Nicolás Tobar
University of Chile
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Featured researches published by Nicolás Tobar.
Molecular and Cellular Biochemistry | 2010
Nicolás Tobar; Victor Villar; Juan F. Santibanez
TGF-β1 has been postulated as a pro-oncogenic factor in the late step of the tumoral progression. In transformed cells, TGF-β1 enhances the capacity to degrade the extracellular matrix, cell invasiveness and epithelial-mesenchymal transition, which are crucial steps for metastasis. Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) are critical components in cell migration and invasion induced by TGF-β1, however, the exact mechanism by which TGF-β1 regulates uPA and MMP-9 is not well elucidated so far. In the present study, we analyzed the role of ROS-NFκΒ, signal as mediator in the cell malignity enhancement by TGF-β1. We found that TGF-β1 activates NFκΒ, through Rac1-NOXs-ROS-dependent mechanism. Our results shows that TGF-β1 stimulation of uPA and MMP-9 expression involve NOXs-dependent ROS and NFκΒ, activation, demonstrated by using DPI, NOXs inhibitor, ROS scavenger N-acetylcysteine and SN50, an NFkb inhibitor. Furthermore, we found that the inhibition of ROS and NFκΒ, abrogates TGF-β1 stimulation of EMT, cell motility and invasion. Thus, ROS-NFκΒ acts as the crucial signal in TGF-β1-induced uPA and MMP-9 expression thereby mediating the enhancement of cellular malignity by TGF-β1.
British Journal of Cancer | 2010
Nicolás Tobar; Javier Guerrero; Patricio C. Smith; Jorge Martínez
Background:The influence of the stromal microenvironment on the progression of epithelial cancers has been demonstrated. Unravelling the mechanisms by which stromal cells affect epithelial behaviour will contribute in understanding cellular malignancy. It has been proposed that redox environment has a role in the acquisition of malignancy. In this work, we studied the influence of epithelial cells on the stromal redox status and the consequence of this phenomenon on MCF-7 cell motility.Methods:We analysed in a co-culture system, the effect of RMF-EG mammary stromal cells on the migratory capacity of MCF-7 cell line. To test whether the NOX-dependent stromal redox environment influences the epithelial migratory behaviour, we knocked down the expression of NOX4 using siRNA strategy. The effect of TGF-β1 on NOX4 expression and activity was analysed by qPCR, and intracellular ROS production was measured by a fluorescent method.Results:Migration of MCF-7 breast epithelial cells was stimulated when co-cultured with RMF-EG cells. This effect depends on stromal NOX4 expression that, in turn, is enhanced by epithelial soluble factors. Pre-treatment of stromal cells with TGF-β1 enhanced this migratory stimulus by elevating NOX4 expression and intracellular ROS production. TGF-β1 seems to be a major component of the epithelial soluble factors that stimulate NOX4 expression.Conclusions:Our results have identified that an increased stromal oxidative status, mainly provided by an elevated NOX4 expression, is a permissive element in the acquisition of epithelial migratory properties. The capacity of stromal cells to modify their intracellular ROS production, and accordingly, to increase epithelial motility, seems to depend on epithelial soluble factors among which TGF-β1 have a decisive role.
Cancer Letters | 2008
Nicolás Tobar; Mónica Cáceres; Juan Francisco Santibáñez; Patricio C. Smith; Jorge Martínez
In the present study, we demonstrated that changes in Rac1 activity associated with the production of intracellular ROS modulate the migratory properties in MCF-7 and T47D human mammary cell lines. We also described that the NFkappaB pathway exerts a downstream control on the expression of the ROS-dependent cellular migratory potential. These results emphasize the importance of redox balance in the acquisition of malignancy and support previous data sustaining that an oxidative environment predisposes cells to activate signal-transduction pathways actively involved in cellular oncogenesis. Our data also provides evidence that NADPH oxidase could constitute the main source of intracellular ROS in response to changes in Rac1 activity. We suggest that Rac1 plays a role in cellular migration not only limited to its known function in reorganization of the actin cytoskeleton, but also as part of the intracellular machinery that controls the redox balance.
Molecular and Cellular Endocrinology | 2012
Mariana Cifuentes; Cecilia Fuentes; Nicolás Tobar; Ingrid Acevedo; Elisa Villalobos; Eric R. Hugo; Nira Ben-Jonathan; Marcela Reyes
The proinflammatory status of adipose tissue has been linked to the metabolic and cardiovascular consequences of obesity. Human adipose cells express the calcium sensing receptor (CaSR), and its expression is elevated in inflammatory states, such as that associated with obesity. Given the CaSRs association with inflammation in other tissues, we evaluated its role elevating the adipose expression of inflammatory factors. The CaSR activation by the calcimimatic cinacalcet (5μM) in adipose tissue and in vitro cultured LS14 adipose cells elicited an elevation in the expression of the proinflammatory cytokines IL6, IL1β, TNFα, and the chemoattractant CCL2. This was in part reverted by SN50, an inhibitor of the inflammatory mediator nuclear factor kappa B (NFκB). Our observations suggest that CaSR activation elevates cytokine and chemokine production, partially mediated by NFκB. These findings support the relevance of the CaSR in the pathophysiology of obesity-induced adipose tissue dysfunction, with an interesting potential for pharmacological manipulation.
Breast Cancer Research and Treatment | 2010
Javier Guerrero; Nicolás Tobar; Mónica Cáceres; Lorena Espinoza; Paula Escobar; Javier Dotor; Patricio C. Smith; Jorge Martínez
In carcinomas such as those of breast, pancreas, stomach, and colon, cancer cells support the expansion of molecular and cellular stroma in a phenomenon termed desmoplasia, which is characterized by a strong fibrotic response. In the case of breast tissue, in which stroma is mainly a fatty tissue, this response presumably occurs at the expense of the adipose cells, the most abundant stromal phenotype, generating a tumoral fibrous structure rich in fibroblast-like cells. In this study, we aimed to determine the cellular mechanisms by which factors present in the media conditioned by MDA-MB-231 and MCF-7 human breast cancer cell lines induce a reversion of adipose cells to a fibroblastic phenotype. We demonstrated that soluble factors generated by these cell lines stimulated the reversion of mammary adipose phenotype evaluated as intracellular lipid content and expression of C/EBPα and PPARγ. We also demonstrated that exogenous TGF-β1 and TNF-α exerts a similar function. The participation of both growth factors, components of media conditioned by tumoral mammary cells, on the expression and nuclear translocation of C/EBPα and PPARγ was tested in 3T3-L1 cells by interfering with the inhibitory effects of media with agents that block the TGF-β1 and TNF-α activity. These results allow us to postulate that TGF-β1 and TNF-α present in this media are in part responsible for this phenotypic reversion.
Archives of Biochemistry and Biophysics | 2010
Mariana Cifuentes; Cecilia Fuentes; Pamela Mattar; Nicolás Tobar; Eric R. Hugo; Nira Ben-Jonathan; Cecilia Rojas; Jorge Martínez
Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.
Journal of Dental Research | 2013
R. Arancibia; Cristian Maturana; D. Silva; Nicolás Tobar; C. Tapia; Juan C. Salazar; Jorge Martínez; Patricio C. Smith
Chitosan is a naturally derived polymer with antimicrobial and anti-inflammatory properties. However, studies evaluating the role of chitosan in the control of periodontal pathogens and the responses of fibroblasts to inflammatory stimuli are lacking. In the present study, we analyzed whether chitosan particles may inhibit the growth of periodontal pathogens and modulate the inflammatory response in human gingival fibroblasts. Chitosan particles were generated through ionic gelation. They inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans at 5 mg/mL. Conversely, IL-1β strongly stimulated PGE2 protein levels in gingival fibroblasts, and chitosan inhibited this response at 50 µg/mL. IL-1β–stimulated PGE2 production was dependent on the JNK pathway, and chitosan strongly inhibited this response. IL-1β stimulated NF-κB activation, another signaling pathway involved in PGE2 production. However, chitosan particles were unable to modify NF-κB signaling. The present study shows that chitosan exerts a predominantly anti-inflammatory activity by modulating PGE2 levels through the JNK pathway, which may be useful in the prevention or treatment of periodontal inflammation.
Journal of Periodontology | 2013
R. Arancibia; Alejandro Oyarzún; Daniel Silva; Nicolás Tobar; Jorge Martínez; Patricio C. Smith
BACKGROUND Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components. METHODS Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-β1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-β1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-β1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. RESULTS TGF-β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-β1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-β1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. CONCLUSIONS TNF-α inhibits several cell responses induced by TGF-β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.
Carcinogenesis | 2014
Nicolás Tobar; M.Celeste Avalos; Nicolás Méndez; Patricio C. Smith; Carmelo Bernabeu; Miguel Quintanilla; Jorge Martínez
It has been proposed that epithelial cells can acquire invasive properties through exposure to paracrine signals originated from mesenchymal cells within the tumor microenvironment. Transforming growth factor-β (TGF-β) has been revealed as an active factor that mediates the epithelial-stroma cross-talk that facilitates cell invasion and metastasis. TGF-β signaling is modulated by the coreceptor Endoglin (Eng), which shows a tumor suppressor activity in epithelial cells and regulates the ALK1-Smad1,5,8 as well as the ALK5-Smad2,3 signaling pathways. In the current work, we present evidence showing that cell surface Eng abundance in epithelial MCF-7 breast cancer cells is inversely related with cell motility. Shedding of Eng in MCF-7 cell surface by soluble matrix metalloproteinase-14 (MMP-14) derived from the HS-5 bone-marrow-derived cell line induces a motile epithelial phenotype. On the other hand, restoration of full-length Eng expression blocks the stromal stimulus on migration. Processing of surface Eng by stromal factors was demonstrated by biotin-neutravidin labeling of cell surface proteins and this processing generated a shift in TGF-β signaling through the activation of Smad2,3 pathway. Stromal MMP-14 abundance was stimulated by TGF-β secreted by MCF-7 cells acting in a paracrine manner. In turn, the stromal proteolytic activity of soluble MMP-14, by inducing Eng shedding, promoted malignant progression. From these data, and due to the capacity of TGF-β to regulate malignancy in epithelial cancer, we propose that stromal-dependent epithelial Eng shedding constitutes a putative mechanism that exerts an environmental control of cell malignancy.
BMC Cancer | 2014
Nicolás Tobar; Marcela Toyos; Carla Urra; Nicolás Méndez; R. Arancibia; Patricio C. Smith; Jorge Martínez
BackgroundHard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-β1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts.MethodsWe used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-β1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-β1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation.ResultsTGF-β1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-β1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-β1-dependent expression of CTGF, NOX4 and α-SMA.ConclusionsHuman mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.