CD8+ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen-presenting cells that induce their proliferation. Here, we show that thapsigargin-induced LAD2 mast cell (MC) line-released products can impair the ability of monocyte-derived DCs to induce CD8+ T-cell proliferation and the generation of Th1 cytokine-producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA-DR. However, thapsLAD2-matured DCs produced no detectable TNFα or IL-12 during the maturation. In addition, although their surface expression of PD-L1 was comparable with the immature or TLR7/8-agonist (R848)-matured DCs, their TIM-3 expression was significantly higher than in immature DCs and even much higher than in R848-matured DCs. In addition, contrary to R848-matured DCs, the thapsLAD2-matured DCs only tended to induce enhanced proliferation of CD4+ T cells than immature DCs. For CD8+ T cells, this tendency was not even detected because thapsLAD2-matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848-matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ- and IFNγ/TNFα-producing T cells. These findings show a novel mechanism of MC-mediated regulation of adaptive immune responses.

• MeSH
• aktivace lymfocytů * účinky léků   imunologie   MeSH
• buněčná diferenciace * účinky léků   MeSH
• buněčné linie MeSH
• buněčný receptor 2 viru hepatitidy A metabolismus   MeSH
• CD8-pozitivní T-lymfocyty * imunologie   účinky léků   MeSH
• cytokiny metabolismus   MeSH
• dendritické buňky * imunologie   účinky léků   metabolismus   MeSH
• imidazoly farmakologie   MeSH
• lidé MeSH
• mastocyty * imunologie   účinky léků   metabolismus   MeSH
• monocyty imunologie   účinky léků   metabolismus   MeSH
• proliferace buněk * účinky léků   MeSH
• thapsigargin * farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Dysregulation of iron homeostasis is one of the important processes in the development of many oncological diseases, such as pancreatic cancer. Targeting it with specific agents, such as an iron chelator, are promising therapeutic methods. In this study, we tested the cytotoxicity of novel azulene hydrazide-hydrazone-based chelators against pancreatic cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1). All prepared chelators (compounds 4-6) showed strong cytotoxicity against pancreatic cancer cell lines and high selectivity for cancer cell lines compared to the healthy line. Their cytotoxicity is lower than thiosemicarbazone-based chelators Dp44mT and DpC, but significantly higher than hydroxamic acid-based chelator DFO. The chelator tested showed mitochondrial and lysosomal co-localization and its mechanism of action was based on the induction of hypoxia-inducible factor-1-alpha (HIF-1α), N-myc downstream-regulated gene-1 (NDRG1) and transferrin receptor 1 (TfR1). This strongly implies that the cytotoxic effect of tested chelators could be associated with mitophagy induction. Lipinski's rule of five analyses was performed to determine whether the prepared compounds had properties ensuring their bioavailability. In addition, the drug-likeness and drug-score were calculated and discussed.

• MeSH
• azuleny MeSH
• chelátory železa farmakologie   MeSH
• hydraziny MeSH
• hydrazony farmakologie   MeSH
• kyseliny hydroxamové MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory slinivky břišní * farmakoterapie   MeSH
• receptory transferinu MeSH
• thiosemikarbazony * farmakologie   MeSH
• železo MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

• MeSH
• aorta MeSH
• bradykinin * farmakologie   MeSH
• cévní endotel MeSH
• krysa rodu rattus MeSH
• lipopolysacharidy farmakologie   MeSH
• membránové potenciály MeSH
• potkani Sprague-Dawley MeSH
• potkani transgenní MeSH
• receptor bradykininu B1 * genetika   MeSH
• techniky in vitro MeSH
• thapsigargin farmakologie   MeSH
• toll-like receptor 4 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Sesquiterpene lactone helenalin is used as an antiphlogistic in European and Chinese folk medicine. The pharmacological activities of helenalin have been extensively investigated, yet insufficient information exists about its metabolic properties. The objectives of the present study were (1) to investigate the in vitro NADPH-dependent metabolism of helenalin (5 and 100 μM) using human and rat liver microsomes and liver cytosol, (2) to elucidate the role of human cytochrome P450 (CYP) enzymes in its oxidative metabolism, and (3) to study the inhibition of human CYPs by helenalin. Five oxidative metabolites were detected in NADPH-dependent human and rat liver microsomal incubations, while two reduced metabolites were detected only in NADPH-dependent human microsomal and cytosolic incubations. In human liver microsomes, the main oxidative metabolite was 14-hydroxyhelenalin, and in rat liver microsomes 9-hydroxyhelenalin. The overall oxidation of helenalin was several times more efficient in rat than in human liver microsomes. In humans, CYP3A4 and CYP3A5 followed by CYP2B6 were the main enzymes responsible for the hepatic metabolism of helenalin. The extrahepatic CYP2A13 oxidized helenalin most efficiently among CYP enzymes, possessing the Km value of 0.6 μM. Helenalin inhibited CYP3A4 (IC50 = 18.7 μM) and CYP3A5 (IC50 = 62.6 μM), and acted as a mechanism-based inhibitor of CYP2A13 (IC50 = 1.1 μM, KI = 6.7 μM, and kinact = 0.58 ln(%)/min). It may be concluded that the metabolism of helenalin differs between rats and humans, in the latter its oxidation is catalyzed by hepatic CYP2B6, CYP3A4, CYP3A5, and CYP3A7, and extrahepatic CYP2A13.

• MeSH
• druhová specificita MeSH
• inhibiční koncentrace 50 MeSH
• inhibitory cytochromu P450 aplikace a dávkování   metabolismus   farmakologie   MeSH
• jaterní mikrozomy metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• NADP metabolismus   MeSH
• potkani Wistar MeSH
• seskviterpeny guajanové aplikace a dávkování   metabolismus   farmakologie   MeSH
• systém (enzymů) cytochromů P-450 účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The preparation of dendritic cells (DCs) for adoptive cellular immunotherapy (ACI) requires the maturation of ex vivo-produced immature(i) DCs. This maturation ensures that the antigen presentation triggers an immune response towards the antigen-expressing cells. Although there is a large number of maturation agents capable of inducing strong DC maturation, there is still only a very limited number of these agents approved for use in the production of DCs for ACI. In seeking novel DC maturation agents, we used differentially activated human mast cell (MC) line LAD2 as a cellular adjuvant to elicit or modulate the maturation of ex vivo-produced monocyte-derived iDCs. We found that co-culture of iDCs with differentially activated LAD2 MCs in serum-containing media significantly modulated polyinosinic:polycytidylic acid (poly I:C)-elicited DC maturation as determined through the surface expression of the maturation markers CD80, CD83, CD86, and human leukocyte antigen(HLA)-DR. Once iDCs were generated in serum-free conditions, they became refractory to the maturation with poly I:C, and the LAD2 MC modulatory potential was minimized. However, the maturation-refractory phenotype of the serum-free generated iDCs was largely overcome by co-culture with thapsigargin-stimulated LAD2 MCs. Our data suggest that differentially stimulated mast cells could be novel and highly potent cellular adjuvants for the maturation of DCs for ACI.

• MeSH
• adjuvancia imunologická farmakologie   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné kultury metody   MeSH
• dendritické buňky cytologie   účinky léků   imunologie   MeSH
• imunoterapie adoptivní * MeSH
• kokultivační techniky MeSH
• lidé MeSH
• mastocyty cytologie   účinky léků   imunologie   MeSH
• monocyty cytologie   účinky léků   imunologie   MeSH
• prezentace antigenu účinky léků   imunologie   MeSH
• thapsigargin farmakologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.

• MeSH
• Apiaceae chemie   metabolismus   MeSH
• butyráty chemie   izolace a purifikace   MeSH
• furany chemie   izolace a purifikace   MeSH
• fytogenní protinádorové látky chemie   izolace a purifikace   MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádory farmakoterapie   patologie   MeSH
• ovoce chemie   metabolismus   MeSH
• oxid uhličitý chemie   MeSH
• rostlinné extrakty chemie   MeSH
• sarkoplazmatická Ca2+-ATPáza antagonisté a inhibitory   metabolismus   MeSH
• superkritická fluidní chromatografie metody   MeSH
• techniky syntetické chemie * MeSH
• thapsigargin analogy a deriváty   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

• MeSH
• apoptóza MeSH
• autokrinní signalizace MeSH
• biologické markery MeSH
• epidermální růstový faktor metabolismus   MeSH
• kardiomyocyty účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• membránové glykoproteiny metabolismus   MeSH
• myši MeSH
• náchylnost k nemoci MeSH
• nádorové proteiny metabolismus   MeSH
• parakrinní signalizace MeSH
• proteom * MeSH
• proteomika * metody   MeSH
• sarkoplazmatické retikulum metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula * účinky léků   MeSH
• thapsigargin farmakologie   MeSH
• vápník metabolismus   MeSH
• vápníková signalizace účinky léků   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Sesquiterpene lactones are bioactive natural compounds with anticancer, antiprotozoal, immunomodulatory, antibacterial and antiviral activity which have potential for drug development. The aim of this review article is to provide a brief insight into the field of sesquiterpene lactones: the main mechanisms of their biological actions, as well as particular compounds, are described, some of which have already become a basis of a drug development and are being tested in clinical trials.

• MeSH
• artemisininy MeSH
• etnofarmakologie MeSH
• farmakognozie MeSH
• fytogenní protinádorové látky izolace a purifikace   MeSH
• imunomodulace MeSH
• inhibitory enzymů MeSH
• laktony * farmakokinetika   farmakologie   terapeutické užití   MeSH
• seskviterpeny guajanové farmakokinetika   farmakologie   terapeutické užití   MeSH
• seskviterpeny farmakokinetika   farmakologie   terapeutické užití   MeSH
• Tanacetum parthenium chemie   MeSH
• thapsigargin MeSH
• Publikační typ
• práce podpořená grantem MeSH

OBJECTIVE: To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue samples from patients with RA and patients with osteoarthritis (OA) were stained for PTPN2. Synovial fibroblasts were stimulated with tumor necrosis factor (TNF) and interleukin-1β (IL-1β), lipopolysaccharide (LPS), TRAIL, or thapsigargin. The expression of PTPN2 in synovial fibroblasts and peripheral blood mononuclear cells (PBMCs) was analyzed by real-time polymerase chain reaction and Western blotting. Cell death, the release of IL-6 and IL-8, and the induction of autophagy were analyzed after PTPN2 silencing. Methylated DNA immunoprecipitation analysis was used to evaluate DNA methylation-regulated gene expression of PTPN2. RESULTS: PTPN2 was significantly overexpressed in synovial tissue samples from RA patients compared to OA patients. Patients receiving anti-TNF therapy showed significantly reduced staining for PTPN2 compared with patients treated with nonbiologic agents. PTPN2 expression was higher in RA synovial fibroblasts (RASFs) than in OASFs. This differential expression was not regulated by DNA methylation. PTPN2 was further up-regulated after stimulation with TNF, TNF combined with IL-1β, or LPS. There was no significant difference in basal PTPN2 expression in PBMCs from patients with RA, ankylosing spondylitis, or systemic lupus erythematosus or healthy controls. Most interestingly, PTPN2 silencing in RASFs significantly increased the production of the inflammatory cytokine IL-6 but did not affect levels of IL-8. Moreover, functional analysis showed that high PTPN2 levels contributed to the increased apoptosis resistance of RASFs and increased autophagy. CONCLUSION: This is the first study of PTPN2 in RASFs showing that PTPN2 regulates IL-6 production, cell death, and autophagy. Our findings indicate that PTPN2 is linked to the pathogenesis of RA via synovial fibroblasts.

• MeSH
• apoptóza účinky léků   MeSH
• autofagie účinky léků   MeSH
• biologické přípravky farmakologie   MeSH
• fibroblasty účinky léků   metabolismus   patologie   MeSH
• interleukin-1beta farmakologie   MeSH
• interleukin-6 metabolismus   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipopolysacharidy farmakologie   MeSH
• osteoartróza metabolismus   patologie   MeSH
• protein TRAIL farmakologie   MeSH
• revmatoidní artritida metabolismus   patologie   MeSH
• senioři MeSH
• synoviální membrána účinky léků   metabolismus   patologie   MeSH
• thapsigargin farmakologie   MeSH
• TNF-alfa antagonisté a inhibitory   farmakologie   MeSH
• tyrosinfosfatasa nereceptorového typu 2 metabolismus   MeSH
• upregulace účinky léků   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. METHODS: Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. RESULTS: Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1-24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. CONCLUSIONS: Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

• MeSH
• buněčná diferenciace * MeSH
• endoplazmatické retikulum účinky léků   metabolismus   MeSH
• fosforylace MeSH
• fyziologický stres * MeSH
• lidé MeSH
• lipidy biosyntéza   MeSH
• signální dráha UPR MeSH
• thapsigargin farmakologie   MeSH
• tukové buňky cytologie   MeSH
• tunikamycin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH