24 research outputs found
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
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Proline Rich Polypeptide (PRP-1) Increases the Superoxide-Producing and Ferrihemoglobin Reducing Activities of Cytochrome B558 Isoforms from Human Lymphosarcoma Tissue Cells
The two cytochromes (cyt) b558 of acidic nature, one—95–100 kDa and another one, 60–70 kDa were isolated for the first time from the human’s lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O2
−-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 μg) with cytochrome b558, caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b558. PRP-1 activated both NADPH dependent O2
−-producing and ferriHb-reducing activities of the cyt b1
558 and cyt b2
558, obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b558 on the surface of the tumor cells by increasing both NADPH dependent O2
−-producing and ferriHb-reducing activities of cyt b558, increases the oxidation- reduction status. Changing the oxidation–reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine
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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
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Effect of hypothalamic proline-rich peptide (PRP-1) on neuronal and bone marrow cell apoptosis
The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we investigated the influence of PRP-1 on staurosporine-induced apoptosis of postnatal hippocampal cells and on doxorubicin-induced bone marrow granulocyte- and monocyte apoptosis. The intention was to further characterize the effect of PRP-1 on the survival rate of neurons and in context with myelopoiesis. We demonstrate that PRP-1 significantly reduced apoptosis of postnatal hippocampal cells induced by staurosporine. The protective effect of PRP-1 against apoptotic cell death was shown to be both time- and dose-dependent. Neuroprotection was more pronounced after prolonged pretreatment of the cells with PRP-1 before the induction of apoptosis with staurosporine. The related peptide [arg(8)]vasopressin did not reveal neuroprotection. PRP-1 also significantly reduced apoptosis of bone marrow monocytes and granulocytes induced by doxorubicin. This protective effect lasted for 2-4 h and was not detectable anymore after 24 h when PRP-1 and doxorubicin were added simultaneously. Previously obtained data and results of the current studies suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate myelopoiesis and neuron survival as we provide evidence that PRP can differentially reduce both staurosporine- and doxorubicin-induced hippocampal and bone marrow cell apoptosis
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Morpho-functional study of the hypothalamic proline-rich polypeptide apoptotic activity against mouse Ehrlich ascites carcinoma
A new type of bioactive polypeptides of the neurosecretory hypothalamus called proline-rich peptides (PRPs), which are isolated from bovine neurosecretory granules of the neurohypophysis, are synthesized in the form of a common precursor protein (neurophysin vasopressin-associated glycoprotein). Proline-rich polypetide 1 (PRP-1; also known as galarmin) is comprised of 15 amino acids residues, and has been suggested to possess anti-neurodegenerative, immunoregulatory, hematopoietic, antimicrobial and antitumor properties. The cytostatic, antiproliferative effect of PRP-1 was demonstrated in the human chondrosarcoma JJ012 and triple negative breast carcinoma MDA MB 231 cell lines. PRP-1 action is disease and tissue specific. To further explore the antitumorigenic and possible cytotoxic effects of PRP-1, a morpho-functional study on the effect of PRP-1 on a mouse Ehrlich ascites carcinoma (EAC) model was conducted. The PRP-1-induced morphological features of EAC cells confirmed the apoptotic nature of PRP-1, as manifested by cell shrinkage, membrane blebbing, chromosome condensation (pyknosis) and nuclear fragmentation (karyorrhexis). The effect of PRP-1 on the number of tumor cells incubated for 24 h and their viability in trypan blue-stained samples lead to a 44% reduction in the number of viable cells on day 11 post-inoculation vs. 22% inhibition of viable cells after PRP-1 treatment (0.1 µg/ml) on day 7 post-inoculation. Apoptosis experiments using an Annexin V-cyanine 3 apoptosis detection kit indicated that 24 h incubation with 0.1 µg/ml PRP-1 caused a significant increase in the number of apoptotic cells, reaching 50.33%, compared to 8.33% in the sample control on day 7 post-inoculation
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Cancer stem cells as a therapeutic target in 3D tumor models of human chondrosarcoma: An encouraging future for proline rich polypeptide‑1
Chondrosarcoma is a malignant bone neoplasm that is refractory to chemotherapy and radiation. With no current biological treatments, mutilating surgical resection is the only effective treatment. Proline rich polypeptide 1 (PRP‑1), which is a 15‑amino acid inhibitor of mammalian target of rapamycin complex‑1 (mTORC1), has been indicated to exert cytostatic and immunomodulatory properties in human chondrosarcoma cells in a monolayer. The aim of the present study was to evaluate the effects of PRP‑1 on an in vitro 3D chondrosarcoma tumor model, known as spheroids, and on the cancer stem cells (CSCs) which form spheroids. JJ012 cells were cultured and treated with PRP‑1. An ALDEFLUOR™ assay was conducted (with N,N‑diethylaminobenzaldehyde as the negative control) to assess aldehyde dehydrogenase (ALDH) activity (a recognized CSC marker), and bulk JJ012, ALDHhigh and PRP‑1 treated ALDHlow cells were sorted using flow cytometry. Colony formation and spheroid formation assays of cell fractions, including CSCs, were used to compare the PRP‑1‑treated groups with the control. CSCs were assessed for early apoptosis and cell death with a modified Annexin V/propidium iodide assay. Western blotting was used to identify mesenchymal stem cell markers (STRO1, CD44 and STAT3), and spheroid self‑renewal assays were also conducted. A clonogenic dose‑response assay demonstrated that 20 µg/ml PRP‑1 was the most effective dose for reducing colony formation capacity. Furthermore, CSC spheroid growth was significantly reduced with increasing doses of PRP‑1. Annexin V analysis demonstrated that PRP‑1 induced CSC cell death, and that this was not attributed to apoptosis or necrosis. Western blot analysis confirmed the expression of mesenchymal markers, and the spheroid self‑renewal assay confirmed the presence of self‑renewing CSCs. The results of the present study demonstrate that PRP‑1 eliminates anchorage independent CSC growth and spheroid formation, indicating that PRP‑1 likely inhibits tumor formation in a murine model. Additionally, a decrease in non‑CSC bulk tumor cells indicates an advantageous decline in tumor stromal cells. These findings confirm that PRP‑1 inhibits CSC proliferation in a 3D tumor model which mimics the behavior of chondrosarcoma in vivo