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1.
EMBO J ; 38(21): e101346, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31566767

RESUMO

The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. Here, we show that regulation of the number of enterocyte-committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase-mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, we found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. We also found that antagonistic interplay between Notch and EGFR signalling governs enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours.


Assuntos
Apoptose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Enterócitos/patologia , Receptores ErbB/metabolismo , Intestinos/patologia , Receptores de Peptídeos de Invertebrados/metabolismo , Receptores Notch/metabolismo , Células-Tronco/patologia , Animais , Caspases , Diferenciação Celular , Linhagem da Célula , Proteínas de Drosophila/genética , Enterócitos/metabolismo , Receptores ErbB/genética , Feminino , Homeostase , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Receptores de Peptídeos de Invertebrados/genética , Receptores Notch/genética , Transdução de Sinais , Células-Tronco/metabolismo
2.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614718

RESUMO

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Pterocarpanos/farmacologia , Antineoplásicos/toxicidade , Movimento Celular , Proliferação de Células , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pterocarpanos/toxicidade , Esferoides Celulares/efeitos dos fármacos , Survivina/genética , Survivina/metabolismo , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
3.
Biol Pharm Bull ; 42(10): 1720-1725, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31378747

RESUMO

Lung cancer is the most common cause of cancer death, approximately 85% of which are non-small cell lung cancer (NSCLC). Here we found that artemether (ART), a natural derivative of artemisinin, significantly inhibits the proliferation of NSCLC cells in a dose- and time-dependent manner. We also demonstrated that high concentration of ART induces apoptosis in NSCLC cells through down-regulating the level of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2). While low concentration of ART inhibits the mRNA level of cell cycle related genes including cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 6 (CDK6), cyclin A2, cyclin B1 and cyclin D1, leading to cell cycle arrest in NSCLC cells. Moreover, we confirmed that low concentration of ART induces DNA double-stranded breaks (DSBs), as well as promoting cellular senescence in NSCLC cells by up-regulating the mRNA and protein level of p16. Taken together, ART represents a promising new anti-NSCLC drug candidate that could attenuate progression of NSCLC cells in a p53-independent manner through inducing apoptosis, cell cycle arrest and promoting cellular senescence.


Assuntos
Apoptose/efeitos dos fármacos , Artemeter/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Células A549 , Artemeter/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Dano ao DNA , Progressão da Doença , Regulação para Baixo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
4.
J Ovarian Res ; 12(1): 76, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412918

RESUMO

BACKGROUND: Granulosa cell tumors (GCTs) are derived from proliferating granulosa cells of the ovarian follicle. They are known for their late recurrence and most patients with an aggressive form die from their disease. There are no treatment options for this slowly proliferating tumor besides surgery and chemotherapy. In a number of tumors, analogs of the second mitochondria-derived activator of caspases (SMAC), alone or in combination with other molecules, such as TNFα, are evolving as new treatment options. SMAC mimetics block inhibitor of apoptosis proteins (IAPs), which bind caspases (e.g. XIAP), or activate the pro-survival NF-κB pathway (e.g. cIAP1/2). Expression of IAPs by GCTs is yet not fully elucidated but recently XIAP and its inhibition by SMAC mimetics in a combination therapy was described to induce apoptosis in a GCT cell line, KGN. We evaluated the expression of cIAP1 in GCTs and elucidated the effects of the SMAC mimetic BV-6 using KGN as a model. RESULTS: Employing immunohistochemistry, we observed cIAP1 expression in a tissue microarray (TMA) of 42 GCT samples. RT-PCR confirmed expression of cIAP1/2, as well as XIAP, in primary, patient-derived GCTs and in KGN. We therefore tested the ability of the bivalent SMAC mimetic BV-6, which is known to inhibit cIAP1/2 and XIAP, to induce cell death in KGN. A dose response study indicated an EC50 ≈ 8 µM for both, early (< 8) and advanced (> 80) passages, which differ in growth rate and presumably aggressiveness. Quantitative RT-PCR showed upregulation of NF-κB regulated genes in BV-6 stimulated cells. Blocking experiments with the pan-caspase inhibitor Z-VAD-FMK indicated caspase-dependence. A concentration of 20 µM Z-VAD-FMK was sufficient to significantly reduce apoptosis. This cell death was further substantiated by results of Western Blot studies. Cleaved caspase 3 and cleaved PARP became evident in the BV-6 treated group. CONCLUSIONS: Taken together, the results show that BV-6 is able to induce apoptosis in KGN cells. This approach may therefore offer a promising therapeutic avenue to treat GCTs.


Assuntos
Tumor de Células da Granulosa/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Tumor de Células da Granulosa/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/genética
5.
Life Sci ; 234: 116788, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445935

RESUMO

Livin is an important member of the human inhibitor of apoptosis proteins (IAPs) family. IAPs are proteins with antiapoptotic abilities, and their functions are different from the Bcl-2 (B-cell lymphoma-2) family proteins. However, the precise role of Livin in colon cancer progression remains unclear. The purpose of this study is to assess the effect of overexpression Livin in colon cancer cells and to examine its molecular mechanism. We demonstrated that Livin induced a colon cancer phenotype, including proliferation and migration, by regulating H2A.XY39ph (histone family 2A variant (H2AX) phosphorylated on the 39th serine site). We elucidated that Livin degraded Jumonji-C domain-containing 6 protein (JMJD6), which was mediated by the proteasome murine double minute 2 (MDM2), thereby regulating H2A.XY39ph. Above all, the overexpression of JMJD6 recovered H2A.XY39ph in colon cancer cells with a high level of Livin, thus inhibiting colon cancer malignancy progression. These results reveal a previously unrecognized role for Livin in regulating the tumor-initiating capacity in colon cancer and provide a novel treatment strategy in cancer via the interruption of H2A.XY39ph function and the interaction between H2A.XY39ph and JMJD6.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo/patologia , Histonas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interação de Proteínas , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Neoplasias/genética , Proteólise
6.
Bioengineered ; 10(1): 365-373, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446833

RESUMO

The inhibitor of apoptosis proteins (IAPs) played important roles in inhibiting the apoptosis of tumor cells by regulating caspase activity in mammals. In this study, we first cloned the full-length cDNA sequence of IAPs gene (designated as Hs-IAPs) in Hyriopsis schlegelii. The Hs-IAPs gene contained an open reading frame of 1719 nucleotides, encoding a predicted protein of 572 amino acids. qRT-PCR assay indicated that the Hs-IAPs gene was ubiquitously expressed in different tissues, and the highest expression level was in gills. Furthermore, we purified and obtained the recombinant protein of Hs-IAPs which showed a molecular weight of 82.5 kDa. We used H2O2 stimulation experiment to explore the possible function of Hs-IAPs. The results showed that the percentage of viable cells significantly increased following the Hs-IAPs concentration. These indicated that the Hs-IAPs may play a role in anti-oxidation causing by H2O2, and its anti-oxidative may be crucial in the process of apoptosis.


Assuntos
Bivalves/genética , Brânquias/metabolismo , Hepatopâncreas/metabolismo , Proteínas Inibidoras de Apoptose/genética , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Bivalves/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Água Doce , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Brânquias/química , Gônadas/química , Gônadas/metabolismo , Células HeLa , Hepatopâncreas/química , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Peso Molecular , Músculos/química , Músculos/metabolismo , Fases de Leitura Aberta , Especificidade de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
7.
Life Sci ; 232: 116590, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228514

RESUMO

Endothelial cell (EC) apoptosis is fundamental for the pathophysiology of atherosclerosis, in which microRNAs (miRNAs) emerge as critical regulators. miR-122 has been shown to regulate the apoptosis of various cell types, however, whether miR-122 is associated with atherosclerosis and EC apoptosis remains unknown. In this study, we found that miR-122 expression was increased in the aortic ECs of ApoE-/- mice fed with a high-fat diet (HFD), as compared to normal-diet (ND), implying a potential association between miR-122 elevation and atherogenesis. In addition, in vitro, miR-122 expression was also induced in human aortic ECs (HAECs) by the treatment of oxidized low-density lipoprotein (ox-LDL), a common atherogenic factor. Functionally, miR-122 knockdown suppressed ox-LDL-induced apoptosis of HAECs, suggesting a pro-apoptotic role of miR-122 in HAECs under this pro-atherogenic condition. Further evidence revealed that the X-linked inhibitor-of-apoptosis protein (XIAP) was directly targeted and suppressed by miR-122 in HAECs, and more importantly, XIAP knockdown diminished miR-122 effect on apoptosis, thus establishing XIAP as a prominent target that mediates miR-122 regulation of the apoptosis of HAECs. Together, these results may identify miR-122 as a novel regulator in EC apoptosis, which offers it as a possible target for therapeutic interventions of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , MicroRNAs/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Aorta/metabolismo , Apolipoproteínas E/metabolismo , Apoptose/fisiologia , Aterosclerose/genética , Aterosclerose/patologia , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
8.
Oxid Med Cell Longev ; 2019: 6581217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31205587

RESUMO

MicroRNAs (miRNAs) have emerged as key modulators in the pathophysiologic processes of cardiovascular diseases. However, its function in cardiac injury induced by obstructive sleep apnea (OSA) remains unknown. The aim of the current study was to identify the effect and potential molecular mechanism of miR-146a-5p in intermittent hypoxia(IH)- induced myocardial damage. We exposed H9c2 cells to IH condition; the expression levels of miR-146a-5p were detected by RT-qPCR. Cell viability, cell apoptosis, and the expressions of apoptosis-associated proteins were assessed via Cell Counting Kit-8 (CCK-8), flow cytometry, and western blotting, respectively. Target genes of miR-146a-5p were confirmed by dual-luciferase reporter assay. IH remarkably lowered viability but enhanced cell apoptosis. Concomitantly, the miR-146a-5p expression level was increased in H9c2 cells after IH. Subsequent experiments showed that IH-induced injury was alleviated through miR-146a-5p silence. X-linked inhibitor of apoptosis protein (XIAP) was predicted by bioinformatics analysis and further confirmed as a direct target gene of miR-146a-5p. Surprisingly, the effect of miR-146a-5p inhibition under IH may be reversed by downregulating XIAP expression. In conclusion, our results demonstrated that miR-146a-5p could attenuate viability and promote the apoptosis of H9c2 by targeting XIAP, thus aggravating the H9c2 cell injury induced by IH, which could enhance our understanding of the mechanisms for OSA-associated cardiac injury.


Assuntos
Apoptose , Hipóxia Celular , Regulação da Expressão Gênica , Proteínas Inibidoras de Apoptose/metabolismo , MicroRNAs/genética , Miócitos Cardíacos/patologia , Animais , Sobrevivência Celular , Células Cultivadas , Proteínas Inibidoras de Apoptose/genética , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais
9.
Drug Discov Today Technol ; 31: 35-42, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31200857

RESUMO

The induction of protein degradation by chimeric small molecules represented by proteolysis-targeting chimeras (PROTACs) is an emerging approach for novel drug development. We have developed a series of chimeric molecules termed specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to effect targeted degradation. Unlike the chimeric molecules that recruit von Hippel-Lindau and cereblon ubiquitin ligases, SNIPERs induce simultaneous degradation of IAPs such as cIAP1 and XIAP along with the target proteins. Because cancer cells often overexpress IAPs-a mechanism involved in the resistance to cancer therapy-SNIPERs could be used to kill cancer cells efficiently.


Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Proteólise , Animais , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Neoplasias/metabolismo
10.
Chem Biol Interact ; 308: 332-338, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170386

RESUMO

BACKGROUND: Sevoflurane (sevo) has been reported to be an effective neuroprotective agent in cerebral ischemia/reperfusion injury (CIRI). However, the precise molecular mechanism underlying sevo preconditioning in CIRI remains largely unknown. METHODS: A middle cerebral artery occlusion (MCAO) rat model and primary cortical neurons after oxygen-glucose deprivation and reoxygenation (OGDR) were used as the in vivo and in vitro models of CIRI. The expression profiles of miR-181a and X chromosome-linked inhibitor-of-apoptosis protein (XIAP) in the cerebral cortex of rats and in cortical neurons were examined by qRT-PCR and Western blot, respectively. The infarct volumes were measured by TTC staining and neurological deficits in rats was determined by Zea-Longa scoring criteria. The cell viability, lactate dehydrogenase (LDH) release and apoptotic rate were detected in cortical neurons by MTT assay, LDH analysis and flow cytometry. Western blot analysis was performed to assess the expression of apoptosis-related protein. Luciferase reporter assay was used to confirm the interaction between miR-181a and XIAP. RESULTS: miR-181a was upregulated and XIAP was downregulated in rats after MCAO. Sevo preconditioning attenuated miR-181a expression and promoted XIAP level in a rat model of CIRI. Sevo preconditioning ameliorated anti-miR-181a-mediated protective effects on cerebral ischemia in rat model of CIRI, presented as the decrease of infarct volume, neurological deficit and apoptosis. Moreover, sevo pretreatment abated miR-181a-induced cellular injury in primary cortical neurons after OGD, embodied by the increase of cell viability, the reduction of LDH release and the decline of apoptosis. Furthermore, miR-181a suppressed XIAP expression by binding to its 3'UTR in cortical neurons, and sevo-mediated increase on XIAP expression was counteracted by miR-181 overexpression in OGDR-treated neurons. CONCLUSION: Sevo preconditioning protected against CIRI in vitro and in vivo possibly by inhibiting miR-181a and facilitating XIAP.


Assuntos
MicroRNAs/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Sevoflurano/uso terapêutico , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Apoptose/efeitos dos fármacos , Sequência de Bases , Regulação para Baixo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/complicações , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia , Alinhamento de Sequência , Sevoflurano/farmacologia
11.
Drug Des Devel Ther ; 13: 1373-1388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118573

RESUMO

Purpose: Mimetics based on Smac, the native inhibitor of XIAP, are promising drug-candidates for the treatment of cancer. Bivalent Smac mimetics inhibit XIAP with even higher potency than monovalent mimetics, but how to optimize the linker that tethers the two monovalent binding motifs remains controversial. Methods: To construct an ensemble of bivalent complex structures for evaluating various linkers, we propose herein a workflow, named TwistDock, consisting of steps of monovalent docking and linker twisting, in which the degrees of freedom are sampled focusing on the rotation of single bonds of the linker. Results: The obtained conformations of bivalent complex distribute randomly in the conformational space with respect to two reaction coordinates introduced by the linker, which are the distance of the two binding motifs and the dihedral angle of the two planes through the linker and each of the binding motifs. Molecular dynamics starting from 10 conformations with the lowest enthalpy of every complex shows that the conformational tendency of the complex participated by compound 9, one of the compounds with the largest binding affinity, is distinct from others. By umbrella sampling of the complex, we find its global minimum of the free energy landscape. The structure shows that the linker favors a compact conformation, and the two BIR domains of XIAP encompass the ligand on the opposite sides. Conclusion: TwistDock can be used in fine-tuning of bivalent ligands targeting XIAP and similar receptors dimerized or oligomerized.


Assuntos
Materiais Biomiméticos/farmacologia , Oligopeptídeos/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteína 3 com Repetições IAP de Baculovírus/antagonistas & inibidores , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Materiais Biomiméticos/química , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Ligantes , Modelos Moleculares , Conformação Molecular , Oligopeptídeos/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
12.
Mol Med Rep ; 19(6): 5079-5086, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059038

RESUMO

The antitumor effects of SM­164 and adriamycin (ADM) on human osteosarcoma U2­OS cells, the underlying mechanism are yet to be investigated. In the present study, U2­OS cells were divided into control, ADM, SM­164, and ADM + SM­164 groups. In addition, cells treated with both SM­164 and ADM were further divided into three subgroups: SM­164 + ADM, SM­164 + ADM + vector and SM­164 + ADM + X­linked inhibitor of apoptosis protein (XIAP) silencing groups. XIAP expression was achieved via transfection with shRNA lentiviral vectors. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the expression of caspases­7, ­9, and ­3, poly ADP­ribose polymerase (PARP), XIAP, cellular inhibitor of apoptosis protein­1 (cIAP­1) and survivin. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. Compared with the control group, cell viability decreased, while apoptosis was increased in the ADM and SM­164­treatment group. ADM and SM­164 treatment promoted the expression of caspases­7, ­9 and ­3, and PARP, but reduced the expression of XIAP, survivin and cIAP­1. Compared with ADM + SM­164 group, XIAP silencing with ADM + SM­164 treatment further reduced cell viability, promoted apoptosis, increased caspase­7, ­9 and ­3, and PARP expression; however the expression of survivin and cIAP­1 were reduced. Combined ADM and SM­164 treatment may be considered as potential therapeutic agent in the treatment of osteosarcoma, possibly via reductions XIAP expression.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Triazóis/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Survivina/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
13.
PLoS One ; 14(5): e0216553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31091257

RESUMO

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Metabolismo Energético , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteossarcoma/patologia , Neoplasias Ovarianas/patologia , Proteínas Quinases Ativadas por AMP/genética , Autofagossomos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosforilação , Transdução de Sinais , Células Tumorais Cultivadas
14.
J Mol Neurosci ; 68(2): 261-274, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949956

RESUMO

The level of miR-181a decreases rapidly in N2a cells following oxygen-glucose deprivation/reperfusion, but its role in this process is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-181a. We hypothesized that miR-181a reduces neuronal apoptosis and protects neurons by targeting reelin. Second mitochondria-derived activator of caspases (Smac) is a protein located in mitochondria that regulates apoptosis. The pro-apoptotic effect of Smac is achieved by reversing the effects of apoptosis-inhibiting proteins (IAPs), particularly X-linked inhibitor of apoptosis (XIAP). We also evaluated the effect of miR-181a on the Smac/IAP signaling pathway after oxygen-glucose deprivation and reperfusion in N2a cells. The miR-181a level, apoptosis rate, and the levels of reelin mRNA and protein, Smac, and XIAP were assessed in N2a cells subjected to oxygen-glucose deprivation for 4 h and reperfusion for 0, 4, 12, or 24 h with/without an miR-181a mimic, or mismatched control. Direct targeting of reelin by miR-181a was assessed in vitro by dual luciferase assay and immunoblotting. Pre-treatment with miR-181a mimicked the increase in the miR-181a level in N2a cells after oxygen-glucose deprivation/reperfusion, resulting in a significant decrease in the apoptosis rate. Changes in the miR-181a level in N2a cells were inversely correlated with reelin protein expression. Direct targeting of the reelin 3' untranslated region by miR-181a was verified by dual luciferase assay, which showed that miR-181a significantly inhibited luciferase activity. The Smac level was significantly lower in the miR-181a mimics than the normal control and mimics-cont groups (P < 0.01), whereas the level of XIAP was increased slightly. These findings suggest that miR-181a protects neurons from apoptosis by inhibiting reelin expression and regulating the Smac/IAP signaling pathway after oxygen-glucose deprivation/reperfusion injury.


Assuntos
Apoptose , MicroRNAs/genética , Neurônios/metabolismo , Oxigênio/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Glucose/deficiência , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
15.
Ann Clin Lab Sci ; 49(1): 16-22, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30814073

RESUMO

OBJECTIVE: This study aims to investigate the role of X-linked inhibitor of apoptosis protein (XIAP) in the pathogenesis of premature ovarian failure (POF) and the effects of the Modified Bazhen Decoction (MBD) in the treatment of POF. MATERIALS AND METHODS: Twenty-four eight-week-old Sprague Dawley (SD) rats were randomly divided into four groups: control group, POF group, MBD treatment group, and Fufang Ejiao Syrup (FES) treatment group. After adaptive feeding for one week, 18 SD rats in the POF, MBD and FES groups were subcutaneously injected with D-galactose (dissolved in saline) at the back of neck for eight weeks (150 mg/kg/day) to establish the POF model. Six SD rats in the control group received equal volumes of subcutaneous injection of saline. Tail blood was collected, and the concentration of follicle stimulating hormones (FSHs) and estradiol (E2) was measured, in order to evaluate the success of the POF model. SD rats in the MBD and FES treatment groups were intragastrically administered with MBD (10 ml/kg/day) and FES (10 ml/kg/day), respectively. Rats in the control and POF groups were intragastrically administered with saline (10 ml/kg/day). After four weeks of intragastrical administration with different medicines and saline, ovarian tissues were collected; and the expression level of XIAP, miR-23a and miR-27a were measured and compared among different groups. RESULTS: Compared with the control group, XIAP expression was significantly lower, and miR-23a and miR-27a expression significantly higher in the POF group. Furthermore, XIAP expression was significantly higher, and miR-23a and miR-27a expression was significantly lower in the MBD group. CONCLUSION: XIAP is involved in the regulation of oocyte and granulosa cells via the cysteinyl aspartate specific proteinase (caspase) pathway, and plays an important role in POF. MBD can dramatically activate XIAP, but inhibit the expression of miR-23a and miR-27a; preventing the apoptosis of oocyte and granulosa cells. Our study suggests that MBD may be a useful traditional Chinese medicine for the treatment of POF.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Células da Granulosa/efeitos dos fármacos , Fitoterapia , Insuficiência Ovariana Primária/tratamento farmacológico , Animais , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Proteínas Inibidoras de Apoptose/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/patologia , Ratos , Ratos Sprague-Dawley
16.
Anticancer Res ; 39(3): 1197-1204, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30842150

RESUMO

BACKGROUND/AIM: Triple-negative breast cancers (TNBC) lack expression of three important receptors, and have limited treatment options. High expression of junctional adhesion molecule-A (JAM-A) has been linked with aggressive tumor phenotypes including TNBC. This study aimed to evaluate the bioactivity of a JAM-A-down-regulating compound, Tetrocarcin-A, in TNBC. MATERIALS AND METHODS: TNBC cell viability, colony formation and xenograft growth were examined in Tetrocarcin-A-treated HCC38 human cells, 4T1 mouse cells or patient-derived primary cells. Protein expression of cell fate signaling effectors was examined by immunoblotting (versus transient JAM-A gene silencing). Apoptotic pathways were investigated in parallel. RESULTS: Tetrocarcin-A reduced TNBC cell viability in vitro and in an in ovo/semi-in vivo xenograft model. Tetrocarcin-A-induced JAM-A down-regulation and reduced ERK phosphorylation, followed by c-FOS phosphorylation on its transcription-regulating residue, which down-regulated several inhibitor of apoptosis (IAP) proteins and induced caspase-dependent intrinsic pathway of apoptosis. CONCLUSION: Tetrocarcin-A merits further investigation as a novel anti-tumor agent in TNBC.


Assuntos
Aminoglicosídeos/farmacologia , Antineoplásicos/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide , Regulação para Baixo , Inativação Gênica , Humanos , Molécula A de Adesão Juncional/genética , Camundongos , RNA Interferente Pequeno/genética , Neoplasias de Mama Triplo Negativas/genética
17.
Chem Pharm Bull (Tokyo) ; 67(3): 165-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30827996

RESUMO

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.


Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Oncogenes , Antineoplásicos/uso terapêutico , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
18.
Phytother Res ; 33(5): 1384-1393, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30887612

RESUMO

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF-induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl-2 family members (upregulation of Bax and downregulation of Bcl-2), and activated caspase-3, -8, and -9. Morin also enhances AF-induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase-dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl-2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.


Assuntos
Auranofina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Flavonoides/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
19.
ACS Chem Biol ; 14(3): 342-347, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30807093

RESUMO

The impact of covalent binding on PROTAC-mediated degradation of BTK was investigated through the preparation of both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. It was determined that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC. These observations were consistently found when PROTACs that were able to recruit either IAP or cereblon E3 ligases were employed. Proteomics analysis determined that the use of a covalently bound PROTAC did not result in the degradation of covalently bound targets, while degradation was observed for some reversibly bound targets. This observation highlights the importance of catalysis for successful PROTAC-mediated degradation and highlights a potential caveat for the use of covalent target binders in PROTAC design.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteólise , Pirazóis/metabolismo , Pirimidinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
20.
Cell Oncol (Dordr) ; 42(3): 319-329, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30778852

RESUMO

BACKGROUND: The X-linked inhibitor of apoptosis (XIAP) is a potent cellular inhibitor of apoptosis, based on its unique capability to bind and to inhibit caspases. However, XIAP is also involved in a number of additional cellular activities independent of its caspase inhibitory function. The aim of this study was to investigate whether modulation of XIAP expression affects apoptosis-independent functions of XIAP in melanoma cells, restores their sensitivity to apoptosis and/or affects their invasive and metastatic capacities. METHODS: XIAP protein levels were analyzed by immunohistochemical staining of human tissues and by Western blotting of melanoma cell lysates. The effects of pharmacological inhibition or of XIAP down-regulation were investigated using ex-vivo and transwell invasion assays. The biological effects of XIAP down-regulation on melanoma cells were analyzed in vitro using BrdU/PI, nucleosome quantification, adhesion and migration assays. In addition, new XIAP binding partners were identified by co-immunoprecipitation followed by mass spectrometry. RESULTS: Here we found that the expression of XIAP is increased in metastatic melanomas and in invasive melanoma-derived cell lines. We also found that the bivalent IAP antagonist birinapant significantly reduced the invasive capability of melanoma cells. This reduction could be reproduced by downregulating XIAP in melanoma cells. Furthermore, we found that the migration of melanoma cells and the formation of focal adhesions at cellular borders on fibronectin-coated surfaces were significantly reduced upon XIAP knockdown. This reduction may depend on an altered vimentin-XIAP association, since we identified vimentin as a new binding partner of XIAP. As a corollary of these molecular alterations, we found that XIAP down-regulation in melanoma cells led to a significant decrease in invasion of dermal skin equivalents. CONCLUSION: From our data we conclude that XIAP acts as a multifunctional pro-metastatic protein in skin melanomas and, as a consequence, that XIAP may serve as a therapeutic target for these melanomas.


Assuntos
Melanoma/metabolismo , Vimentina/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica , Ligação Proteica , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
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