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1.
Cancer Invest ; 38(6): 349-355, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32441531

RESUMO

Background: Meningiomas represent ∼30% of primary central nervous system (CNS) tumors. Although advances in surgery and radiotherapy have significantly improved survival, there remains an important subset of patients whose tumors have more aggressive behavior and are refractory to conventional therapy. Recent advances in molecular genetics and epigenetics suggest that this aggressive behavior may be due to the deletion of the DNA repair and tumor suppressor gene, CHEK2, neurofibromatosis Type 2 (NF2) mutation on chromosome 22q12, and genetic abnormalities in multiple RTKs including FGFRs. Management of higher-grade meningiomas, such as anaplastic meningiomas (AM: WHO grade III), is truly challenging and there isn't an established chemotherapy option. We investigate the effect of active multi tyrosine receptor kinase inhibitor Dovitinib at stopping AM cell growth in in vitro with either frequent codeletion or mutated CHEK2 and NF2 gene.Methods: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot analysis, caspases assay, and DNA fragmentation assay.Results: Treatment of CH157MN and IOMM-Lee cells with Dovitinib suppressed multiple angiokinases-mainly FGFRs, leading to suppression of downstream signaling by RAS-RAF-MAPK molecules and PI3K-AKT molecules which are involved in cell proliferation, cell survival, and tumor invasion. Furthermore, Dovitinib induced apoptosis via downregulation of survival proteins (Bcl-XL), and over-expression of apoptotic factors (Bax and caspase-3) regardless of CHEK2 and NF2 mutation status.Conclusions: This study establishes the groundwork for the development of Dovitinib as a therapeutic agent for high-grade AM with either frequent codeletion or mutated CHEK2 and NF2, an avenue with high translational potential.


Assuntos
Benzimidazóis/farmacologia , Quinase do Ponto de Checagem 2/genética , Meningioma/tratamento farmacológico , Neurofibromina 2/genética , Quinolonas/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meningioma/genética , Meningioma/patologia , Mutação/genética , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína bcl-X/genética
2.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(1): 20-26, 2020 Jan 30.
Artigo em Chinês | MEDLINE | ID: mdl-32376564

RESUMO

OBJECTIVE: To investigate the effect of overexpression of leukemia inhibitory factor (LIF) on cisplatin and paclitaxel resistance of endometrial cancer cells in vitro. METHODS: Endometrial cancer cell lines HEC-1B and RL95-2 were infected with a recombinant lentivirus to overexpress LIF, and the changes in LIF expression was verified using RT-qPCR and ELISA. The viability of the LIF-overexpressing cells was assessed using CCK-8 assay, and the cell apoptosis and changes in mitochondrial membrane potential in response to cisplatin or paclitaxel treatment were analyzed with annexin V-FITC/PI staining and JC-1 assay, respectively. The effect of LIF overexpression on the expressions of Bcl-2 family proteins and STAT3 pathway was evaluated using Western blotting; dual-luciferase reporter gene assay was employed to detect the transcriptional activity of STAT3. The effect of STAT3 silencing on apoptosis of the LIF-overexpressing cells induced by cisplatin or paclitaxel was investigated. RESULTS: The cell lines infected with the recombinant lentivirus showed significantly increased mRNA and protein levels of LIF (P < 0.05) without obvious changes in the cell viability (P>0.05). LIF overexpression significantly attenuated cisplatin-or paclitaxel-induced apoptosis of the endometrial cancer cells (P < 0.05) and markedly increased mitochondrial membrane potential of the cells (P < 0.05). The expressions of Bcl-2, Bcl-xL and p-STAT3 proteins increased obviously while the expressions of Bax, Bad and STAT3 either decreased or showed no obvious changes in the LIF-overexpressing cells. Overexpressing LIF significantly enhanced the transcriptional activity of STAT3 (P < 0.05), and silencing STAT3 obviously enhanced apoptosis of the endometrial cancer cells overexpressing LIF (P < 0.05). CONCLUSIONS: s Overexpression of LIF can enhance cisplatin and paclitaxel resistance to endometrial cancer cells in vitro.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias do Endométrio/genética , Fator Inibidor de Leucemia/genética , Paclitaxel/farmacologia , Transdução de Sinais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína bcl-X/metabolismo
3.
Life Sci ; 254: 117760, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32418889

RESUMO

AIM: The present study focused on the possible underlying protective mechanisms of UDCA against GNT-induced hepatic injury. METHODS: For achieving this goal, adult male rats were allocated into 4 groups: normal control (received vehicle), GNT (100 mg/kg, i.p. for 8 days), UDCA (60 mg/kg, P.O. for 15 days), and GNT + UDCA (received UDCA for 15 days and GNT started from the 7th day and lasted for 8 days). RESULTS: The results revealed that UDCA significantly improved GNT-induced hepatic injury, oxidative stress, apoptosis, and inflammatory response. Interestingly, UDCA inhibited apoptosis by marked down-regulation of the Bax gene, Caspase-3, and cleaved Caspase-3 protein expressions while the level of Bcl-xL gene significantly increased. Moreover, UDCA strongly inhibited the inflammatory response through the down-regulation of both NF-κB-p65 and TNF-α accompanied by IL-10 elevation. Furthermore, the obtained results ended with the restored of mitochondria function that confirmed by electron microscopy. Histological analysis showed that UDCA remarkably ameliorated the histopathological changes induced by GNT. SIGNIFICANCE: UDCA may be a promising agent that can be used to prevent hepatotoxicity observed in GNT treatment. This effect could be attributed to, at least in part, the ability of UDCA to modulate NF-κB-p65/TNF-α, Bax/Bcl-xl/Caspase-3, and eNOS/iNOS signaling pathways.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Gentamicinas/antagonistas & inibidores , Gentamicinas/toxicidade , Hepatócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido Ursodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Interações Medicamentosas , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
4.
Nature ; 580(7804): 542-547, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322059

RESUMO

Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.


Assuntos
Apoptose , Mitose , Separase/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Especificidade por Substrato , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo
5.
Gene ; 741: 144552, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32165297

RESUMO

Hypoxia, as a form of stress, plays a critical role in oncogenesis, including metabolic reprogramming. Mitochondrial, the centers of energy production, re-balance mitochondria dynamic to maintain cell survival during high levels of environmental stresses. NDRG1 is a hypoxia-inducible protein that is involved in various human cancers, including HCC. However, little is known about whether NDRG1 participants in the quality control of mitochondrial in times of stress. Here, we firstly showed that how NDRG1 exerted its role through mediating mitochondrial dynamic in HCC cells under hypoxia. Initially, we identified that NDRG1 expression varies with oxygen content. NDRG1 silencing notably induced cell apoptosis under hypoxia, while no obviously change of wildtype cells in hypoxia compared with that in normoxia. Further analysis revealed that NDRG1 silencing in HCC cells led to increase of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx, which meant mitochondrial damage were induced. In the analysis of mitochondria, we found that more released cytochrome c located in cytosolic with NDRG1 knockdown in hypoxia, which may be due to mitochondria division. And the following experiment proved that more fragmented mitochondria were presented in NDRG1 silencing cells, as well as destroyed mitochondrial membrane potential with evidence by JC-1 was verified. Moreover, these trends could be reversed by Mdivi1. Further research showed that NDRG1 silencing disrupt hypoxia-enhanced aerobic glycolysis through effectively decreased glucose uptake, lactate output and ECAR value. In sum, we provide the first direct evidence that NDRG1-driven change in mitochondrial dynamics and aerobic glycolysis maintain cells survival in HCC during hypoxia.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Reprogramação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Apoptose/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Citocromos c/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/genética , Dinâmica Mitocondrial/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Hipóxia Tumoral/genética , Proteína bcl-X/genética
6.
Nat Commun ; 11(1): 752, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029722

RESUMO

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.


Assuntos
Testes Genéticos/métodos , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular , Células Clonais , Técnicas de Inativação de Genes , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Análise de Célula Única , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência , Proteína bcl-X/genética
7.
Eur J Cancer ; 126: 93-103, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31927215

RESUMO

INTRODUCTION: Uveal melanoma (UM) is a rare and malignant intraocular tumour with a dismal prognosis. Despite a good control of the primary tumour by radiation or surgery, up to 50% of patients subsequently develop metastasis for which no efficient treatment is yet available. METHODOLOGY: To identify therapeutic opportunities, we performed an in vitro screen of 30 combinations of different inhibitors of pathways that are dysregulated in UM. Effects of drug combinations on viability, cell cycle and apoptosis were assessed in eight UM cell lines. The best synergistic combinations were further evaluated in six UM patient-derived xenografts (PDXs). RESULTS: We demonstrated that the Bcl-2/XL/W inhibitor (ABT263) sensitised the UM cell lines to other inhibitors, mainly to mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase (MEK) and murine double minute 2 (MDM2) inhibitors. mTOR (RAD001) and MEK1/2 (trametinib) inhibitors were efficient as single agents, but their combinations with ABT263 displayed no synergism in UM PDXs. In contrast, the combination of ABT263 with MDM2 inhibitor (HDM201) showed a trend for a synergistic effect. CONCLUSION: We showed that inhibition of Bcl-2/XL/W sensitised the UM cell lines to other treatments encouraging investigation of the underlying mechanisms. Furthermore, our findings highlighted Bcl-2/XL/W and MDM2 co-inhibition as a promising strategy in UM.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Melanoma/tratamento farmacológico , Neoplasias Uveais/tratamento farmacológico , Compostos de Anilina/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Combinação de Medicamentos , Everolimo/administração & dosagem , Humanos , Imidazóis/administração & dosagem , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas/administração & dosagem , Pirróis/administração & dosagem , Sulfonamidas/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
8.
Biochim Biophys Acta Biomembr ; 1862(4): 183190, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935366

RESUMO

A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.


Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Canal de Ânion 1 Dependente de Voltagem/genética , Fenômenos Biofísicos , Membrana Celular/química , Membrana Celular/genética , Humanos , Proteínas de Membrana/genética , Fosfolipídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Canal de Ânion 1 Dependente de Voltagem/química , Proteína bcl-X/química , Proteína bcl-X/genética
9.
Nat Commun ; 11(1): 259, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937780

RESUMO

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.


Assuntos
Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
10.
Int J Radiat Oncol Biol Phys ; 106(4): 867-877, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31786278

RESUMO

PURPOSE: The incidence of mesothelioma continues to rise and prognosis remains dismal owing to resistance to conventional therapies and few novel treatment options. Failure to activate apoptotic cell death is a resistance mechanism that may be overcome by inhibition of antiapoptotic Bcl-2 proteins using BH3-mimetic drugs. We investigated the role of antiapoptotic proteins in the radioresistance of mesothelioma, identifying clinically relevant targets for radiosensitization and evaluating the activity of BH3-mimetics alone and in combination with radiation therapy in preclinical models. METHODS, MATERIALS AND RESULTS: Mesothelioma cell lines 211H, H2052, and H226 exposed to BH3-mimetics demonstrated Bcl-xL dependence that correlated with protein expression and was confirmed by genetic knockdown. The Bcl-xL inhibitor A1331852 exhibited cytotoxic (EC50, 0.13-1.42 µmol/L) and radiosensitizing activities (sensitizer enhancement ratios, 1.3-1.8). Cytotoxicity was associated with induction of mitochondrial outer membrane permeabilization and caspase-3/7 activation. Efficacy was maintained in a 3-dimensional model in which combination therapy completely eradicated mesothelioma spheroids. Clinical applicability was confirmed by immunohistochemical analysis of Bcl-2 proteins in patient samples and radiosensitizing activity of A1331852 in primary patient-derived mesothelioma cells. CONCLUSIONS: Mesothelioma cells exhibit addiction to the antiapoptotic protein Bcl-xL, and their intrinsic radioresistance can be overcome by small molecule inhibition of this novel therapeutic target.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Mesotelioma/patologia , Fragmentos de Peptídeos , Peptidomiméticos/farmacologia , Proteínas Proto-Oncogênicas , Radiossensibilizantes/farmacologia , Proteína bcl-X/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos
11.
Acta Haematol ; 143(1): 51-59, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31434075

RESUMO

BACKGROUND: SAM domain- and HD domain-containing protein 1 (SAMHD1) is a cellular enzyme which is responsible for blocking replication in viruses and participates in the progression of many cancers. OBJECTIVE: The aim of this study was to correlate the expression level of SAMHD1 with other apoptotic and autophagic genes in acute myeloid leukemia (AML) patients. METHODS: In the present study, mRNA levels of SAMHD1 with other apoptotic and autophagic-related genes were evaluated in patients who were newly diagnosed with AML. RESULTS: SAMHD1, Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in the AML group compared to the non-AML group (p < 0.05). SAMHD1 expression did not correlate with the other genes, while most apoptotic genes were positively correlated with each other. SAMHD1 expression was not associated with the blood routine or blast percentage of the AML patients, while Bax, Bak, cIAP2, and LC3 were significantly correlated with white blood cells. No statistically significant differences were found between the studied genes and prognosis stratifications, but Bcl-xl, Bak, cIAP1, and Mcl-1, LC3 were expressed at lower levels in the unfavorable AML group compared to the controls. CONCLUSION: SAMHD1 and Bcl-xl, Bax, Bak, XIAP, and cIAP1 were downregulated in AML patients, while there were no significant differences in the clinical characteristics and prognosis with reference to SAMHD1 expression.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Reguladoras de Apoptose/genética , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucócitos/citologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína 1 com Domínio SAM e Domínio HD/genética , Taxa de Sobrevida , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
12.
Immunity ; 52(1): 96-108.e9, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31810881

RESUMO

Although type 1 innate lymphoid cells (ILC1s) have been originally found as liver-resident ILCs, their pathophysiological role in the liver remains poorly investigated. Here, we demonstrated that carbon tetrachloride (CCl4) injection into mice activated ILC1s, but not natural killer (NK) cells, in the liver. Activated ILC1s produced interferon-γ (IFN-γ) and protected mice from CCl4-induced acute liver injury. IFN-γ released from activated ILC1s promoted the survival of hepatocytes through upregulation of Bcl-xL. An activating NK receptor, DNAM-1, was required for the optimal activation and IFN-γ production of liver ILC1s. Extracellular adenosine triphosphate accelerated interleukin-12-driven IFN-γ production by liver ILC1s. These findings suggest that ILC1s are critical for tissue protection during acute liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Hepatócitos/metabolismo , Interferon gama/imunologia , Fígado/citologia , Linfócitos/imunologia , Proteína bcl-X/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Feminino , Subunidade p35 da Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/lesões , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
13.
Biochim Biophys Acta Gene Regul Mech ; 1863(1): 194475, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870784

RESUMO

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Proteína bcl-X/genética , Apoptose , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Células U937 , Proteína bcl-X/metabolismo
14.
Biomed Pharmacother ; 121: 109641, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31751869

RESUMO

Glioma is an aggressive and lethal type of brain tumor that originates from glial cells. Glioblastoma cells confer considerable resistance to induction of apoptosis, which may be due to overexpression of anti-apoptotic proteins, or the reduction of the level of some pro-apoptotic proteins. MicroRNAs (miRNAs) can affect the cell biology pathways, including replication, autophagy, necrosis, and apoptosis by regulating gene expression. In this study, using bioinformatics methods, we selected the anti-apoptotic genes, BCL2L1 and MCL1, and microRNA that targeted them (miR-342). In the next step, the Lentiviral particles that contain miR-342 (LV-miR-342) were synthesized in HEK293T cell lines. Glioblastoma cell lines, U251 and U87, were transduced with LV-miR-342. The gene expression and apoptosis induction were then assayed by real-time PCR and flow cytometry respectively. The present study showed that increasing the expression of miR-342 reduced the expression of the anti-apoptotic genes, BCL2L1 and MCL1. The results of luciferase assay reports confirmed that miR-342 targeted BCL2L1 and MCL1. In addition, flow cytometry analysis indicated that miR-342 overexpression induced apoptosis in glioblastoma cells. As well as, Western blotting results confirmed a decrease in BCL2L1 protein following overexpression of miR-342 in glioblastoma cells. These findings may provide a novel therapeutic target for the treatment of glioblastoma.


Assuntos
Apoptose/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , MicroRNAs/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína bcl-X/genética , Regiões 3' não Traduzidas/genética , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Morte Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Células HEK293 , Humanos
15.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801186

RESUMO

Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/genética , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Prednisona/uso terapêutico , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirimidinas/farmacologia , Rituximab/uso terapêutico , Transdução de Sinais , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Resultado do Tratamento , Vincristina/uso terapêutico , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
16.
Nat Med ; 25(12): 1938-1947, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31792461

RESUMO

B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.


Assuntos
Compostos de Anilina/farmacologia , Sulfonamidas/farmacologia , Trombocitopenia/tratamento farmacológico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína bcl-X/genética , Compostos de Anilina/química , Animais , Antineoplásicos/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Proteólise , Sulfonamidas/química , Trombocitopenia/genética , Trombocitopenia/patologia , Proteína bcl-X/antagonistas & inibidores
17.
Cell Mol Biol (Noisy-le-grand) ; 65(7): 127-131, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31880530

RESUMO

To investigate the relationship between the Erk1/2 signal pathway and neuronal apoptosis in ischemic stroke rats. Male SD(Sprague Dawley)  rats (n = 24) were randomly divided into three groups, each containing 8 rats: sham-operated group, MCAO(Midle cerebral artery oclusion)  group, and MCAO + U0126 intervention group (U0126 group). In in vitro trial, primary cortical nerve cells were divided into three groups: control group, OGD(Oxygen and glucose deprivation)  group, and U0126 intervention group (U0126 group). In vivo protein expression levels of Erk1/2, p-Erk1/2 and Bcl-2 were determined using western blot. The expressions of Bcl-2, Bcl-xl and Bax were assayed using immunohistochemical staining. Nerve cell mortality in cerebral tissue was detected using TUNEL staining. In in vitro trials, cell apoptosis was assayed with flow cytometry and LDH release. The activity of caspase-3 was determined. Nerve cell apoptosis was determined using Hoechst33258 staining method. In in vivo trial, it was found that the protein expression level of p-ERK1/2 in cerebral tissue in the MCAO group was significantly increased, when compared with that of the sham-operated group, while the protein expression level of p-Erk1/2 in the U0126 group was significantly lower than that in the MCAO group. The expression levels of Bcl-2 and Bcl-xl in the MCAO group were significantly lower than the corresponding expression levels in the sham-operated group, while the expressions of Bcl-2 and Bcl-xl in the U0126 group were significantly lower than those in MCAO group. In MCAO group, the expression of Bax was significantly higher than that in the sham-operated group, while Bax expression was higher in U0126 than in MCAO group. There were significantly higher number of dead nerve cells in MCAO group than in the sham-operated group, while nerve cell mortality in U0126 group was significantly lower than in MCAO group. In in vitro trials, flow cytometry revealed significantly higher apoptosis of OGD-treated nerve cells, relative to the control group. Nerve cells exposed to U0126 and treated with ODR (Oxygen-dependent repressor)  were significantly decreased in population, when compared with single OGD treatment group. The LDH release level of nerve cells treated OGD was significantly increased, when compared with that of the control group. However, LDH release level of nerve cells treated with OGD after U0126 intervention was significantly decreased, relative to the single OGD treatment group. The dilution of nerve cell nucleus after OGD treatment was significantly increased, when compared with that of the control group. For nerve cells treated with ODR after U0126 intervention, the nuclear dilution was significantly decreased, relative to that of nerve cell nucleus in the single OGD treatment group. The OGD treatment led to significant increase in nerve cell caspase-3 activity, relative the control group. However, the caspase-3 activity of nerve cells treated with ODR after U0126 intervention was significantly decreased, when compared with single OGD treatment group. The activation of Erk1/2 signal pathway during ischemic stroke promotes apoptosis of nerve cells. Based on these findings, it can be reasonably inferred that the ERK1/2 signal pathway may be an important target for treating ischemic stroke.


Assuntos
Isquemia Encefálica/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Isquemia Encefálica/patologia , Butadienos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Nitrilos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
18.
Nat Commun ; 10(1): 5167, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727888

RESUMO

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.


Assuntos
Apoptose , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Camundongos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteína bcl-X/metabolismo
19.
Fitoterapia ; 139: 104421, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31730794

RESUMO

Three new prenyloxy chromanone derivatives, aucherine A-C (6, 7 and 9) as well as six known prenylated phloroglucinols (1-5 and 8) were isolated from the aerial parts of Hypericum aucheri Jaub. Et Spach. The structures of the isolated compounds were established by means of spectral techniques (HRESIMS, 1D and 2D NMR). The new compounds were tested on а panel of human tumor cell line using MTT assay. All tested compounds exerted moderate cytotoxicity with IC50 values ranging from 19.6 to 57.8 µM. The influence of the new compounds on some key signaling molecules (procaspase-9 and Bcl-xL), implicated in the regulation of programmed cell death was assessed by Western blot analysis.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cromonas/farmacologia , Hypericum/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose , Bulgária , Caspase 9/metabolismo , Linhagem Celular Tumoral , Cromonas/isolamento & purificação , Humanos , Estrutura Molecular , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Componentes Aéreos da Planta/química , Prenilação , Proteína bcl-X/metabolismo
20.
Int J Mol Sci ; 20(21)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671779

RESUMO

Androgen receptor (AR) stimulators, such as androgen and Tip60, play a pivotal role in prostatic carcinogenesis as androgen receptor signaling is critical for the growth and transformation of the prostate gland. Moreover, androgen and Tip60 promotes HIF-1α activation, involved in metabolic reprogramming by increasing glycolysis, a hallmark in cancer initiation and development. In this study we evaluated the effect of androgen and Tip60 stimulus in AR pathway activation and HIF-1α stabilization, in terms of proliferation and cell metabolism in androgen-sensitive LNCaP cells. The protective role of the bioactive compounds sulforaphane and capsaicin against the effect of these stimuli leading to pro-carcinogenic features was also addressed. Sulforaphane and capsaicin decreased nuclear AR, prostate specific antigen and Bcl-XL levels, and cell proliferation induced by androgen and Tip60 in LNCaP cells. These bioactive compounds prevented the increase in glycolysis, hexokinase and pyruvate kinase activity, and reduced HIF-1α stabilization induced by androgen and Tip60 in LNCaP cells. The protective role of sulforaphane and capsaicin on prostate cancer may rely on mechanisms involving the inhibition of Tip60, AR and HIF-1α effects.


Assuntos
Androgênios/metabolismo , Capsaicina/farmacologia , Isotiocianatos/farmacologia , Lisina Acetiltransferase 5/metabolismo , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Capsaicina/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isotiocianatos/química , Lisina Acetiltransferase 5/genética , Masculino , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína bcl-X/metabolismo
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