19 research outputs found
Juvenile Huntington’s disease skin fibroblasts respond with elevated parkin level and increased proteasome activity as a potential mechanism to counterbalance the pathological consequences of mutant Huntingtin protein
Huntington’s disease (HD) is an inherited neurodegenerative disorder, caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Mitochondrial dysfunction and impairment of the ubiquitin-proteasome system (UPS) are hallmarks of HD neurons. The extraneural manifestations of HD are still unclear. We investigated the crosstalk between mitochondria and proteolytic function in skin fibroblasts from juvenile HD patients. We found reduced mitosis, increased cell size, elevated ROS and increased mitochondrial membrane potential in juvenile HD fibroblasts, while cellular viability was maintained. Mitochondrial OXPHOS analysis did not reveal significant differences compared to control. However, the level of mitochondrial fusion and fission proteins was significantly lower and branching in the mitochondria network was reduced. We hypothesized that juvenile HD fibroblasts counterbalance cellular damage and mitochondrial network deficit with altered proteasome activity to promote cell survival. Our data reveal that juvenile HD fibroblasts exhibit higher proteasome activity, which was associated with elevated gene and protein expression of parkin. Moreover, we demonstrate elevated proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells compared to control cells. Our data suggest that juvenile HD fibroblasts respond to mutant polyQ expansion of Htt with enhanced proteasome activity and faster turnover of specific UPS substrates to protect cells
Targeting Nuclear NAD+ Synthesis Inhibits DNA Repair, Impairs Metabolic Adaptation and Increases Chemosensitivity of U-2OS Osteosarcoma Cells
Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS
treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin +
methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically
improved overall survival of OS patients. However, further research is needed to identify new
therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme,
nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon
exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage
response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin,
doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of
NMNAT1-/- cells showed features of both apoptosis and necroptosis, as indicated by the protective
effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of
the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of
NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage
(phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1-/- cells
to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important
for chemosensitization. Cisplatin-induced cell death of NMNAT1-/- cells was also characterized
by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity,
highlighting the central role of compromised cellular bioenergetics in chemosensitization byNMNAT1
inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when
grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is
a promising therapeutic target for osteosarcoma and possibly other tumors as well
Targeting nuclear NAD+ synthesis inhibits DNA repair, impairs metabolic adaptation increases chemosensitivity of U-2OS osteosarcoma cells
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Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling
Anti-counterfeiting protection, personalized medicines − Development of 2D identification methods using laser technology
Redox Profiling Reveals Clear Differences between Molecular Patterns of Wound Fluids from Acute and Chronic Wounds
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The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC
Despite recent advances in the development of novel personalized therapies, breast cancer continues to challenge physicians with resistance to various advanced therapies. The anticancer action of the anti-HER2 antibody, trastuzumab, involves antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells. Here, we report a repurposing screen of 774 clinically used compounds on NK-cell + trastuzumab-induced killing of JIMT-1 breast cancer cells. Using a calcein-based high-content screening (HCS) assay for the image-based quantitation of ADCC that we have developed and optimized for this purpose, we have found that the multitargeted tyrosine kinase inhibitor sunitinib inhibits ADCC in this model. The cytoprotective effect of sunitinib was also confirmed with two other assays (lactate dehydrogenase release, and electric cell substrate impedance sensing, ECIS). The drug suppressed NK cell activation as indicated by reduced granzyme B deposition on to the target cells and inhibition of interferon-γ production by the NK cells. Moreover, sunitinib induced downregulation of HER2 on the target cells' surface, changed the morphology and increased adherence of the target cells. Moreover, sunitinib also triggered the autophagy pathway (speckled LC3b) as an additional potential underlying mechanism of the cytoprotective effect of the drug. Sunitinib-induced ADCC resistance has been confirmed in a 3D tumor model revealing the prevention of apoptotic cell death (Annexin V staining) in JIMT-1 spheroids co-incubated with NK cells and trastuzumab. In summary, our HCS assay may be suitable for the facile identification of ADCC boosting compounds. Our data urge caution concerning potential combinations of ADCC-based immunotherapies and sunitinib. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00262-022-03146-z
PARP1 facilitates EP300 recruitment to the promoters of the subset of RBL2-dependent genes
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PARP1-LSD1 functional interplay controls transcription of SOD2 that protects human pro-inflammatory macrophages from death under an oxidative condition
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