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Salado C.,Innoprot SL | Olaso E.,University of the Basque Country | Gallot N.,Pharmakine Ltd. | Valcarcel M.,Pharmakine Ltd. | And 3 more authors.
Journal of Translational Medicine | Year: 2011

Background: Implantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis.Methods: Two experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18thhour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied.Results: Resveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells.Conclusions: These results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver. © 2011 Salado et al; licensee BioMed Central Ltd.

Badiola I.,Innoprot SL | Olaso E.,University of the Basque Country | Crende O.,University of the Basque Country | Friedman S.L.,Mount Sinai School of Medicine | Vidal-Vanaclocha F.,University of San Pablo - CEU
Gut | Year: 2012

Background: The transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a major mechanism for stroma development in hepatic metastasis, but their regulatory pathways remain unclear. Transdifferentiated HSCs from fibrotic liver express high levels of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2), but it is unclear if DDR2 plays a direct profibrogenic role in the tumour microenvironment. Aim: To assess the impact of DDR2 on the prometastatic role of HSC-derived myofibroblasts. Methods: Hepatic metastases were induced in DDR2-/- and DDR2+/+ mice by intrasplenic injection of MCA38 colon carcinoma cells, and their growth and features were characterised. Stromagenic, angiogenic and cancer cell proliferation responses were quantified in metastases by immunohistochemistry. The adhesion-, migration- and proliferation-stimulating activities of supernatants from primary cultured DDR2-/- and DDR2+/+ HSCs, incubated in MCA38 cell-conditioned medium, were evaluated in primary cultured liver sinusoidal endothelium cells (LSECs) and MCA38 cells. Gene expression signatures from freshly isolated DDR2-/- and DDR2 +/+ HSCs were compared and DDR2-regulated genes were studied by RT-PCR under basal conditions and after stimulation with MCA38 tumour-conditioned media. Results: Metastases were increased three fold in DDR2-/- livers, and contained a higher density of α-smooth muscle actin-expressing myofibroblasts, CD31-expressing microvessels and Ki67-expressing MCA38 cells than metastases in DDR2+/+ livers. Media conditioned by MCA38-activated DDR2-/- HSCs significantly increased adhesion, migration and proliferation of LSECs and MCA38 cells, compared with DDR2+/+ HSCs. DDR2 deficiency in HSCs led to decreased gene expression of interferon γ-inducing factor interleukin (IL)-18 and insulin-like growth factor-I; and increased gene expression of prometastatic factors IL-10, transforming growth factor (TGF)β and vascular endothelial growth factor (VEGF), bone morphogenetic protein-7 and syndecan-1. MC38 tumour-conditioned media further exacerbated expression changes in DDR2-dependent IL-10, TGFβ and VEGF genes. Conclusion: DDR2 deficiency fosters the myofibroblast transdifferentiation of tumour-activated HSCs, generating a prometastatic microenvironment in the liver via HSC-derived factors. These findings underscore the role of stromal cells in conditioning the hepatic microenvironment for metastases through altered receptor-stroma interactions.

Valcarcel M.,Innoprot SL | Carrascal T.,University of the Basque Country | Crende O.,University of the Basque Country | Vidal-Vanaclocha F.,University of San Pablo - CEU
Journal of Investigative Dermatology | Year: 2014

Very late antigen-4 (VLA-4) is frequently overexpressed on melanoma cells contributing to inflammation-dependent metastasis. Melanoma cell adhesion to endothelium via VLA-4-vascular cell adhesion molecule-1 (VCAM-1) interaction was used to study VLA-4 activation during melanoma cell response to inflammation. Cooperation among major inflammatory mediators was analyzed in melanoma cells exposed to single inflammatory factors in the presence of inhibitors for other assayed mediators. A stepwise cascade of hierarchized molecules heterogeneously made and used during melanoma response to IL-18, induced hydrogen peroxide (H 2 O 2), in turn activating VLA-4 and melanoma cell adhesion to endothelium. The cascade involved prostaglandin E2 (PGE2) production from melanoma induced by IL-18-dependent tumor necrosis factor-α (TNFα); next, PGE2-induced IL-1β via vascular endothelial growth factor (VEGF) secretion, which in turn induced VLA-4 activation via cyclooxygenase 2-dependent H 2 O 2. This sequence operated in IL-18R/VLA-4/VEGF-expressing murine (B16) and human (A375 and 883) melanomas, but not in those without this phenotype. Separation of active VLA-4-expressing B16 melanoma cells through immobilized VCAM-1 verified their higher IL-18R/TNFR1/VEGFR2 expression and metastatic growth than inactive VLA-4-expressing cells. However, cooperation among melanoma cell sub-populations with heterogeneous cytokine receptor levels may occur through VLA-4-stimulating factors, leading to intratumoral amplification of metastatic potential. Therefore, expression of the VLA-4-stimulating factor sequence may help to predict melanoma prometastatic risk, and offers therapeutic targets for metastatic melanoma deactivation through VLA-4 activation blockade. © 2014 The Society for Investigative Dermatology.

Valcarcel M.,Innoprot SL | Mendoza L.,University of the Basque Country | Hernandez J.,University of the Basque Country | Carrascal T.,University of the Basque Country | And 3 more authors.
Journal of Translational Medicine | Year: 2011

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown.Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied.Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion- and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFα induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1.Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion- and proliferation-stimulating effects of TNFα, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing melanoma cells. These data suggest COX-2 neutralization as a potential anti-metastatic therapy in melanoma patients at high risk of systemic and bone dissemination due to intercurrent infectious and inflammatory diseases. © 2011 Valcárcel et al; licensee BioMed Central Ltd.

Crende O.,University of the Basque Country | Sabatino M.,U.S. National Institutes of Health | Valcarcel M.,Innoprot SL | Carrascal T.,University of the Basque Country | And 7 more authors.
American Journal of Pathology | Year: 2013

IL-18 is an immune-stimulating cytokine that promotes experimental melanoma metastasis via vascular endothelial growth factor (VEGF)-induced very late antigen (VLA)-4. We studied genes associated with the ability of melanoma cells to allow metastasis under IL-18 effects, and we verified their expression in metastatic lesions from patients with melanoma. Human melanoma cell lines with and without the IL-18 receptor (IL-18R)/VEGF/VLA-4-expressing phenotype were identified, and their metastatic potential was studied in nude mice. RNA from untreated and IL-18-treated melanoma phenotypes was hybridized to a cDNA microarray, and their signature genes were studied. RNA from primary and metastatic lesions from patients with melanoma was hybridized to a cDNA microarray to identify lesions with the transcript patterns of melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype. IL-18R/VEGF/VLA-4-expressing A375 and 1182 melanoma cells produced a higher metastasis number than 526 and 624.28 melanoma cells, not using this prometastatic pathway. Melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype had distinct transcript patterns. However, the type I transcriptional cluster, including cutaneous and lymph node metastases, but not the type II cluster, not including cutaneous metastases, had signature genes from IL-18-treated melanoma cells with, but not without, the IL-18R/VEGF/VLA-4 phenotype. Metastatic melanoma lesions with and without IL-18-dependent genes were identified, suggesting that melanoma metastasis developed via inflammation-dependent and inflammation-independent mechanisms. Signature genes from melanomas with and without the IL-18R/VEGF/VLA-4 phenotype may serve as diagnostic biomarkers of melanoma predisposition to prometastatic effects of IL-18. © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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