Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1) activity and increase airway smooth muscle contraction in asthma

Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms

Matricellular Proteins Produced by Melanocytes and Melanomas: In Search for Functions

Matricellular proteins are modulators of cell-matrix interactions and cellular functions. The group includes thrombospondin, osteopontin, osteonectin/SPARC, tenascin, disintegrins, galectins and CCN proteins. The production of matricellular proteins such as osteopontin, SPARC or tenascin is highly upregulated in melanoma and other tumors but little is known about their functions in tumor growth, survival, and metastasis. The distribution pattern of CCN3 differs from most other matricellular proteins, such that it is produced abundantly by normal melanocytes, but is not significantly expressed in melanoma cells. CCN3 is known to inhibit melanocyte proliferation and stimulate adhesion to collagen type IV, the main component of the basement membrane. CCN3 has a unique role in securing adhesion of melanocytes to the basement membrane distinct from other melanoma-produced matricellular proteins which act as de-adhesive molecules and antagonists of focal adhesion. Qualitative and quantitative changes in matricellular protein expression contribute to melanoma progression similar to the E-cadherin to N-cadherin class switch, allowing melanoma cells to escape from keratinocyte control

Cytostatic effect of novel mTOR inhibitor, PRP-1 (galarmin) in MDA 231 (ER-) breast carcinoma cell line. PRP-1 inhibits mesenchymal tumors

Activation of the PI3K-Akt-mTOR pathway is implicated both in the establishment of tumors and as well as a target for therapy in many types of solid malignancy, its blockade represents an opportunity to improve outcomes in patients with tumors that are associated with poor prognosis. Our experimental data indicates that proline-rich polypeptide-1 (PRP-1, galarmin) is immunomodulator cytokine, produced by hypothalamic neurosecretory cells and exerts its antiproliferative effect on the tumor cells of mesenchymal origin via inhibiting mTOR kinase activity and repressing cell cycle progression. The goal of these investigations was to elucidate the antiproliferative action of PRP-1 on the breast carcinoma cell line MDA 231 (ER-) and to compare PRP-1 action previously reported on other mesenchymal tumors. These experiments confirmed maximum inhibition of cell growth at 0.5 and 1 μg/ml PRP-1 (71% and 63%, respectively) and inhibition at 10 μg/ml of 44%. There was no inhibitory effect observed on luminal T47-D (ER+) cells. Videomicroscopy results demonstrated dividing cells in the cytokine-treated MDA 231 (ER-), suggesting that the cells were not in the state of dormancy. The flow cytometry experiments confirmed that PRP-1-treated cells were accumulated in S phase. No apoptosis, caspase activation, or senescence was detected after treatment with this cytokine. Experiments with mTOR with PRP-1 (10 μg/ml) indicated statistically significant 40% inhibition of mTOR kinase activity in immunoprecipitates of the MDA 231 (ER-) cell line. PRP-1 is a novel mTOR inhibitor with strong antiproliferative action in mesenchymal tumors mostly resistant to radiation and chemotherapy

Antioxidant and Electron Donating Function of Hypothalamic Polypeptides: Galarmin and Gx-NH2

Chemical mechanisms of antioxidant and electron donating function of the hypothalamic proline-rich polypeptides have been clarified on the molecular level. The antioxidant-chelating property of Galarmin and Gx-NH2 was established by their capability to inhibit copper(II) dichloride catalyzed H2O2 decomposition, thus preventing formation of HO• and HOO• radicals. The antiradical activity of Galarmin and Gx-NH2 was determined by their ability to react with 2,2-diphenyl-1-picrylhydrazyl radical applying differential pulse voltammetry and UV–Vis spectrophotometry methods. Galarmin manifest antiradical activity towards 2,2-diphenyl-1-picrylhydrazyl radical, depending on the existence of phenolic OH group in tyrosine residue at the end of the molecule. The presence of antiradical activity and reduction properties of Galarmin are confirmed by the existence of an oxidation specific peak in voltammograms made by differential pulse voltammetry at E ∘ = 0.795 V vs. Ag/Ag+ aq

PRP-1 significantly decreases the [ALDH.sup.high] cancer stem cell population and regulates the aberrant Wnt/[beta]-catenin pathway in human chondrosarcoma JJ012 cells

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline-rich poly-peptide 1 (PRP-1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP-1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/[beta]-catenin signaling. A significant decrease in [ALDH.sup.high] CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP-1. For [RT.sup.2] profiler PCR array analysis of Wnt/[beta]-catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) [ALDH.sup.high] cells sorted from untreated JJ012 cells ([ALDH.sup.high-untreated]); and iii) [ALDH.sup.low] cells sorted from PRP-1-treated JJ012 cells ([ALDH.sup.low-PRP-1]). The expression levels of Wnt/[beta]-catenin signaling genes were determined to be downregulated in the [ALDH.sup.high-untreated] cells and upregulated in [ALDH.sup.low-PRP-1] cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be down-regulated in the [ALDH.sup.low-PRP-1] cells. Immunocytochemistry demonstrated the localization of [beta]-catenin in the nuclei of the PRP-1-treated cells. Western blotting indicated increased [beta] -catenin expression in the [ALDH.sup.low-PRP-1] cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP-1 and [beta]-catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of [beta]-catenin following PRP-1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP-1 resulted in a significant decrease in cytoplasmic [beta]-catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of [beta]-catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/[beta]-catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.Academi

Utility of quantitative computerized pain drawings in a sample of spinal stenosis patients

To evaluate the utility of quantitative computerized pain drawings (CPDs) in a sample of spine patients before and after surgery. Analysis of changes in quantified CPDs, the Oswestry Disability Index (ODI), the Short Form-36 Health Survey Questionnaire (SF-36), and numerical ratings of pain intensity before and after surgery. Private clinic in large metropolitan area. Patients. Forty-six patients with spinal stenosis. Interventions. Surgery for the relief of pain due to spinal stenosis. A total points (TP) score was calculated from the CPD that reflected the total number of pixels filled by the patient, and the percentage of total pain area indicated as aching, stabbing, numbness, pins and needles, burning, and other, were each calculated separately. CPD scores, ODI score, Physical Components Summary (PCS) and Mental Components Summary scores of the SF-36, and pain intensity ratings (0-10 scale) were all recorded before and after surgical intervention. Results. After surgery, patients showed significant improvements in the extent of shaded pain area of the CPD, pain intensity ratings, ODI, and SF-36 PCS scores (paired t-test, P < or = 0.01). Changes in TP scores calculated from the CPDs were significantly correlated (P < or = 0.05) with changes in ODI scores (r = 0.34) and pain intensity ratings (r = 0.37). Changes in the percentage of total pain area covered by specific qualities of pain were not significant. Results from the present study provide initial support for the use of automated quantified data collected from CPDs to evaluate treatment interventions and to serve the clinician as a record of changes in spatial location, radiation or extent of pain, and the sensory quality of pain when evaluating individual patient needs