Browsing by Author "Leyton, Lisette"
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Item Caveolin-1-Mediated Tumor Suppression Is Linked to Reduced HIF1α S-Nitrosylation and Transcriptional Activity in Hypoxia(2020) Sanhueza, Carlos; Castillo Bennett, Jimena; Valenzuela-Valderrama, Manuel; Contreras, Pamela; Lobos-González, Lorena; Campos, América; Wehinger, Sergio; Lladser, Álvaro; Kiessling, Rolf; Leyton, Lisette; Quest, Andrew F. G.Caveolin-1 (CAV1) is a well-established nitric oxide synthase inhibitor, whose function as a tumor suppressor is favored by, but not entirely dependent on, the presence of E-cadherin. Tumors are frequently hypoxic and the activation of the hypoxia-inducible factor-1α (HIF1α) promotes tumor growth. HIF1α is regulated by several post-translational modifications, including S-nitrosylation. Here, we evaluate the mechanisms underlying tumor suppression by CAV1 in cancer cells lacking E-cadherin in hypoxia. Our main findings are that CAV1 reduced HIF activity and Vascular Endothelial Growth Factor expression in vitro and in vivo. This effect was neither due to reduced HIF1α protein stability or reduced nuclear translocation. Instead, HIF1α S-nitrosylation observed in hypoxia was diminished by the presence of CAV1, and nitric oxide synthase (NOS) inhibition by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced HIF1α transcriptional activity in cells to the same extent as observed upon CAV1 expression. Additionally, arginase inhibition by (S)-(2-Boronoethyl)-L-cysteine (BEC) partially rescued cells from the CAV1-mediated suppression of HIF1α transcriptional activity. In vivo, CAV1-mediated tumor suppression was dependent on NOS activity. In summary, CAV1-dependent tumor suppression in the absence of E-cadherin is linked to reduced HIF1α transcriptional activity via diminished NOS-mediated HIF1α S-nitrosylation.Item Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation(Multidisciplinary Digital Publishing Institute (MDPI), 2019) Arce, Maximiliano; Pinto, Mauricio; Galleguillos, Macarena; Muñoz, Catalina; Lange, Soledad; Ramirez, Carolina; Erices, Rafaela; González, Pamela; Velásquez, Ethel; Tempio, Fabián; López, Mercedes; Salazar-Onfray, Flavio; Cautivo, Kelly; Kalergis, Alexis; Cruz, Sebastián; Lobos-González, Lorena; Lladser, Álvaro; Valenzuela, Guillermo; Olivares, Nixa; Sáez, Claudia; Koning, Tania; Sánchez, Fabiola; Fuenzalida, Patricia; Godoy, Alejandro; Contreras, Pamela; Leyton, Lisette; Lugano, Roberta; Dimberg, Anna; Quest, Andrew; Owen, GarethHypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.Item Extracellular Vesicles as Mediators of Cancer Disease and as Nanosystems in Theranostic Applications(2021) Burgos, Renato; Campos, América; Díaz, Magda; González, María Fernanda; León, Daniela; Lobos-González, Lorena; Leyton, Lisette; Kogan, Marcelo; Quest, AndrewCancer remains a leading cause of death worldwide despite decades of intense efforts to understand the molecular underpinnings of the disease. To date, much of the focus in research has been on the cancer cells themselves and how they acquire specific traits during disease development and progression. However, these cells are known to secrete large numbers of extracellular vesicles (EVs), which are now becoming recognized as key players in cancer. EVs contain a large number of different molecules, including but not limited to proteins, mRNAs, and miRNAs, and they are actively secreted by many different cell types. In the last two decades, a considerable body of evidence has become available indicating that EVs play a very active role in cell communication. Cancer cells are heterogeneous, and recent evidence reveals that cancer cell-derived EV cargos can change the behavior of target cells. For instance, more aggressive cancer cells can transfer their “traits” to less aggressive cancer cells and convert them into more malignant tumor cells or, alternatively, eliminate those cells in a process referred to as “cell competition”. This review discusses how EVs participate in the multistep acquisition of specific traits developed by tumor cells, which are referred to as “the hallmarks of cancer” defined by Hanahan and Weinberg. Moreover, as will be discussed, EVs play an important role in drug resistance, and these more recent advances may explain, at least in part, why pharmacological therapies are often ineffective. Finally, we discuss literature proposing the use of EVs for therapeutic and prognostic purposes in cancer.Publication Helicobacter pylori outer membrane vesicles induce astrocyte reactivity through nuclear factor-κappa B activation and cause neuronal damage in vivo in a murine model(2023) Palacios, Esteban; Lobos-González, Lorena; Guerrero, Simón; Kogan, Marcelo; Shao, Baohai; Heinecke, Jay; Quest, Andrew; Leyton, Lisette; Valenzuela, ManuelBackground: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. Methods: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. Results: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVβ3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. Conclusions: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.Publication Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models(2023) Lobos-González, Lorena; Oróstica, Lorena; Díaz, Natalia; Rojas, Victoria; Campos, America; Duran, Eduardo; Farfán, Nicole; Farfán, Nicole; Leyton, Lisette; Quest, AndrewCaveolin-1 (CAV1) is a membrane-bound protein that suppresses tumor development yet also promotes metastasis. E-cadherin is important in CAV1-dependent tumor suppression and prevents CAV1-enhanced lung metastasis. Here, we used murine B16F10 and human A375 melanoma cells with low levels of endogenous CAV1 and E-cadherin to unravel how co-expression of E-cadherin modulates CAV1 function in vitro and in vivo in WT C57BL/6 or Rag-/- immunodeficient mice and how a pro-inflammatory environment generated by treating cells with prostaglandin E2 (PGE2) alters CAV1 function in the presence of E-cadherin. CAV1 expression augmented migration, invasion, and metastasis of melanoma cells, and these effects were abolished via transient co-expression of E-cadherin. Importantly, exposure of cells to PGE2 reverted the effects of E-cadherin expression and increased CAV1 phosphorylation on tyrosine-14 and metastasis. Moreover, PGE2 administration blocked the ability of the CAV1/E-cadherin complex to prevent tumor formation. Therefore, our results support the notion that PGE2 can override the tumor suppressor potential of the E-cadherin/CAV1 complex and that CAV1 released from the complex is phosphorylated on tyrosine-14 and promotes migration/invasion/metastasis. These observations provide direct evidence showing how a pro-inflammatory environment caused here via PGE2 administration can convert a potent tumor suppressor complex into a promoter of malignant cell behavior.Item Src-family kinase inhibitors block early steps of caveolin-1-enhanced lungmetastasis by melanoma cells(2020) Ortiz, Rina; Díaz, Jorge; Díaz-Valdivia, Natalia; Martínez, Samuel; Simón, Layla; Contreras, Pamela; Lobos-González, Lorena; Guerrero, Simón; Leyton, Lisette; Quest, Andrew F.G.In advanced stages of cancer disease, caveolin-1 (CAV1) expression increases and correlates with increased migratory and invasive capacity of the respective tumor cells. Previous findings from our laboratory revealed that specific ECM-integrin interactions and tyrosine-14 phosphorylation of CAV1 are required for CAV1-enhanced melanoma cell migration, invasion and metastasis in vivo. In this context, CAV1 phosphorylation on tyrosine-14 mediated by non-receptor Src-family tyrosine kinases seems to be important; however, the effect of Src-family kinase inhibitors on CAV1-enhanced metastasis in vivo has not been studied. Here, we evaluated the effect of CAV1 and c-Abl overexpression, as well as the use of the Src-family kinase inhibitors, PP2 and dasatinib (more specific for Src/Abl) in lung metastasis of B16F10 melanoma cells. Overexpression of CAV1 and c-Abl enhanced CAV1 phosphorylation and the metastatic potential of the B16F10 murine melanoma cells. Alternatively, treatment with PP2 or dasatinib for 2 h reduced CAV1 tyrosine-14 phosphorylation and levels recovered fully within 12 h of removing the inhibitors. Nonetheless, pre-treatment of cells with these inhibitors for 2 h sufficed to prevent migration, invasion and trans-endothelial migration in vitro. Importantly, the transient decrease in CAV1 phosphorylation by these kinase inhibitors prevented early steps of CAV1-enhanced lung metastasis by B16F10 melanoma cells injected into the tail vein of mice. In conclusion, this study underscores the relevance of CAV1 tyrosine-14 phosphorylation by Src-family kinases during the first steps of the metastatic sequence promoted by CAV1. These findings open up potential options for treatment of metastatic tumors in patients in which Src-family kinase activation and CAV1 overexpression favor dissemination of cancer cells to secondary sites.