ORTLOFF TRAUTMANN, ALEXANDER
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Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation
GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor ? (ER?)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ER?-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ER?-positive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca2+]i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca2+]i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca2+]i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ER? expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ER?/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen. © 2020 Elsevier B.V., All rights reserved.
Mitochondrial transfer balances cell redox, energy and metabolic homeostasis in the osteoarthritic chondrocyte preserving cartilage integrity
Osteoarthrosis (OA) is a leading cause of disability and early mortality, with no disease modifying treatment. Mitochondrial (MT) dysfunction and changes in energy metabolism, leading to oxidative stress and apoptosis, are main drivers of disease. In reaction to stress, mesenchymal stromal/stem cells (MSCs) donate their MT to damaged tissues. Methods: To evaluate the capacity of clinically validated MSCs to spontaneously transfer their MT to human OA chondrocytes (OA-Ch), primary cultured Ch isolated from the articular cartilage of OA patients were co-cultured with MT-labeled MSCs. MT transfer (MitoT) was evidenced by flow cytometry and confocal microscopy of MitoTracker-stained and YFP-tagged MT protein. MT persistence and metabolic analysis on target cells were assessed by direct transfer of MSC-derived MT to OA-Chs (Mitoception), through SNP-qPCR analysis, ATP measurements and Seahorse technology. The effects of MitoT on MT dynamics, oxidative stress and cell viability were gauged by western blot of fusion/fission proteins, confocal image analysis, ROS levels, Annexin V/7AAD and TUNEL assays. Intra-articular injection of MSC-derived MT was tested in a collagenase-induced murine model of OA. Results: Dose-dependent cell-to-cell MitoT from MSCs to cultured OA-Chs was detected starting at 4 hours of co-culture, with increasing MT-fluorescence levels at higher MSC:Ch ratios. PCR analysis confirmed the presence of exogenous MSC-MT within MitoT+ OA-Chs up to 9 days post Mitoception. MitoT from MSCs to OA-Ch restores energetic status, with a higher ATP production and metabolic OXPHOS/Glycolisis ratio. Significant changes in the expression of MT network regulators, increased MFN2 and decreased p-DRP1, reveal that MitoT promotes MT fusion restoring the MT dynamics in the OA-Ch. Additionally, MitoT increases SOD2 transcripts, protein, and activity levels, and reduces ROS levels, confering resistance to oxidative stress and enhancing resistance to apoptosis. Intra-articular injection of MSC-derived MT improves histologic scores and bone density of the affected joints in the OA mouse model, demonstrating a protective effect of MT transplantation on cartilage degradation. Conclusion: The Mitochondria transfer of MSC-derived MT induced reversal of the metabolic dysfunction by restoring the energetic status and mitochondrial dynamics in the OA chondrocyte, while conferring resistance to oxidative stress and apoptosis. Intra-articular injection of MT improved the disease in collagenase-induced OA mouse model. The restoration of the cellular homeostasis and the preclinical benefit of the intra-articular MT treatment offer a new approach for the treatment of OA. © 2024 Elsevier B.V., All rights reserved.
Aspergillus fumigatus-sensitive IgE is associated with bronchial hypersensitivity in a murine model of neutrophilic airway inflammation
Neutrophils are the predominant inflammatory cells that infiltrate airways during acute exacerbation of asthma. The importance of A. fumigatus sensitization, and IgE response in the airways in patients with acute asthma is unclear. Rockefeller (RK) mice were sensitized with A. fumigatus extract protein. The animals were subsequently challenged with different degrees of A. fumigatus contamination in the cage bedding. All groups of mice were euthanized to obtain bronchoalveolar lavage fluid (BALF) for cytological and Elisa assays, and lung tissue for histological analysis. Moreover, several bioassays were conducted to determine whether BALF IgE antibodies can activate mast cells. In this study, we demonstrated that exposure of sensitized mice to a known concentration of A. fumigatus conidia produces bronchial hyperreactivity with marked neutrophilic bronchial infiltration and increased BALF IgE, capable of triggering mast cell degranulation. This study suggests that IgE may play a role in bronchial hyperreactivity associated to A. fumigatus exposure in mice. Mice sensitized and challenged with this fungus showed characteristics of severe asthma, with an increase of BALF neutrophils, histological changes consistent with severe asthma and an increase of IgE capable of triggering type I hypersensitivity. © 2019 Elsevier B.V., All rights reserved.