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1.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445118

RESUMO

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cisteína/metabolismo , Inibidores de Proteases/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Linhagem Celular Tumoral , Células Clonais , Granzimas/metabolismo , Humanos , Células Jurkat , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/fisiologia , Linfócitos T Citotóxicos/metabolismo
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1123-1128, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362491

RESUMO

OBJECTIVE: To observe the effects of down-regulation of long non-coding RNA HOX antisense intergenic RNA myeloid 1 (LncRNA-HOTAIRM1) to the proliferation and apoptosis of Jurkat in human leukemia T lymphocytes, and explore its mechanism. METHODS: Jurkat cells were cultured in vitro and randomly divided into control group, HOTAIRM1 siRNA-NC group and HOTAIRM1 siRNA group; the expressions of LncRNA-HOTAIRM1 mRNA, KIT receptor tyrosine kinase (KIT) mRNA and serine threonine kinase (AKT) mRNA in Jurkat cells were detected by real-time fluorescence quantification (RT-qPCR); the proliferation of Jurkat cells in each groups was detected by CCK-8 method; the apoptosis of Jurkat cells in each groups was detected by Annexin V-FITC/PI double staining; the expressions of KIT, AKT, p-KIT, p-AKT, B-lymphoma-2 gene (BCL-2) and Caspase-3 were detected by Western blot. RESULTS: Compared with the cells in the control group and HOTAIRM1 siRNA-NC group, the expression level of LncRNA-HOTAIRM1 mRNA, cell survival rate, expression levels of KIT mRNA, AKT mRNA, p-KIT, p-AKT and BCL-2 proteins in Jurkat cells in HOTAIRM1 siRNA group were significantly lower (P<0.05), while the expression level of Cleared Caspase-3 protein and Jurkat cell apoptosis rate were significantly higher (P<0.05). CONCLUSION: LncRNA-HOTAIRM1 may inhibit Jurkat cell proliferation and induce apoptosis through KIT/AKT signaling pathway.


Assuntos
RNA Longo não Codificante , Apoptose , Proliferação de Células , Regulação para Baixo , Humanos , Células Jurkat , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética
3.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445394

RESUMO

Cytotoxic effects of cannabidiol (CBD) and tamoxifen (TAM) have been observed in several cancer types. We have recently shown that CBD primarily targets mitochondria, inducing a stable mitochondrial permeability transition pore (mPTP) and, consequently, the death of acute lymphoblastic leukemia (T-ALL) cells. Mitochondria have also been documented among cellular targets for the TAM action. In the present study we have demonstrated a synergistic cytotoxic effect of TAM and CBD against T-ALL cells. By measuring the mitochondrial membrane potential (ΔΨm), mitochondrial calcium ([Ca2+]m) and protein-ligand docking analysis we determined that TAM targets cyclophilin D (CypD) to inhibit mPTP formation. This results in a sustained [Ca2+]m overload upon the consequent CBD administration. Thus, TAM acting on CypD sensitizes T-ALL to mitocans such as CBD by altering the mitochondrial Ca2+ homeostasis.


Assuntos
Cálcio/metabolismo , Canabidiol/farmacologia , Ciclofilina D/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Tamoxifeno/farmacologia , Linhagem Celular Tumoral , Ciclofilina D/química , Sinergismo Farmacológico , Homeostase/efeitos dos fármacos , Humanos , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Conformação Proteica
4.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443436

RESUMO

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.


Assuntos
Antígeno B7-H1/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica
5.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445489

RESUMO

An original synthetic route was developed for the preparation of previously unknown unsaturated polyaromatic macrolactones containing a 1Z,5Z-diene moiety in 48-71% yields and with >98% stereoselectivity. The method is based on intermolecular cyclocondensation of aromatic dicarboxylic acids with α,ω-alka-nZ,(n+4)Z-dienediols (1,12-dodeca-4Z,8Z-dienediol, 1,14-tetradeca-5Z,9Z-dienediol, 1,18-octadeca-7Z,11Z-dienediol) mediated by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/4-dimethylaminopyridine (DMAP). The unsaturated diols were prepared by successive homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp2TiCl2 catalyst (10 mol.%) and subsequent treatment with 0.1 equiv. of para-toluenesulfonic acid of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The resulting cyclophanes exhibited high cytotoxic activity in vitro against Jurkat, K562, U937, and HL60 cancer lines. Additionally, the synthesized products were studied for their effect on mitochondria, ability to induce apoptosis, and influence on the cell cycle using modern flow cytometry methods.


Assuntos
Antineoplásicos/síntese química , Éteres Cíclicos/síntese química , Compostos Organometálicos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reação de Cicloadição , Éteres Cíclicos/química , Éteres Cíclicos/farmacologia , Citometria de Fluxo , Células HL-60 , Humanos , Células Jurkat , Células K562 , Estrutura Molecular
6.
Nat Commun ; 12(1): 4821, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376672

RESUMO

Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease.


Assuntos
Genoma Viral/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Adulto , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Evolução Clonal/genética , Células Clonais/metabolismo , Células Clonais/patologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Mutação , RNA-Seq/métodos , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T/genética , Fator de Transcrição STAT3/genética
7.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281255

RESUMO

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 µM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 µM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.


Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Midazolam/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Citocinas/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Relação Dose-Resposta a Droga , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Cinética , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Microscopia Confocal , Midazolam/administração & dosagem , Técnicas de Patch-Clamp , Fito-Hemaglutininas/farmacologia , Linfócitos T/imunologia
8.
Nat Commun ; 12(1): 4197, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234139

RESUMO

Single cell RNA sequencing (scRNA-seq) is a powerful tool in detailing the cellular landscape within complex tissues. Large-scale single cell transcriptomics provide both opportunities and challenges for identifying rare cells playing crucial roles in development and disease. Here, we develop GapClust, a light-weight algorithm to detect rare cell types from ultra-large scRNA-seq datasets with state-of-the-art speed and memory efficiency. Benchmarking on diverse experimental datasets demonstrates the superior performance of GapClust compared to other recently proposed methods. When applying our algorithm to an intestine and 68 k PBMC datasets, GapClust identifies the tuft cells and a previously unrecognised subtype of monocyte, respectively.


Assuntos
Algoritmos , RNA-Seq/métodos , Análise de Célula Única/métodos , Conjuntos de Dados como Assunto , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Células Jurkat , Software
9.
Biomed Pharmacother ; 139: 111656, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243603

RESUMO

INTRODUCTION: Amyrins are triterpenes that have attractive pharmacological potential; however, their low water solubility and erratic stomach absorption hinders their use as a drug. The aim of this paper was to develop a novel α-amyrin-loaded nanocapsule for intestinal delivery and evaluate, preliminarily, its cytotoxic ability against leukemic cells. MATERIAL AND METHODS: Five nanocapsule formulations were designed by the solvent displacement-evaporation method. Poly-ε-caprolactone, Eudragit® E100, and Kollicoat® Mae 100 P were used as film-former materials. Particle size, polydispersity index (PdI), zeta potential, and the pH of all formulations were measured. The cytotoxic potential of the nanocapsules was evaluated in vitro using different leukemic lineages RESULTS: Nanocapsules coated with Kollicoat® Mae 100 P presented the smallest particle size (130 nm), the lowest zeta-potential (-38 mV), and the narrowest size distribution (PdI = 0.100). The entrapment efficiency was 65.47%, while the loading capacity was 2.40%. Nanocapsules release 100% of α-amyrin in 40 min (pH 7.4), by using a possible mechanism of swelling-diffusion. The formulation showed excellent on-shelf physicochemical stability during one year. Additionally, nanocapsules produced a selective cytotoxic effect on a human leukemia lineage Kasumi-1, an acute myeloid leukemia cell line, and produced cell death by apoptosis CONCLUSION: α-amyrin-loaded nanocapsules appear to be a promising nanoformulation that could be used against leukemia.


Assuntos
Leucemia/tratamento farmacológico , Nanocápsulas/química , Triterpenos Pentacíclicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caproatos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Células Jurkat , Células K562 , Lactonas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Tamanho da Partícula , Ácidos Polimetacrílicos/química
10.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199262

RESUMO

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.


Assuntos
Técnicas de Cultura de Células/métodos , Microvasos/fisiologia , Engenharia Tecidual/métodos , Simulação de Ausência de Peso , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Células Jurkat , Teste de Materiais , Microvasos/efeitos dos fármacos , Células THP-1
11.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203724

RESUMO

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Morte Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismo
12.
Molecules ; 26(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207168

RESUMO

Xanthone derivatives have shown promising antitumor properties, and 1-carbaldehyde-3,4-dimethoxyxanthone (1) has recently emerged as a potent tumor cell growth inhibitor. In this study, its effect was evaluated (MTT viability assay) against a new panel of cancer cells, namely cervical cancer (HeLa), androgen-sensitive (LNCaP) and androgen-independent (PC-3) prostate cancer, and nonsolid tumor derived cancer (Jurkat) cell lines. The effect of xanthone 1 on macrophage functions was also evaluated. The effect of xanthone 1-conditioned THP-1 human macrophage supernatants on the metabolic viability of cervical and prostate cancer cell lines was determined along with its interference with cytokine expression characteristic of M1 profile (IL-1 ≤ ß; TNF-α) or M2 profile (IL-10; TGF-ß) (PCR and ELISA). Nitric oxide (NO) production by murine RAW264.7 macrophages was quantified by Griess reaction. Xanthone 1 (20 µM) strongly inhibited the metabolic activity of the cell lines and was significantly more active against prostate cell lines compared to HeLa (p < 0.05). Jurkat was the cell most sensitive to the effect of xanthone 1. Compound 1-conditioned IL-4-stimulated THP-1 macrophage supernatants significantly (p < 0.05) inhibited the metabolic activity of HeLa, LNCaP, and PC-3. Xanthone 1 did not significantly affect the expression of cytokines by THP-1 macrophages. The inhibiting effect of compound 1 observed on the production of NO by RAW 264.7 macrophages was moderate. In conclusion, 1-carbaldehyde-3,4-dimethoxyxanthone (1) decreases the metabolic activity of cancer cells and seems to be able to modulate macrophage functions.


Assuntos
Antineoplásicos/farmacologia , Macrófagos/efeitos dos fármacos , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Xantonas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Células HeLa , Papillomavirus Humano 18/patogenicidade , Humanos , Células Jurkat , Macrófagos/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Células PC-3 , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Células RAW 264.7 , Células THP-1 , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
13.
Lab Chip ; 21(16): 3112-3127, 2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34286793

RESUMO

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation cell sorters, microfluidic fluorescence-activated cell sorting (µ-FACS) devices have been developed to avoid cross-contamination, minimize device footprint, and eliminate bio-aerosols. However, due to highly precise flow control, the achievable throughput of the µ-FACS system is generally lower than the throughput of conventional FACS devices. Here, we report a fully integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting (µ-CFACS) system for the enrichment of clinical rare cells. A microfluidic sorting cartridge has been developed for enriching samples through a sequential sorting process, which was further realized by the integration of both fast amplified piezoelectrically actuated on-chip valves and compact pneumatic cylinders actuated on-chip valves. At an equivalent throughput of ∼8000 events per second (eps), the purity of rare fluorescent microparticles has been significantly increased from ∼0.01% to ∼27.97%. An enrichment of ∼9400-fold from 0.009% to 81.86% has also been demonstrated for isolating fluorescently labelled MCF-7 breast cancer cells from Jurkat cells at an equivalent sorting throughput of ∼6400 eps. With the advantages of high throughput and contamination-free design, the proposed integrated µ-CFACS system provides a new option for the enrichment of clinical rare cells.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Separação Celular , Citometria de Fluxo , Humanos , Células Jurkat , Células MCF-7
14.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
15.
Molecules ; 26(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299644

RESUMO

Metallodrugs form a large family of therapeutic agents against cancer, among which is cisplatin, a paradigmatic member. Therapeutic resistance and undesired side effects to Pt(II) related drugs, prompts research on different metal-ligand combinations with potentially enhanced biological activity. We present the synthesis and biological tests of novel palladium(II) complexes containing bisdemethoxycurcumin (BDMC) 1 and 2. Complexes were fully characterized and their structures were determined by X-ray diffraction. Their biological activity was assessed for several selected human tumor cell lines: Jurkat (human leukaemic T-cell lymphoma), HCT-116 (human colorectal carcinoma), HeLa (human cervix epitheloid carcinoma), MCF-7 (human breast adenocarcinoma), MDA-MB-231 (human mammary gland adenocarcinoma), A549 (human alveolar adenocarcinoma), Caco-2 (human colorectal carcinoma), and for non-cancerous 3T3 cells (murine fibroblasts). The cytotoxicity of 1 is comparable to that of cisplatin, and superior to that of 2 in all cell lines. It is a correlation between IC50 values of 1 and 2 in the eight studied cell types, promising a potential use as anti-proliferative drugs. Moreover, for Jurkat cell line, complexes 1 and 2, show an enhanced activity. DFT and docking calculations on the NF-κB protein, Human Serum Albumin (HSA), and DNA were performed for 1 and 2 to correlate with their biological activities.


Assuntos
Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação , Citotoxinas , DNA de Neoplasias , Diarileptanoides , Simulação de Acoplamento Molecular , Paládio , Células 3T3 , Animais , Células CACO-2 , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Citotoxinas/química , Citotoxinas/farmacologia , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Diarileptanoides/química , Diarileptanoides/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Células HeLa , Humanos , Células Jurkat , Células MCF-7 , Camundongos , Paládio/química , Paládio/farmacologia
16.
Nat Commun ; 12(1): 4628, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330905

RESUMO

Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the "tag" and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.


Assuntos
Azepinas/metabolismo , Cisplatino/metabolismo , Espaço Intracelular/metabolismo , Espectrometria de Massa de Íon Secundário/métodos , Triazóis/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Azepinas/farmacocinética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cisplatino/farmacocinética , Citoplasma/metabolismo , Células HeLa , Humanos , Células Jurkat , Microscopia Confocal , Triazóis/farmacocinética
17.
Front Immunol ; 12: 615859, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220794

RESUMO

Purpose: Systemic lupus erythematosus (SLE) is a serious autoimmune disease. Its molecular pathogenesis, especially the long non-coding RNA (lncRNA) function, remains unclear. We want to investigate the lncRNA dysregulation profile and their molecular mechanisms in SLE. Methods: In this study, we analyzed the transcriptome profiles (RNA-seq) of peripheral blood mononuclear cells (PBMCs) from SLE patients and two published transcriptome datasets to explore lncRNA profiles. The differentially expressed lncRNAs were confirmed by quantitative real-time PCR in another set of female patients. We constructed the lncRNA-mRNA regulatory networks by performing weighted gene co-expression network analysis (WGCNA). Dysregulated lncRNA AC007278.2 was repressed by short hairpin RNA (shRNA) in Jurkat cells. Dual-luciferase reporter gene assay was performed to investigate the regulatory mechanism of AC007278.2 on target gene CCR7. Results: We observed dominant up-regulation of transcripts, including mRNAs and lncRNAs, in SLE patients. By WGCNA method, we identified three modules that were highly related to SLE. We then focused on one lncRNA, AC007278.2, with a T-helper 1 lineage-specific expression pattern. We observed consistently higher AC007278.2 expression in SLE patients. Co-expression network revealed that AC007278.2 participated in the innate immune response and inflammatory bowel disease pathways. By knocking down AC007278.2 expression, we found that AC007278.2 could regulate the expression of inflammatory and cytokine stimulus response-related genes, including CCR7, AZU1, and TNIP3. AC007278.2 inhibits the functional CCR7 promoter to repress its transcription, thereby regulating autoimmunity and follicular T-helper cell differentiation. Conclusion: In summary, our study indicated the important regulatory role of lncRNAs in SLE. AC007278.2 may be treated as a novel biomarker for SLE diagnosis and treatment.


Assuntos
Centro Germinativo/imunologia , Lúpus Eritematoso Sistêmico/genética , RNA Longo não Codificante/genética , Receptores CCR7/metabolismo , Células Th1/fisiologia , Autoimunidade/genética , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Marcadores Genéticos/genética , Humanos , Células Jurkat , Lúpus Eritematoso Sistêmico/diagnóstico , RNA Interferente Pequeno/genética , Receptores CCR7/genética , Transcriptoma , Regulação para Cima
18.
FEBS Lett ; 595(16): 2127-2146, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34160065

RESUMO

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.


Assuntos
Membrana Celular/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Agregados Proteicos , Linfócitos T/citologia , Humanos , Células Jurkat , Transdução de Sinais
19.
Biochim Biophys Acta Proteins Proteom ; 1869(10): 140684, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34146732

RESUMO

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 ß-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.


Assuntos
Substituição de Aminoácidos , Amino Açúcares/metabolismo , Galectinas/química , Galectinas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Difusão Dinâmica da Luz , Galectinas/genética , Humanos , Células Jurkat , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em Folha beta , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
20.
Genes (Basel) ; 12(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067766

RESUMO

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.


Assuntos
Processamento Alternativo , Metilação de DNA , Código das Histonas , Antígenos Comuns de Leucócito/genética , RNA Antissenso/genética , Fator de Ligação a CCCTC/metabolismo , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/metabolismo , Ligação Proteica , Pseudogenes , RNA Antissenso/metabolismo
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