ObjectiveTo investigate the role of death receptor 3 (DR-3) and its ligand tumor necrosis factor–like molecule 1A (TL1A) in the early stages of inflammatory arthritis.MethodsAntigen-induced arthritis (AIA) was generated in C57BL/6 mice deficient in the DR-3 gene (DR3−/−) and their DR3+/+ (wild-type) littermates by priming and intraarticular injection of methylated bovine serum albumin. The joints were sectioned and analyzed histochemically for damage to cartilage and expression of DR3, TL1A, Ly-6G (a marker for neutrophils), the gelatinase matrix metalloproteinase 9 (MMP-9), the aggrecanase ADAMTS-5, and the neutrophil chemoattractant CXCL1. In vitro production of MMP-9 was measured in cultures from fibroblasts, macrophages, and neutrophils following the addition of TL1A and other proinflammatory stimuli.ResultsDR3 expression was up-regulated in the joints of wild-type mice following generation of AIA. DR3−/− mice were protected against cartilage damage compared with wild-type mice, even at early time points prior to the main accumulation of Teff cells in the joint. Early protection against AIA in vivo correlated with reduced levels of MMP-9. In vitro, neutrophils were major producers of MMP-9, while neutrophil numbers were reduced in the joints of DR3−/− mice. However, TL1A neither induced MMP-9 release nor affected the survival of neutrophils. Instead, reduced levels of CXCL1 were observed in the joints of DR3−/− mice.ConclusionDR-3 drives early cartilage destruction in the AIA model of inflammatory arthritis through the release of CXCL1, maximizing neutrophil recruitment to the joint and leading to enhanced local production of cartilage-destroying enzymes.
The essential oils from Commiphora species have for centuries been recognized to possess medicinal properties. Here, we performed gas chromatography-mass spectrometry on the essential oil from opoponax (Commiphora guidotti) and identified bisabolene isomers as the main constituents of this essential oil. Opoponax essential oil, a chemical component; β-bisabolene and an alcoholic analogue, α-bisabolol, were tested for their ability to selectively kill breast cancer cells. Only β-bisabolene, a sesquiterpene constituting 5% of the essential oil, exhibited selective cytotoxic activity for mouse cells (IC50 in normal Eph4: >200 µg/ml, MG1361: 65.49 µg/ml, 4T1: 48.99 µg/ml) and human breast cancer cells (IC50 in normal MCF-10A: 114.3 µg/ml, MCF-7: 66.91 µg/ml, MDA-MB-231: 98.39 µg/ml, SKBR3: 70.62 µg/ml and BT474: 74.3 µg/ml). This loss of viability was because of the induction of apoptosis as shown by Annexin V-propidium iodide and caspase-3/7 activity assay. β-bisabolene was also effective in reducing the growth of transplanted 4T1 mammary tumours in vivo (37.5% reduction in volume by endpoint). In summary, we have identified an anti-cancer agent from the essential oil of opoponax that exhibits specific cytotoxicity to both human and murine mammary tumour cells in vitro and in vivo, and this warrants further investigation into the use of β-bisabolene in the treatment of breast cancers.
We evaluated the pharmacodynamic effects of the O 6 -methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O 6 -methylguanine (O 6 -meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O 6 -meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O 6 -meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O 6 -meG in PBMC DNA during treatment.
BackgroundThe clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged.MethodsWe used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively.ResultsTRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling.ConclusionThese data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0478-y) contains supplementary material, which is available to authorized users.
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