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1.
Life Sci ; 242: 117228, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31881227

RESUMO

AIMS: Berberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. MAIN METHODS: P-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6-8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. KEY FINDINGS: BBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SIGNIFICANCE: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Berberina/farmacologia , Células Jurkat/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células U937/efeitos dos fármacos , Animais , Western Blotting , Citometria de Fluxo , Imunofluorescência , Humanos , Células Jurkat/metabolismo , Leucemia Experimental/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Reação em Cadeia da Polimerase em Tempo Real , Células U937/metabolismo , Ubiquitinação
2.
Mediators Inflamm ; 2018: 3286905, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30581368

RESUMO

Titanium and its alloys have been widely used in dental and orthopedic implants. Owing to the biotribocorrosion behavior of implants in simulated oral environment, Ti(IV) ions could be released into surrounding tissues. Current studies have found that Ti(IV) ions could affect the biological activities of immune cells in adjacent tissues and subsequently jeopardize the long-term performance of implant prostheses. However, the potential mechanism underlying its immunomodulatory properties remains unclear. Calcium signaling has been confirmed to be involved in regulation of lymphocyte immune function. Therefore, we hypothesize that Ti(IV) ions modulated T cell function through the change of intracellular calcium concentrations. This study is aimed at exploring the role of intracellular calcium responses in the modulatory effect of Ti(IV) ions on unactivated and phytohemagglutinin-activated Jurkat T cells. Here, we confirmed that Ti(IV) ions within a certain concentration range induced CD69 expression on both unactivated and activated T cells in our study. Additionally, the combined stimulation with Ti(IV) ions and PHA increased expression of IL-1ß, TNF-α, and RANKL. Furthermore, we found that treatment with Ti(IV) induced a transitory increase in the levels of [Ca2+]i in activated Jurkat cells, dependent on the presence of exogenous calcium. Treatment with different doses of Ti(IV) for 24 h significantly increased the levels of [Ca2+]i in the activated Jurkat cells in a dose-dependent manner, but had little effect in the unactivated cells. Treatment with Ti(IV) did not significantly affect the PLCγ1 activation and inositol-1,4,5-trisphosphate (IP3) secretion in Jurkat cells. Taken together, these data indicated that Ti(IV) enhanced calcium influx during the T cell activation, independent of IP3-mediated intracellular calcium release. Our work provides insights into the mechanism involved in the regulation of lymphocyte behaviors under the effect of Ti(IV) ions, which may help to develop therapeutic strategies for dental implant failures.


Assuntos
Adjuvantes Imunológicos/farmacologia , Cálcio/metabolismo , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Titânio/farmacologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Microscopia Confocal
3.
Microbiol Immunol ; 62(4): 229-242, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29350405

RESUMO

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV-infected lesions has yet to be established, it is here shown that substances produced by HCMV-infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV-associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV-infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX-2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP-ribose polymerase-1 (PARP-1)-mediated cell death, or parthanatos. HCMVAIS-induced cell death is accompanied by PARP-1 activation in a caspase-independent manner, nuclear translocation of apoptosis-inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS-induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV-positive foci in serial section samples of a large intestine with HCMV infection was TUNEL-positive, cleaved caspase 3-negative and CD45-positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV-infected cells through ROS-induced parthanatos by HCMVAIS.


Assuntos
Citomegalovirus/metabolismo , Espécies Reativas de Oxigênio , Proteínas Virais/farmacologia , Animais , Fator de Indução de Apoptose , Linfócitos T CD4-Positivos/efeitos dos fármacos , Caspase 3 , Morte Celular/efeitos dos fármacos , Linhagem Celular , Citomegalovirus/patogenicidade , Dano ao DNA/efeitos dos fármacos , Feminino , Humanos , Evasão da Resposta Imune , Intestino Grosso/patologia , Intestino Grosso/virologia , Células Jurkat/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Poli(ADP-Ribose) Polimerases/farmacologia , Células THP-1/efeitos dos fármacos
4.
Int J Mol Sci ; 18(12)2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29244717

RESUMO

Since interferon-γ (IFN-γ) tunes both innate and adaptive immune systems, it was expected to enter clinical practice as an immunomodulatory drug. However, the use of IFN-γ has been limited by its dose-dependent side effects. Low-dose medicine, which is emerging as a novel strategy to treat diseases, might circumvent this restriction. Several clinical studies have proved the efficacy of therapies with a low dose of cytokines subjected to kinetic activation, while no in vitro data are available. To fill this gap, we investigated whether low concentrations, in the femtogram range, of kinetically activated IFN-γ modulate the behavior of Jurkat cells, a widely used experimental model that has importantly contributed to the present knowledge about T cell signaling. In parallel, IFN-γ in the nanogram range was used and shown to activate Signal transducer and activator of transcription (STAT)-1 and then to induce suppressor of cytokine signaling-1 (SOCS-1), which inhibits downstream signaling. When added together, femtograms of IFN-γ interfere with the transduction cascade activated by nanograms of IFN-γ by prolonging the activation of STAT-1 through the downregulation of SOCS-1. We conclude that femtograms of IFN-γ exert an immunomodulatory action in Jurkat cells.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Linfócitos T/efeitos dos fármacos , Imunidade Adaptativa/genética , Relação Dose-Resposta a Droga , Humanos , Imunidade Inata/genética , Imunomodulação/efeitos dos fármacos , Interferon gama/administração & dosagem , Células Jurkat/efeitos dos fármacos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/imunologia , Linfócitos T/imunologia
5.
Carbohydr Res ; 451: 59-71, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-28965067

RESUMO

The convergent synthesis of broussonetinines related congeners 3 and 4 with the simple C13 alkyl side chain and differently configured pyrrolidine skeleton has been achieved. Our approach relied on the [3,3]-sigmatropic rearrangements of chiral allylic substrates derived from d-xylose. Cross metathesis of the common oxazolidinone intermediates 7 and 8 with tridec-1-ene followed by alkylative cyclization completed the construction of both C-alkyl iminosugars. The targeted compounds 3 and 4 were screened for antiproliferative/cytotoxic activities against multiple cancer cell lines by MTT assay. Compound 3 exhibited very good in vitro potency on Caco-2 and Jurkat cell lines with IC50 value of 5.1 µM and 5.8 µM, respectively.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Humanos , Células Jurkat/efeitos dos fármacos , Oxazolidinonas/química , Pirrolidinas/química , Relação Estrutura-Atividade
6.
Adv Clin Exp Med ; 26(3): 379-385, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28791810

RESUMO

BACKGROUND: The majority of the clinical trials with poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors were conducted or are ongoing in patients with solid tumors, while trials with leukemia patients are less frequent. Surprisingly scarce data is available on the combinatory effects of PARP inhibitors with DNA damaging antitumor drugs in leukemic cells (primary cells or established lines). OBJECTIVES: The aim of the present study was to assess the effect of PJ-34 (PARP-1 inhibitor) on the cytotoxicity of different antileukemic drugs with different DNA damaging mechanisms and potency (doxorubicin, etoposide, cytarabine and chlorambucil) in human leukemic Jurkat and HL-60 cells. MATERIAL AND METHODS: Different exposure scenarios were applied: 1) 72 h simultaneous incubation with PJ-34 (2.5 or 5 µM for Jurkat and HL-60 cells, respectively) and a drug used at a wide concentration range; 2) preincubation of the cells with PJ-34 for 24 h and then with a combination of PJ-34 + drug for an additional 48 h; 3) preincubation of the cells with the drug for 24 h with a subsequent incubation with a combination of PJ-34 + drug for an additional 48 h. Cytotoxicity was assessed using a WST-1 reduction test. RESULTS: It was determined that PJ-34, when used in all 3 scenarios, did not induce any significant enhancement of cytotoxicity of the drugs either in Jurkat or in HL-60 cells. CONCLUSIONS: Although the results do not confirm the beneficial effects of PARP inhibition in combination treatment of the leukemic cells, we propose that future studies including an additional step with the inhibition of DNA repair by homologous recombination should provide promising results.


Assuntos
Antineoplásicos/farmacologia , Células HL-60/efeitos dos fármacos , Células Jurkat/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Adenosina Difosfato Ribose , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Fenantrenos
7.
Appl Microbiol Biotechnol ; 101(19): 7227-7238, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28801829

RESUMO

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K m , Vmax, and k cat values were 5.6 mM, 2.2 µmol/min, and 1.96 × 103 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC50 of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.


Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Proteínas de Bactérias/farmacologia , Halomonas/enzimologia , Animais , Asparaginase/genética , Asparagina/metabolismo , Proteínas de Bactérias/genética , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetulus , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamina/metabolismo , Halomonas/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Células Jurkat/efeitos dos fármacos , Peso Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Células U937
8.
Virulence ; 8(8): 1732-1743, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-28762863

RESUMO

Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.


Assuntos
Síndrome de Imunodeficiência Adquirida/metabolismo , Células Dendríticas/imunologia , HIV-1/fisiologia , Células Jurkat/virologia , Fator de Necrose Tumoral alfa/farmacologia , Latência Viral , Síndrome de Imunodeficiência Adquirida/genética , Síndrome de Imunodeficiência Adquirida/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Células Dendríticas/citologia , HIV-1/genética , Humanos , Células Jurkat/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ativação Viral
9.
Eur J Med Chem ; 137: 139-155, 2017 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-28582670

RESUMO

The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein perforin have been explored. Perforin is a key component of the human immune response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.


Assuntos
Perforina/antagonistas & inibidores , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Estrutura Molecular , Perforina/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
10.
Acta Pharmacol Sin ; 38(8): 1171-1183, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28603286

RESUMO

T-cell acute lymphoblastic leukaemia (T-ALL) is a challenging malignancy with a high relapse rate attributed to drug resistance. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid extracted from a Chinese herb, is a potential anti-cancer and anti-leukaemic drug. In this study we investigated the mechanisms of TET resistance in T-ALL cells in vitro. Among the four T-ALL cell lines tested, Jurkat and CEM cells exhibited the lowest and highest resistance to TET with IC50 values at 24 h of 4.31±0.12 and 16.53±3.32 µmol/L, respectively. When treated with TET, the activity of transcription factor activator protein 1 (AP-1) was significantly decreased in Jurkat cells but nearly constant in CEM cells. To avoid cell-specific variation in drug resistance and transcription factor activities, we established a TET-R Jurkat subclone with the estimated IC50 value of 10.90±.92 µmol/L by exposing the cells to increasing concentrations of TET. Interestingly, when treated with TET, TET-R Jurkat cells exhibited enhanced AP-1 and NF-κB activity, along with upregulation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways, whereas the expression of P-gp was not altered. Selective inhibition of JNK but not ERK suppressed AP-1 activity and TET resistance in TET-R Jurkat cells and in CEM cells. These results demonstrate that Jurkat cells acquire TET resistance through activation of the JNK/AP-1 pathway but not through P-gp expression. The JNK/AP-1 pathway may be a potential therapeutic target in relapsed T-ALL.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzilisoquinolinas/farmacologia , Sistema de Sinalização das MAP Quinases , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Western Blotting , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Humanos , Células Jurkat/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo
11.
Free Radic Biol Med ; 106: 134-147, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28189848

RESUMO

Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.


Assuntos
Aminoglicosídeos/administração & dosagem , Antibióticos Antineoplásicos/administração & dosagem , Células Jurkat/metabolismo , Leucemia/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Apoptose/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/metabolismo , Doxorrubicina/administração & dosagem , Humanos , Peróxido de Hidrogênio/toxicidade , Células Jurkat/efeitos dos fármacos , Células Jurkat/patologia , Leucemia/metabolismo , Leucemia/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptomyces/química , Superóxidos/toxicidade
12.
J Pharm Biomed Anal ; 138: 100-108, 2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28189890

RESUMO

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.


Assuntos
Azatioprina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Jurkat/efeitos dos fármacos , Células Jurkat/fisiologia , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Fator de Transferência/farmacologia , Animais , Linhagem Celular Tumoral , Análise Custo-Benefício/métodos , Humanos , Camundongos , Reprodutibilidade dos Testes
13.
J Pept Sci ; 23(7-8): 574-580, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28078743

RESUMO

Interaction of CXCR4 with its endogenous ligand, stromal-cell derived factor-1 (SDF-1)/CXCL12, induces various physiological functions involving chemotaxis. Bivalent ligands with a polyproline helix bearing a cyclic pentapeptide, FC131, were previously shown to have higher binding affinities for CXCR4 than the corresponding monovalent ligands. Bivalent ligands based on a 14-mer peptide T140 derivative with polyproline linkers have been designed and synthesized. The activity of these peptides as well as the effect of bivalency of the ligand on CXCR4 binding has been assessed. The binding affinity of these series of bivalent ligands is increased as the linker length increases up to the 12-/15-mer proline linker. The inhibitory activity against chemotaxis on Jurkat cells also depends on the linker length. The T140-derived bivalent ligands with the 9- and 12-mer proline linkers showed the most effective inhibition against chemotaxis at 1000 nM, which is even higher than that of known CXCR4 antagonists in the monomer structure. The effective metastatic inhibition by bivalent T140 derivatives indicates the therapeutic potential. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Células Jurkat/efeitos dos fármacos , Peptídeos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Humanos , Peptídeos/química
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(12): 1641-1645, 2016 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-27916097

RESUMO

Objective To investigate the effect of (Pyr1)apelin-13 on the proliferation and activation of Jurkat T lymphocytes. Methods Jurkat T cells were treated by 0, 10, 50 nmol/L (Pyr1)apelin-13 for 24 hours and cell proliferation ability was detected by CCK-8 assay. The effect of (Pyr1)apelin-13 on the activation of Jurkat T lymphocytes with anti-CD3 and anti-CD28 antibodies was observed and the molecular mechanism was investigated. Quantitative real-time PCR was applied to detect the expressions of CD69, CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) mRNAs. Results The proliferation of Jurkat T cells was not affected by (Pyr1) apelin-13. However, (Pyr1)apelin-13 increased the expressions of CD69 and CD25 and then activated Jurkat T lymphocytes. Furthermore, the expressions of CTLA-4 and PD-1 had no difference between activation group and (Pyr1)apelin-13 group. Conclusion (Pyr1)apelin-13 promotes Jurkat T lymphocyte activation by increasing the expressions of CD69 and CD25.


Assuntos
Proliferação de Células/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células Jurkat/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Apoptose/genética , Complexo CD3/imunologia , Antígeno CTLA-4/genética , Células Cultivadas , Humanos , Células Jurkat/citologia , Células Jurkat/metabolismo , RNA Mensageiro/genética
15.
Proteomics ; 16(23): 2997-3008, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27687999

RESUMO

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.


Assuntos
4-Butirolactona/análogos & derivados , Proteômica/métodos , 4-Butirolactona/imunologia , 4-Butirolactona/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dermatite Alérgica de Contato/etiologia , Eletroforese em Gel Bidimensional , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/imunologia , Células Jurkat/metabolismo , Metaboloma , Dobramento de Proteína/efeitos dos fármacos , Purinas/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Testes de Toxicidade/métodos
16.
Biomacromolecules ; 17(10): 3205-3212, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27599388

RESUMO

A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.


Assuntos
Rastreamento de Células , Técnicas de Transferência de Genes , Polímeros/química , RNA Interferente Pequeno/química , Fluoresceína-5-Isotiocianato/química , Guanidina/química , Humanos , Células Jurkat/efeitos dos fármacos , Polímeros/administração & dosagem , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Transdução Genética
17.
Ann Hematol ; 95(11): 1787-93, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27506924

RESUMO

Although the response rates of chemotherapy in patients with acute T-lymphoblastic leukemia (T-ALL) have improved significantly, the outcome of these patients is still poor. Previous studies suggested that baicalein could inhibit the growth of several cancers, while its effect on T-ALL cells remains unclear. We used Jurkat cells as an in vitro model of T-ALL. Cell counting kit-8 assay and cytometric analysis with Annexin V-FITC/PI double staining were used to investigate the proliferation and apoptosis of Jurkat cells treated with increasing concentration of baicalein for indicated time. RT-PCR and western blotting was used to test the expression of Wnt/ß-catenin associated genes and proteins. In cell viability assay, baicalein could inhibit the proliferation of Jurkat cells both in dose- and time-dependent manners. In cell apoptosis assay, baicalein could stimulate apoptosis of Jurkat cells both in dose- and time-dependent manners. Moreover, we demonstrated that baicalein could down-regulated the mRNA and protein levels of ß-catenin and its widely accepted downstream targets (c-Myc, cyclin D1, and Axin2) in dose-dependent manners. These results proved that baicalein might be a potential choice for the treatment of T-ALL.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Flavanonas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Tempo , beta Catenina/biossíntese , beta Catenina/genética
18.
PLoS One ; 11(8): e0160575, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27500529

RESUMO

The radioprotective capacity of a rationally-designed Mn2+-decapeptide complex (MDP), based on Mn antioxidants in the bacterium Deinococcus radiodurans, was investigated in a mouse model of radiation injury. MDP was previously reported to be extraordinarily radioprotective of proteins in the setting of vaccine development. The peptide-component (DEHGTAVMLK) of MDP applied here was selected from a group of synthetic peptides screened in vitro for their ability to protect cultured human cells and purified enzymes from extreme damage caused by ionizing radiation (IR). We show that the peptides accumulated in Jurkat T-cells and protected them from 100 Gy. MDP preserved the activity of T4 DNA ligase exposed to 60,000 Gy. In vivo, MDP was nontoxic and protected B6D2F1/J (female) mice from acute radiation syndrome. All irradiated mice treated with MDP survived exposure to 9.5 Gy (LD70/30) in comparison to the untreated mice, which displayed 63% lethality after 30 days. Our results show that MDP provides early protection of white blood cells, and attenuates IR-induced damage to bone marrow and hematopoietic stem cells via G-CSF and GM-CSF modulation. Moreover, MDP mediated the immunomodulation of several cytokine concentrations in serum including G-CSF, GM-CSF, IL-3 and IL-10 during early recovery. Our results present the necessary prelude for future efforts towards clinical application of MDP as a promising IR countermeasure. Further investigation of MDP as a pre-exposure prophylactic and post-exposure therapeutic in radiotherapy and radiation emergencies is warranted.


Assuntos
Deinococcus/química , Protetores contra Radiação/química , Protetores contra Radiação/farmacologia , Animais , Antígenos CD34/metabolismo , Antioxidantes/química , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Citocinas/sangue , DNA Ligases/metabolismo , Desenho de Fármacos , Feminino , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/efeitos da radiação , Leucopenia/tratamento farmacológico , Manganês/química , Camundongos Endogâmicos , Peptídeos/química , Lesões por Radiação/prevenção & controle , Radiação Ionizante , Protetores contra Radiação/efeitos adversos , Esplenomegalia/tratamento farmacológico
19.
J Nat Prod ; 79(9): 2304-14, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27571379

RESUMO

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Basidiomycota/química , Naftoquinonas/química , Naftoquinonas/farmacologia , Antineoplásicos/sangue , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Jurkat/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Estrutura Molecular , Naftoquinonas/sangue , Naftoquinonas/síntese química , Naftoquinonas/isolamento & purificação , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR
20.
Apoptosis ; 21(9): 1019-32, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27364951

RESUMO

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E commonly used as a drug delivery agent. Recently, TPGS alone has been reported to induce cell death in lung, breast and prostate cancer. However, the effect of TPGS on cancer cell viability remains unclear. Thus, this study was aimed to evaluate the cytotoxic effect of TPGS on human periphral blood lymphocytes (PBL) and on T cell acute lymphocytic leukemia (ALL) Jurkat clone E6-1 cells and its possible mechanism of action. PBL and Jurkat cells were treated with TPGS (10, 20, 40, 60, and 80 µM), and morphological changes in the cell nucleus, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by immune-fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by immunocytochemistry. In this study, TPGS induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing nuclear DNA fragmentation, increasing cell cycle arrest, and decreasing ΔΨm. Additionally, TPGS increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, in a dose-independent fashion. TPGS increased DJ-1 Cys(106)-sulfonate, as a marker of intracellular stress and induced the activation of NF-κB, p53 and c-Jun transcription factors. Additionally, it increased the expression of apoptotic markers Bcl-2 related pro-apoptotic proteins Bax and PUMAand activated caspase-3. The antioxidant N-acetyl-L-cysteine and known pharmacological inhibitors protected the cells from the TPGS induced effects. In conclusion, TPGS selectively induces apoptosis in Jurkat cells through two independent but complementary H2O2-mediated signaling pathways. Our findings support the use of TPGS as a potential treatment for ALL.


Assuntos
Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Vitamina E/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
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