Dataset Information

In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

ABSTRACT: Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy.

SUBMITTER: Teiti I

PROVIDER: S-EPMC4500923 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (?RB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in ?RB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models. Impact Statement Here we assessed the effects of Rho-associated protein kinase (ROCK) inhibition on mesenchymal stem cell (MSC) chondrogenesis in different culture models, including three-dimensional (3D) microribbon scaffolds, two-dimensional hydrogel substrates, 3D hydrogels, and pellet culture. Our results demonstrate that effects of ROCK inhibition on MSC chondrogenesis differ substantially depending on culture models. Furthermore, MSC chondrogenesis cannot be predicted using ROCK signaling alone. The results from this study fill in a gap of knowledge in the correlation between ROCK signaling and MSC chondrogenesis, which may be used to enhance MSC-based cartilage regeneration in different culture models.

Project description:Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Dataset Information

In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets