• MeSH
• apoptóza účinky záření   MeSH
• Brassicaceae MeSH
• buněčná smrt MeSH
• finanční podpora výzkumu jako téma MeSH
• fytoterapie MeSH
• Jurkat buňky účinky léků   MeSH
• lidé MeSH
• rostlinné extrakty toxicita   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Fisturalines are bromotyrosine compounds isolated from marine sponges. Previous studies have shown antineoplasic, antiviral and antibacterial effects in Vitro; however, the possible effects of these compounds in hematologic malignancies have not been assessed. METHODS: In the present study, the antiproliferative and pro apoptotic effects of Fistularin-3 (F) and 11-Deoxyfistularin-3 (DF) were assessed using the MTT method and annexin V/propidium iodide by flow cytometry using the cell lines: Jurkat E6.1 and U937. In addition, the cell cycle was assessed by flow cytometry. RESULTS: Inhibition of the proliferative response was concentration and time dependent. The IC50 of F was 7.39 and 8.10 µM for Jurkat E6.1 and U937 respectively. At 24 and 48 h, in the U937 cell line, but not in the Jurkat cell line, both compounds induced up to 35% annexin V increase. Necrosis was not observed in any case. Compound F induced, in both cell lines, a decrease in the number of cells in the S phase and increase in the G0/G1 phase. In the Jurkat cell line only, there was an increase in the number of cells in the G2/M phase. Compound DF was not as effective as F. CONCLUSIONS: F is more active than DF in repressing the cell cycle and inducing apoptosis. Both compounds are potentially useful in the development of new drugs to treat hematologic malignancies.

• MeSH
• antibiotika antitumorózní farmakologie   MeSH
• apoptóza účinky léků   MeSH
• biologické přípravky farmakologie   MeSH
• buněčný cyklus účinky léků   MeSH
• hematologické nádory farmakoterapie   MeSH
• inhibiční koncentrace 50 MeSH
• Jurkat buňky MeSH
• lidé MeSH
• proliferace buněk účinky léků   MeSH
• průtoková cytometrie MeSH
• tyrosin analogy a deriváty   farmakologie   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

• MeSH
• analýza nákladů a výnosů metody   MeSH
• azathioprin farmakologie   MeSH
• Jurkat buňky účinky léků   fyziologie   MeSH
• leukocyty účinky léků   fyziologie   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• reprodukovatelnost výsledků MeSH
• transfer faktor farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Programmed cell death (PCD) pathways play a crucial role in the response of cancer cells to treatment. Their dysregulation is one of the cancer hallmarks and one of the reasons of drug resistance. Here, we studied the significance of the individual members of PCD signaling pathways in response to treatment with common anti-cancer drugs using the T-cell leukemia Jurkat cells with single or double knockouts of necroptosis and/or apoptosis genes. We identified apoptosis as the primary cell death pathway upon anti-cancer drugs treatment. The cells with knocked out either Fas-associated protein with death domain (FADD) or all executioner caspases were resistant. This resistance could be partially overcome by induction of RIP1-dependent necroptosis through TNFR1 activation using combined treatment with TNF-α and smac mimetic (LCL161). RIP1 was essential for cellular response to TNF-α and smac mimetic, but dispensable for the response to anti-cancer drugs. Here, we demonstrated the significance of FADD and executioner caspases in carrying out programmed cell death upon anti-cancer drug treatments and the ability of combined treatment with TNF-α and smac mimetic to partially overcome drug resistance of FADD and/or CASP3/7/6-deficient cells via RIP1-dependent necroptosis. Thus, a combination of TNF-α and smac mimetic could be a suitable strategy for overcoming resistance to therapy in cells unable to trigger apoptosis.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   genetika   MeSH
• buněčná smrt účinky léků   MeSH
• chemorezistence účinky léků   fyziologie   MeSH
• Jurkat buňky MeSH
• kaspasy genetika   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• lidé MeSH
• nekroptóza účinky léků   genetika   MeSH
• protein FADD asociující s Fas genetika   MeSH
• proteiny vázající RNA metabolismus   MeSH
• TNF-alfa farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

• MeSH
• alkaloidy amarylkovitých farmakologie   terapeutické užití   MeSH
• antitumorózní látky fytogenní analýza   MeSH
• apoptóza účinky léků   MeSH
• fenantridiny farmakologie   terapeutické užití   MeSH
• fytoterapie * MeSH
• geny p53 MeSH
• inhibitor p16 cyklin-dependentní kinasy metabolismus   MeSH
• Jurkat buňky MeSH
• kaspasy metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• léky antitumorózní - screeningové testy MeSH
• leukemie farmakoterapie   MeSH
• lidé MeSH
• liliovité chemie   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• rostlinné extrakty farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Regulation of Na+/K+-ATPase in bipolar disorder and lithium therapy has been investigated for more than 40 years. Contradictory results in this area may be caused by the difference between acute and long-term Li effects on cell metabolism and variance in responsiveness of different cell types. We compared the time-course of Li action focusing on Na+/K+-ATPase and lipid peroxidation in two widely different cell models-Jurkat and HEK293. Na+/K+-ATPase expression level was determined in cells cultivated in the absence or presence of 1 mM Li for different time spans (1, 7, and 28 days) using [3H] ouabain binding and immunoblot assay of α-subunit. In parallel samples, the formation of malondialdehyde (MDA) was quantified by HPLC, and 4-hydroxy-2-nonenal (4-HNE) protein adducts were determined by immunoblot. Cultivation of Jurkat cells in 1 mM Li medium resulted in downregulation of Na+/K+-ATPase (decrease of [3H] ouabain-biding sites and intensity of immunoblot signals) in all Li-groups. In HEK293 cells, the decrease of Na+/K+-ATPase was observed after the acute, 1-day exposure only. The long-term treatment with Li resulted in Na+/K+-ATPase upregulation. MDA and 4-HNE modified proteins were decreased in Jurkat cells in all Li-groups. On the other hand, in HEK293 cells, MDA concentration was decreased after the acute, 1-day Li exposure only; the long-term cultivations, for 7 or 28 days, resulted in a significant increase of lipid peroxidation products. The Li-induced decrease of lipid peroxidation products was associated with the decrease of Na+/K+-ATPase level and vice versa.

• MeSH
• bipolární porucha farmakoterapie   metabolismus   MeSH
• časové faktory MeSH
• HEK293 buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lipidové peroxidy metabolismus   MeSH
• peroxidace lipidů účinky léků   MeSH
• regulace genové exprese účinky léků   MeSH
• sloučeniny lithia aplikace a dávkování   metabolismus   farmakologie   MeSH
• sodíko-draslíková ATPasa genetika   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4⁺ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.

• Publikační typ
• časopisecké články MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biochemie
• alergologie a imunologie

BACKGROUND: Kaurane-type diterpenoids, obtained from various natural sources, have shown many biological activities, including anti-inflammatory and antitumor effects. Caracasine, an ent-kaurane diterpenoid isolated from the flowers of Croton micans, was shown to induce apoptosis in leukaemia cell lines. OBJECTIVE: The present study aimed to ascertain the compound's mechanism of cell death induction using two leukaemia cell lines, Jurkat E6.1 (T cell) and HL-60 (promyeloblast cells). METHODS: Cell death in Jurkat and HL60 cells were evaluated by flow cytometry for apoptosis with annexin-V/PI, mitochondrial membrane potential disturbance, changes in cell cycle, CD95 expression, caspase activation, Nuclear Factor kappa B inhibition, and differentiation into a neutrophil-like cell (dHL60). RESULTS: Caracasine (10 μM) increased the G0/G1 phase in Jurkat and arrested the cell cycle in the S phase in HL60. Caracasine increased CD95 expression (p<0.01 in Jurkat and p<0.05 in HL60) and caspase-8 activation (p<0.001 in Jurkat and p<0.05 in HL60). Caspase-9 was activated in both cell lines (p<0.001) along with the decline in mitochondrial Δψm (p<0.05 in Jurkat and p<0.001 in HL60). In HL60 cells, the kaurane induced neutrophil differentiation was assessed by CD40 expression and reactive oxygen species production. In Jurkat cells, caracasine inhibited the NF-κB pathway in cells pretreated with PHA to activate the NF-κB pathway, suggesting a possible role in inflammatory diseases. CONCLUSION: Caracasine induced apoptosis through the intrinsic and extrinsic pathways in both cell lines were evaluated which could be the leading structure for new anti-leukemic and anti-inflammatory drugs.

• MeSH
• apoptóza MeSH
• diterpeny kauranové * farmakologie   chemie   MeSH
• diterpeny * farmakologie   MeSH
• HL-60 buňky MeSH
• Jurkat buňky MeSH
• leukemie * farmakoterapie   MeSH
• lidé MeSH
• NF-kappa B metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

• MeSH
• inhibitory proteinkinas farmakologie   MeSH
• ionizující záření MeSH
• jaderné proteiny antagonisté a inhibitory   MeSH
• Jurkat buňky MeSH
• leukemie T-buněčná enzymologie   genetika   patologie   MeSH
• lidé MeSH
• oprava DNA MeSH
• poškození DNA účinky záření   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   MeSH
• rekombinasa Rad51 antagonisté a inhibitory   MeSH
• tyrosinkinasy antagonisté a inhibitory   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH