EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats

Objectives: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. Materials and methods: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. Results: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. Conclusions: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

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.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9 A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2.

Fragment-guided approach to incorporating structural information into a CoMFA study: BACE-1 as an example

Alzheimer`s disease is an ultimately fatal neurodegenerative disease, and BACE-1 has become an attractive validated target for its therapy, with more than a hundred crystal structures deposited in the PDB. In the present study, we present a new methodology that integrates ligand-based methods with structural information derived from the receptor. 128 BACE-1 inhibitors recently disclosed by GlaxoSmithKline R&D were selected specifically because the crystal structures of 9 of these compounds complexed to BACE-1, as well as five closely related analogs, have been made available. A new fragment-guided approach was designed to incorporate this wealth of structural information into a CoMFA study, and the methodology was systematically compared to other popular approaches, such as docking, for generating a molecular alignment. The influence of the partial charges calculation method was also analyzed. Several consistent and predictive models are reported, including one with r (2) = 0.88, q (2) = 0.69 and r (pred) (2) = 0.72. The models obtained with the new methodology performed consistently better than those obtained by other methodologies, particularly in terms of external predictive power. The visual analyses of the contour maps in the context of the enzyme drew attention to a number of possible opportunities for the development of analogs with improved potency. These results suggest that 3D-QSAR studies may benefit from the additional structural information added by the presented methodology.

Learning preferences of TAFE and university students

Using Entwistle and Ramsden's (1983) Approaches to Studying Inventory, Fuller and Chalmers showed little difference between TAFE students and university students both in the factor structures derived through factor analysis, and in a comparison of individual subscale through analysis of variance. Fuller and Chalmers had hypothesised that differences in approaches to study may be identified as a result of the different learning and teaching contexts experienced by the two groups of students. The current study has examined the similarities and differences between students in the two sectors using a learning preferences inventory. Learning preferences were chosen for investigation on the basis of previous theoretical work which suggests that it is preferences that are most influenced by environmental factors. The present study has shown significant similarity between the learning preferences of the two groups of students. However, there were also significant differences which indicate that the Fuller and Chalmers hypothesised differences may be more observable at a preferences level than at an approaches to study level.

Improved glusoce homeostasis and enhanced insulin signalling in Grb 14-deficient mice

Gene targeting was used to characterize the physiological role of growth factor receptor-bound (Grb)14, an adapter-type signalling protein that associates with the insulin receptor (IR). Adult male Grb14^-/- mice displayed improved glucose tolerance, lower circulating insulin levels, and increased incorporation of glucose into glycogen in the liver and skeletal muscle. In ex vivo studies, insulin-induced 2-deoxyglucose uptake was enhanced in soleus muscle, but not in epididymal adipose tissue. These metabolic effects correlated with tissue-specific alterations in insulin signalling. In the liver, despite lower IR autophosphorylation, enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and activation of protein kinase B (PKB) was observed. In skeletal muscle, IR tyrosine phosphorylation was normal, but signalling via IRS-1 and PKB was increased. Finally, no effect of Grb14 ablation was observed on insulin signalling in white adipose tissue. These findings demonstrate that Grb14 functions in vivo as a tissue-specific modulator of insulin action, most likely via repression of IR-mediated IRS-1 tyrosine phosphorylation, and highlight this protein as a potential target for therapeutic intervention.

Low-dose metronomic paclitaxel chemotherapy suppresses breast tumors and metastases in mice

This study investigated the use of low-dose metronomic (LDM) chemotherapy with paclitaxel in a highly metastatic mouse model of 4T1 breast cancers, and compared it with the maximum tolerable dose (MTD) therapy. LDM therapy displayed a stronger anti-tumor activity in suppressing primary and metastatic breast tumors with less degree of side effects, and stronger anti-angiogenic and anti-lymphangiogenic activities than MTD therapy. But MTD therapy showed stronger pro-apoptotic and anti-proliferative activities in situ. Paclitaxel therapy downregulated expression of vascular endothelial growth factor receptor-2 (VEGFR2) and up-regulated expression of thrombospondin-1. The results support the application of paclitaxel LDM therapy to treat advanced breast cancer.

Impact of resistance exercise training on interleukin-6 and JAK/STAT in young men

The JAK/STAT signaling pathway is essential for myogenic regeneration and is regulated by a diverse range of ligands, including interleukin-6 (IL-6) and platelet-derived growth factor-BB (PDGF-BB). Our aim was to evaluate the responsiveness of IL-6 and PDGF-BB to intense exercise, along with STAT3 activation, before and after 12 weeks of resistance training. In young men, IL-6 and PDGF-BB protein concentrations were quantified in biopsied muscle and increased at 3 h post-exercise (17.5-fold and 3-fold, respectively). The response was unaltered by 12 weeks of training. Similarly, STAT3 phosphorylation was elevated post-exercise (12.5-fold), irrespective of training status, as was the expression of downstream targets c-MYC (8-fold), c-FOS (4.5-fold), and SOCS3 (2.3-fold). Thus, intense exercise transiently increases IL-6 and PDGF-BB proteins, and STAT3 phosphorylation is increased. These responses are preserved after intense exercise. This suggests they are not modified by training and may be an essential component of the adaptive responses to intense exercise.

The need to examine metastatic tissue at the time of progression of breast cancer : is re-biopsy a necessity or a luxury?

Knowledge of estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor-2 (HER2) status is necessary for determining the optimal treatment of breast cancer patients. At the same time, the discordance between marker profiles (ER/PR and HER2) of primary and metastatic breast cancer is well documented. Whether discordant cases are secondary to “clonal selection” in the face of targeted anti-estrogen or anti-HER2 therapy or whether they are a laboratory artifact is still debated; both scenarios are likely. This article outlines current modalities for ER, PR, and HER2 testing in primary breast carcinoma and its metastases and reviews prospective and retrospective studies that have addressed these issues, as well as recent advances in the field.

SOCS proteins in development and disease

Cytokine and growth factor signaling mediates essential roles in the differentiation, proliferation, survival and function of a number of cell lineages. This is achieved via specific receptors located on the surface of target cells, with ligand binding activating key intracellular signal transduction cascades to mediate the requisite cellular outcome. Effective resolution of receptor signaling is also essential, with excessive signaling having the potential for pathological consequences. The Suppressor of cytokine signaling (SOCS) family of proteins represent one important mechanism to extinguish cytokine and growth factor receptor signaling. There are 8 SOCS proteins in mammals; SOCS1-7 and the alternatively named Cytokine-inducible SH2-containing protein (CISH). SOCS1-3 and CISH are predominantly associated with the regulation of cytokine receptor signaling, while SOCS4-7 are more commonly involved in the control of Receptor tyrosine kinase (RTK) signaling. Individual SOCS proteins are typically induced by specific cytokines and growth factors, thereby generating a negative feedback loop. As a consequence of their regulatory properties, SOCS proteins have important functions in development and homeostasis, with increasing recognition of their role in disease, particularly their tumor suppressor and anti-inflammatory functions. This review provides a synthesis of our current understanding of the SOCS family, with an emphasis on their immune and hematopoietic roles.

Bovine lactoferrin targets apoptotic and EGFR mechanisms in breast cancer

 This research investigated the anti-cancer effects of milk protein, lactoferrin. It was found that lactoferrin specifically induced cell death in breast cancer cells and was non-toxic to normal mammary gland cells. Key molecular mechanisms targeted by lactoferrin were elucidated in this study which provides important insight into the activity of this protein as an anti-cancer agent.

Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells

This study used the Eri silk nanoparticles (NPs) for delivering apo-bovine lactoferrin (Apo-bLf) (~2% iron saturated) and Fe-bLf (100% iron saturated) in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm) showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR)-positive MDA-MB-231 cells, while transferrin receptor (TfR) and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+) cells when compared to MCF-7 (EGFR-) cells. The expression of a prominent anticancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer.

Expressão do VEGFR-2 em polpas de dentes decíduos e permanentes jovens humanos

O conhecimento dos mecanismos celulares envolvidos nos fenômenos orgânicos fisiológicos e/ou patológicos, poderá conduzir a diagnósticos clínicos mais precisos, tratamentos mais direcionados e prognósticos mais favoráveis. Considerando esta premissa, foi objetivo deste estudo avaliar por meio de reação imunohistoquímica a presença ou ausência do vascular endothelial growth factor receptor-2 (VEGFR-2) em células endoteliais pulpares de dentes decíduos e permanentes jovens humanos, assim como a distribuição da sua imuno-marcação. A amostra foi constituída de nove dentes decíduos e quatro dentes permanentes jovens, selecionados de indivíduos de ambos os sexos, com idade entre 5 e 10 anos e 11 e 14 anos, respectivamente. Os espécimes foram fixados em formol diluído a 10% com tampão fosfato 0,1 M por 24 horas e descalcificados com solução de Ana Morse. Após, os mesmos foram processados e incluídos em parafina. Realizaram-se cortes de 5 µm de espessura. Uma lâmina de cada amostra foi corada pela técnica de hematoxilina e eosina (H&E) para análise histológica e as demais foram submetidas à reação imunohistoquímica enzimática. Nesta, utilizou-se como anticorpo primário o anti-h VEGFR-2 na diluição de 1:100 e anticorpo secundário biotinilado na diluição/proporção pré-estabelecidas pelo fabricante. Após o processamento das lâminas, os campos microscópicos mais significativos foram capturados e analisados qualitativamente quanto à presença ou ausência do VEGFR-2 nas células endoteliais pulpares, assim como à distribuição da imuno-marcação Os resultados observados mostraram que tanto os dentes decíduos quanto os dentes permanentes jovens apresentaram, especificamente, as células endoteliais pulpares imuno-marcadas para o VEGFR-2. Entretanto, verificou-se que nem todas apresentaram a mesma distribuição de marcação, sendo esta nos dentes decíduos mais evidente próxima à região subodontoblástica. As células endoteliais da polpa dos dentes permanentes jovens demonstraram no mesmo espécime estudado, ausência e presença de marcação endotelial, entretanto esta se apresentou de forma mais uniforme. Levando-se em consideração as limitações encontradas neste estudo, sugere-se que os dentes decíduos apresentam uma capacidade de resposta maior das células endoteliais pulpares ao anti-h VEGFR-2 quando comparados aos dentes permanentes.