection of CRF can be blocked by antagonists to CRF, antibodies to opioid peptides, antisense to CRF and opioid receptor-specific antagonists. The release of opioid peptides from lymphocytes is calcium dependent and opioid receptor specific. Furthermore, endogenous sources of opioid peptides produce potent analgesia when implanted into the spinal cord. 5. Activated immune cells migrate directly to inflamed tissue using cell adhesion molecules to adhere to the epithelial surface of the vasculature in inflamed tissue. Lymphocytes that have been activated can express opioid peptides. Memory type T cells that contain opioid peptides are present within inflamed tissue; naive cells are not present in inflamed tissue and do not contain opioid peptides. Inhibiting the migration of memory type T cells into inflamed tissue by blocking selectins results in reduced numbers of beta-endorphin-containing cells, a reduced quantity of beta-endorphin in inflamed paws and reduced stress- and CRF-induced peripheral analgesia. 6. Immunosuppression is associated with increased pain in patients. Moreover, immunosuppression results in decreased lymphocyte numbers as well as decreased analgesia in animal models.

Pain control in inflammation governed by selectins.

Opioid-containing immune cells migrate preferentially to inflamed sites, where they release beta-endorphin which activates peripheral opioid receptors to inhibit pain. Immunocyte recruitment is a multistep, sequential engagement of various adhesion molecules located on immune cells and vascular endothelium. Selectins mediate the initial phase of immunoctye extravasation into inflamed sites. Here we show that anti-selectin treatment abolishes peripheral opioid analgesia elicited either endogenously (by stress) or by corticotropin-releasing factor. This results from a blockade of the infiltration of immunocytes containing beta-endorphin and the consequent decrease of the beta-endorphin content in the inflamed tissue. These findings indicate that the immune system uses mechanisms of cell migration not only to fight pathogens but also to control pain in injured tissue. Thus, pain is exacerbated by measures inhibiting the immigration of opioid-producing cells or, conversely, analgesia might be conveyed by adhesive interactions that recruit those cells to injured tissue.

Morphine has a dual concentration-dependent effect on K(+)-evoked substance P release from rat peripheral airways.

Our previous investigations of possible lung mechanisms underlying the effectiveness of nebulized morphine for the relief of dyspnoea, have shown a high density of non-conventional opioid binding sites in rat airways with similar binding characteristics (opioid alkaloid-sensitive, opioid peptide-insensitive) to that of putative mu 3-opioid receptors on immune cells. To investigate whether these lung opioid binding sites are functional receptors, this study was designed to determine (using superfusion) whether morphine modulates the K(+)-evoked release of the pro-inflammatory neuropeptide, substance P (SP), from rat peripheral airways. Importantly, K(+)-evoked SP release was Ca(2+)-dependent, consistent with vesicular release. Submicromolar concentrations of morphine (1 and 200 nM) inhibited K(+)-evoked SP release from rat peripheral airways in a naloxone (1 microM) reversible manner. By contrast, 1 microM morphine enhanced K(+)-evoked SP release and this effect was not reversed by 1 microM naloxone. However, 100 microM naloxone not only antagonized the facilitatory effect of 1 microM morphine on K(+)-evoked SP release from rat peripheral airways but it inhibited release to a similar extent as 200 nM morphine. It is possible that these latter effects are mediated by non-conventional opioid receptors located on mast cells, activation of which causes naloxone-reversible histamine release that in turn augments the release of SP from sensory nerve terminals in the peripheral airways. Clearly, further studies are required to investigate this possibility.

Immune cell-derived beta-endorphin. Production, release, and control of inflammatory pain in rats.

Localized inflammation of a rat's hindpaw elicits an accumulation of beta-endorphin-(END) containing immune cells. We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking. In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN. Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN. However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells. Accordingly, corticotropin-releasing factor and IL-1beta released significantly more END from noninflamed than from inflamed LN-immune cells. This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release. Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END. Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain. Afterwards they migrate to the regional LN, depleted of the peptide. Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells. Thus, the immune system is important for the control of inflammatory pain. This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS.

Quantitative autoradiography of peripheral opioid binding sites in rat lung.

Previous studies in our laboratory have characterized non-conventional opioid binding sites in membrane preparations from both rat and human lung. The studies described in this paper utilized autoradiography to investigate the regional distribution of these [3H]morphine binding sites within rat lungs. Specific binding of [3H]morphine was saturable and Rosenthal analysis of tissue section wipes revealed the presence of both high-affinity and low-affinity opioid binding sites. The mean +/- S.E.M. binding affinity and the mean +/- S.E.M. density values for the low-affinity binding site (Kd = 217 +/- 160 nM, Bmax = 12 +/- 8 pmol/mg protein) were similar to the values obtained in our previous whole-rat lung membrane binding assays (Kd = 187 +/- 36 nM, Bmax = 13.5 +/- 2 pmol/mg protein) (Cabot, P.J., P.R. Dodd, T. Cramond and M.T. Smith, 1994, Eur. J. Pharmacol. 268, 247). Quantitative autoradiography showed that the highest density of opioid binding sites appeared to be present within the alveolar wall (13.2 +/- 0.8 pmol/mg protein). A significantly lower (P < 0.05) density of binding was also observed in the smooth muscle of the trachea and main bronchi (5.5 +/- 2.1 pmol/mg protein). However, no morphine binding sites were evident in the smooth muscle surrounding the smaller airways and pulmonary vasculature within the lobes of the rat lung. It remains to be investigated whether the opioid binding sites located within the trachea and main bronchi of the rat airways are the prejunctional opioid receptors on C-afferent nerve fibres which modulate the release of potent inflammatory neuropeptides.

Characterization of non-conventional opioid binding sites in rat and human lung.

Indirect evidence suggests that nebulized morphine relieves dyspnoea and bronchoconstriction via opioid receptors within the lung. This study used equilibrium binding studies to characterize opioid binding sites in lung membrane preparations. [3H]Morphine and [3H]naloxone were incubated separately with homogenates of Wistar rat brain and lung, and human lung. Binding affinities for both morphine and naloxone in rat and human lung were two orders of magnitude lower than those in brain. However, opioid binding site densities in lung were up to 100 times greater than that in brain. The addition of Na+ or GTP to lung homogenate preparations caused atypical effects on opioid binding. Na+ (50 mM) decreased the specific binding of [3H]naloxone 50% viz-à-vis a 20% increase in binding in the brain. GTP (100 microM) caused a 200% increase in the apparent capacity of morphine binding in the lung compared with a marked decrease in binding in the brain.