Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells ( an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Aims: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness(1). The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. Methods and results: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. Conclusions: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Role of SOCS-1 Gene on Melanoma Cell Growth and Tumor Development

Melanoma is the most aggressive form of skin cancer, and its incidence has increased dramatically over the years. The murine B16F10 melanoma in syngeneic C57Bl/6 mice has been used as a highly aggressive model to investigate tumor development. Presently, we demonstrate in the B16F10-Nex2 subclone that silencing of SOCS-1, a negative regulator of Jak/Stat pathway, leads to reversal of the tumorigenic phenotype and inhibition of melanoma cell metastasis. SOCS-1 silencing with short hairpin RNA affected tumor growth and cell cycle regulation with arrest at the S phase with large-sized nuclei, reduced cell motility, and decreased melanoma cell invasion through Matrigel. A clonogenic assay showed that SOCS-1 acted as a modulator of resistance to anoikis. In addition, down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor ( mainly the phosphorylated-R), Ins-R alpha, and fibroblast growth factor receptor. In vivo, silencing of SOCS-1 inhibited subcutaneous tumor growth and metastatic development in the lungs. Because SOCS-1 is expressed in most melanoma cell lines and bears a relation with tumor invasion, thickness, and stage of disease, the present results on the effects of SOCS-1 silencing in melanoma suggest that this regulating protein can be a target of cancer therapy.

Pivotal Role for Platelet-Activating Factor Receptor in CD36 Expression and oxLDL Uptake by Human Monocytes/Macrophages

The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined. Copyright (C) 2011 S. Karger AG, Basel

Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel

Cyclooxygenase-2 and vascular endothelial growth factor expression in 5-fluorouracil-induced oral mucositis in hamsters: evaluation of two low-intensity laser protocols

The aim of this study was to investigate the mechanisms whereby low-intensity laser therapy may affect the severity of oral mucositis. A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil followed by surface irritation. Animals were randomly allocated into three groups and treated with a 35 mW laser, 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. This tissue was used for immunohistochemistry for cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and factor VIII (marker of microvessel density) and the resulting staining was quantified. Peak severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW laser and control groups. This reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a significantly lower level of COX-2 staining. The 100 mW laser did not have an effect on the severity of clinical mucositis, but was associated with a decrease in VEGF levels at the later time-points, as compared to the other groups. There was no clear relationship of VEGF levels or microvessel density to clinical mucositis severity. The tissue response to laser therapy appears to vary by dose. Low-intensity laser therapy appears to reduce the severity of mucositis, at least in part, by reducing COX-2 levels and associated inhibition of the inflammatory response.

Fibroblast growth factor 2 restrains ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant YI adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated -galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Yl adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either YI or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Rasdependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RboAGTP. Surprisingly, attempts to select FGF2-resistant cells from the Yl and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Rasdependent malignant cells could rarely overcome.

Keratinocyte growth factor: a new mesothelial targeted therapy to reduce postoperative pericardial adhesions

Background: Several methods have been utilized to prevent pericardial and retrosternal adhesions, but none of them evaluated the mesothelial regenerative hypothesis. There are evidences that the mesothelial trauma reduces pericardial fibrinolytic capability and induces an adhesion process. Keratinocyte growth factor (KGF) has proven to improve mesothelial cells proliferation. This study investigated the influence of keratinocyte growth factor in reducing post-surgical adhesions. Methods: Twelve pigs were operated and an adhesion protocol was employed. Following a stratified randomization, the animals received a topical application of KGF or saline. At 8 weeks, intrapericardial adhesions were evaluated and a severity score was established. The time spent to dissect the adhesions and the amount of sharp dissection used, were recorded. Histological sections were stained with sirius red and morphometric analyses were assessed with a computer-assisted image analysis system. Results: The severity score was lower in the KGF group than in the control group (11.5 vs 17, p = 0.005). The dissection time was lower in the KGF group (9.2 +/- 1.4 min vs 33.9 +/- 9.2 min, p = 0.004) and presented a significant correlation with the severity score (r = 0.83, p = 0.001). A significantly less sharp dissection was also required in the KGF group. Also, adhesion area and adhesion collagen were significantly tower in the KGF group than in the control group. Conclusion: The simulation of pericardial cells with KGF reduced the intensity of postoperative adhesions and facilitated the re-operation. This study suggests that the mesothelial regeneration is the new horizon in anti-adhesion therapies. (C) 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Synergism Between Keratinocyte Growth Factor and Carboxymethyl Chitosan Reduces Pericardial Adhesions

Background. Mesothelial injury is the pivot in the development of adhesions. An increase in the proliferation of mesothelial cells was verified by in vitro studies with the use of keratinocyte growth factor (KGF). This study investigated the influence of KGF associated with thermo-sterilized carboxymethyl chitosan (NOCCts) in the reduction of pericardial adhesions. Methods. An induction model of pericardial adhesion was carried out in 24 pigs. Animals were randomly allocated to receive topical application of KGF, KGF + NOCCts, NOCCts, or saline (control). At 8 weeks, intra-pericardial adhesions were evaluated and a severity score was established. The time spent to dissect the adhesions and the amount of sharp dissection used, were recorded. Histologic sections were stained with sirius red for a morphometric evaluation using a computer-assisted image analysis system. Cytokeratin AE1/AE3 immunostaining were employed to identify mesothelial cells. Results. The severity score expressed in median (minimum to maximum), in relation to the control group (17 [15 to 18]), was lower in the KGF + NOCCts group (7 [6 to 9], p < 0.01) followed by the KGF group (11.5 [9 to 12], 0.01 < p < 0.05) and the NOCCts group (12 [9 to 14], p > 0.05). The dissection time was significantly lower in the KGF + NOCCts group (7.1 +/- 0.6 vs 33.9 +/- 9.2 minutes, p < 0.001). A significantly less sharp dissection was also required in the KGF + NOCCts group. In the adhesion segment, a decreased collagen proportion was found in the KGF + NOCCts group (p < 0.05). Mesothelial cells were present more extensively in groups in which KGF was delivered (p = 0.01). Conclusions. The use of KGF associated with NOCCts resulted in a synergic action that decreases postoperative pericardial adhesions in a highly significant way. (Ann Thorac Surg 2010; 90: 566-72) (C) 2010 by The Society of Thoracic Surgeons

Poly (A)(+) Transcriptome Assessment of ERBB2-Induced Alterations in Breast Cell Lines

We report the first quantitative and qualitative analysis of the poly (A)(+) transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Vandetanib Plus Docetaxel/Prednisolone in Patients with Hormone-Refractory Prostate Cancer

Vandetanib (ZACTIMA(TM)) is a once-daily oral anticancer drug that selectively inhibits vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection signaling. This randomized (1: 1), double-blind study evaluated vandetanib (100mg/day) or placebo in combination with docetaxel (D; 75mg/m(2) every 3 weeks) and prednisolone (P; 2 x 5 mg/day) in 86 patients with metastatic hormone-refractory prostate cancer (mHRPC). The primary assessment was prostate-specific antigen (PSA) response (confirmed reduction of >= 50% from baseline) and a greater number of patients showed a PSA response with placebo + DP (67%) versus vandetanib + DP (40%); hazard ratio = 2.23 (one-sided 80% confidence limit = 2.90; one-sided p = 0.99). More patients experienced progression events (disease progression or death from any cause) with vandetanib + DP (65%) versus placebo + DP (60%); hazard ratio = 1.13 (one-sided 80% confidence limit = 1.44; one-sided p = 0.67). The overall incidence of adverse events was similar in both groups, although more patients experienced adverse events, leading to permanent discontinuation with vandetanib + DP (28%) versus placebo + DP (12%). However, the safety and tolerability profile for vandetanib was similar to that previously reported; adverse events that occurred more frequently in the vandetanib + DP arm were hypertension (14% vs. 2%), erythematous rash (14% vs. 2%), and exfoliative rash (12% vs. 2%). In this study of patients with mHRPC, vandetanib + DP did not demonstrate any efficacy benefit, compared with placebo + DP.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via Fc epsilon RI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express Fc epsilon RI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were Fc epsilon RI, LAT and alpha-galactosyl derivatives of ganglioside GD(1b) mobilized to lipid raft domains following Fc epsilon RI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of beta-hexosaminidase activity after Fc epsilon RI activation. The two mutant cell lines have a reduced release of beta-hexosaminidase activity after Fc epsilon RI stimulation, but not after exposure to calcium ionophore. These results indicate that the alpha-galactosyl derivatives of ganglioside GD(1b) are important in the initial events of Fc epsilon RI signaling upstream of Ca(2+) influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via Fc epsilon RI. (c) 2008 Published by Elsevier Inc.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression

Aims: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects oil bone. Thus. we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. Main methods: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (Phosphate buffer saline Solution (n = 24); (G2) administration of empty liposomes (it = 24): (Q) administration 20 rig of EGF Solution (n = 24): and (G4) 20 ng of EGF-liposomes Solution (it = 24). Each Solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 2 1 after drug administration. 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP) + cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappa B ligand) and epidermal growth factor receptor (EGFR) expression. Key findings: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. Significance: Local delivery of EGF-liposome stimulates, osteoclastogenesis and tooth movement. (C) 2009 Elsevier Inc. All rights reserved.

Vaccination, atherosclerosis and systemic lupus erythematosus

Atherosclerosis is an inflammatory disease, leading to the formation of pro-inflammatory and pro-oxidative lipids that generate an immune response. Several antigens have been shown to activate the immune response and affect the development of atherogenesis. Systemic lupus erythematosus is an autoimmune and inflammatory disease strongly associated with premature development of atherosclerotic plaques. Modulation of the immune system could represent a useful approach to prevent and/or treat atherosclerosis. A vaccination-based approach might be a useful, effective tool in the modern arsenal of cardiovascular therapies and could be used on a large scale at a low cost. In non-systemic lupus erythematosus populations, vaccines against oxidized low-density lipoprotein, beta-2-glycoprotein I, heat shock proteins, lipoproteins, cholesterol, molecules involved in cholesterol metabolism, and other molecules (CD99, vascular endothelial growth factor-receptor, and interleukin-2) have been tested, with promising results. However, there are no studies of vaccination against atherosclerosis in systemic lupus erythematosus. Lupus (2009) 18, 1209-1212.