CD95/Fas ligand (FasL) is a cell death-promoting member of the tumor necrosis factor family with important functions in the regulation of T-cell homeostasis and cytotoxicity. In T cells, FasL expression is tightly regulated on a transcriptional level involving a complex set of different transcription factors. The orphan nuclear receptor liver receptor homolog-1 (LRH-1/NR5a2) is involved in the regulation of development, lipid metabolism and proliferation and is predominantly expressed in epithelial tissues. However, its expression in T lymphocytes has never been reported so far. Based on in silico analysis, we identified potential LRH-1 binding sites within the FASLG promoter. Here, we report that LRH-1 is expressed in primary and secondary lymphatic tissues, as well as in CD4+ and CD8+ T cells. LRH-1 directly binds to its binding sites in the FASLG promoter, and thereby drives FASLG promoter activity. Mutations in the LRH-1 binding sites reduce FASLG promoter activity. Pharmacological inhibition of LRH-1 decreases activation-induced FasL mRNA expression, as well as FasL-mediated activation-induced T-cell apoptosis and T-cell cytotoxicity. In a mouse model of Concanavalin A-induced and FasL-mediated hepatitis pharmacological inhibition of LRH-1 resulted in decreased hepatic FasL expression and a significant reduction of liver damage. In summary, these data show for the first time LRH-1 expression in T cells, its role in FASLG transcription and the potential of pharmacological inhibition of LRH-1 in the treatment of FasL-mediated immunopathologies.
AA amyloidosis belongs to the group of amyloid diseases which can follow chronic inflammatory conditions of various origin. The disease is characterized by the deposition of insoluble amyloid fibrils formed by serum amyloid A1 (SAA1) leading eventually to organ failure. Macrophages are intimately involved in the fibrillogenesis as well as in the clearance of amyloid fibrils. In vivo, macrophages may occur as classically (M1) or alternatively activated (M2) macrophages. We investigate here how SAA1 might affect the macrophage phenotype and function. Gene microarray analysis revealed upregulation of 64 M1-associated genes by SAA1. M1-like polarization was further confirmed by the expression of the M1-marker MARCO, activation of the NF-κB transcription factor, and secretion of the M1-cytokines TNF-α, IL-6, and MCP-1. Additionally, we demonstrate here that M1-polarized macrophages exhibit enhanced fibrillogenic activity towards SAA1. Based on our data, we propose reconsideration of the currently used cellular amyloidosis models towards an in vitro model employing M1-polarized macrophages. Furthermore, the data suggest macrophage repolarization as potential intervention strategy in AA amyloidosis.
Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to meta- or para-substituted derivatives with electron-withdrawing groups -F, -Br, or -CF3 at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound 10 also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.
In vitro analysis of human macrophages is generally hampered by the necessity to differentiate them from peripheral blood monocytes. We have analyzed to which extent noncancerous SC monocytes could be used as an in vitro macrophage model. Macrophages differentiated from peripheral monocytes using standard CSF1 and CSF2 protocols for M2 and M1 precursors, respectively, were compared with THP‐1‐derived macrophages treated with PMA and with SC‐derived macrophages differentiated either by CSF1, CSF2, or PMA according to different protocols. The optimal condition for generation of SC macrophages was treatment with PMA for 3 days, followed by 5‐days culture without PMA and 24‐h polarization with LPS/IFN‐γ or IL‐4/IL‐13. Similar to THP‐1, SC cells do not express the monocyte marker CD14 and differentiation to macrophages results neither in CD68 nor in CD14 expression, both of which were expressed by monocyte‐derived macrophages. Similar to THP‐1‐macrophages, a proportion of SC macrophages can be polarized to the M1‐like subtype that is characterized by higher expression of CD38, CD86, CD80, TNF‐α, and IL‐1ra, whereas treatment with IL4/IL13 did not lead to expression of the M2‐associated receptors CD163, CD206, and only slightly increased the CD200R expression. Still, SC‐M1 express much lower levels of the M1‐associated markers compared with monocyte‐derived M1 and no IL‐1β. The data demonstrate that SC‐derived macrophages differ from monocyte‐derived macrophages in respect of their morphology, expression of important macrophage markers, phagocytosis. Yet, polarized SC‐M1‐like cells may with restrictions serve as a model for M1 macrophages, though this model does not provide significant advantages over already well‐described THP‐1‐M1‐like cells.
This study explores biocompatible amino-functionalized gold nanoparticles (Au-NH 2 ) as nanotherapeutics for the selective eradication of leukemia cells, elucidates the mechanism of cytotoxicity, and it confirms in vivo efficacy of the engineered nanomaterial. Au-NH 2 trigger apoptotic cell death of myeloid leukemia cell lines and primary acute myeloid leukemia (AML) cells by i) inhibition of mitochondrial respiration, ii) ATP depletion, iii) loss of mitochondrial membrane potential, and iv) mitochondrial release of cytochrome c. Au-NH 2 act selectively on leukemia cells inasmuch as the viability of normal peripheral blood mononuclear cells and macrophages as well as the colony formation of hematopoietic stem cells remain basically unaffected. The selectivity of Au-NH 2 for AML cells can be attributed to both the preferential accumulation of AuNH 2 in AML cells and the strong dependence of those cells on mitochondrial oxidative phosphorylation for ATPproduction. Importantly, Au-NH 2 applied either as monotherapy or as a cytarabine combination regimen possess antileukemic efficacy in the absence of adverse events in mice xenografted with primary human AML in vivo. The engineered material may pave the way for a novel nanotherapeutic treatment of AML.
Acute myeloid leukemia (AML) is a malignancy associated with poor prognosis. Particularly, older patients suffer greatly from the standard chemotherapy and have a 5‐year survival of only 4%. Hence, new therapeutic agents with higher specificity and lower general cytotoxicity are urgently required. We have discovered that positively charged amino‐functionalized polystyrene nanoparticles (NP‐PS+) induce accumulation of acidic vesicular organelles with elevated pH and impaired processing of procathepsin B leading to mTOR inhibition, activation of autophagy, and induction of caspase‐dependent apoptosis in leukemia cells, but not in normal human macrophages. The antileukemic effect of NP‐PS+ was also preserved in vivo, where NP‐PS+ inhibited proliferation and induced apoptosis in leukemia xenografts grown on chick chorioallantoic membranes. Similar to polystyrene particles, amino‐functionalized gold nanoparticles (NP‐Au+) exhibited selective cytotoxicity towards AML cell lines as well as primary patient‐derived AML cells. Thus, NP‐Au+ particles induced cell death in primary human leukemia cells and reduced their colony‐forming potential, whereas normal hematopoietic cells remained unaffected by the treatment with NP‐Au+. NP‐Au+ targeted specifically the oxidative mitochondrial respiration, which is, different to normal hematopoietic cells, the main source of energy production in AML blasts and leukemic stem cells. Different to NP‐Au+, conventional chemotherapeutics such ascytarabine act in a cell cycle‐dependent manner and target only on proliferating AML blasts but not on quiescent leukemic stem cells. In agreement with the in vitro data, NP‐Au+ exhibited antileukemic efficacy against primary human AML xenografted into mice applied either as monotherapy or as a cytarabine combination regimen in the absence of detectable adverse events. Thus, this engineered nanomaterial that targets particularly resistant quiescent leukemic stem cells holds great promise as a novel nanotherapeutic for the treatment of acute myeloid leukemia independent of its cytogenetic profile.
In recent years, nanomaterials emerged as versatile tools for biomedical applications. Due to their special physicochemical characteristics, many different types of nanomaterials are under investigation for diagnostic as well as therapeutic purposes. We have previously shown the intrinsic cytotoxic activity of amino‐functionalized polystyrene nanoparticles towards acute myeloid leukemia cells. Here, we developed biocompatible gold nanoparticles with amino‐functionalization and investigated their mechanism of cellular toxicity.Acute myeloid leukemia (AML) is characterized by the proliferation and accumulation of mostly undifferentiated myeloid progenitor cells. In comparison to other leukemias, AML is a disease of the elderly with a mean age at diagnosis of 68 years. Due to severe side effects of the standard therapy, elderly AML patients often receive only palliative care, which results in profoundly reduced survival rates in older patients. Even though, a range of molecular changes in AML has already been investigated in detail, only few therapeutic agents have been approved. One possible explanation for the limited efficacy of targeted agents in AML might be the high level of genetic heterogeneity in myeloid leukemia.For a long time, glycolysis has been considered the major pathway of energy production in cancer cells, also known as the Warburg effect. Recently, however, oxidative phosphorylation emerged as a crucial metabolic pathway for the survival and proliferation of leukemia cells. The high dependence of acute myeloid leukemia cells on oxidative phosphorylation suggests the possibility to target the mitochondrial respiratory chain for antileukemic efficacy.Here, we performed high resolution respirometry using an Oroboros oxygraph and show the enhanced respiratory activity of THP‐1 leukemia cells compared to healthy peripheral mononuclear cells. Furthermore, chromogenic complex activity assays employed to study the effect of gold nanoparticles on respiratory complex activities of isolated mitochondria demonstrated that the amino‐functionalized gold nanoparticles, but not the carboxy‐functionalized control particles, inhibit mitochondrial complex activities. We could further show that such direct inhibitory effect on mitochondrial complex activities also impairs mitochondrial respiration in intact THP‐1 cells. Inhibition of mitochondrial respiration further led to depolarization of the mitochondrial membrane, depletion of ATP, and cell death of THP‐1 cells and primary AML blasts.Hence, our findings confirm the high dependence of AML cells on oxidative phosphorylation. On that basis, our amino‐functionalized gold nanoparticles represent novel nanotherapeutics for a broad application in AML therapy, specifically targeting and inhibiting the sensitive energy production pathway in AML cells.Support or Funding InformationThis work was supported by the Volkswagen Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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