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1.
Curr Med Sci ; 39(3): 442-448, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209817

RESUMO

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitor-of-apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine™ 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified HSP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial cells.


Assuntos
Aldeídos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Apoptose/genética , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Peroxidação de Lipídeos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Pigmentos da Retina/metabolismo , Transdução de Sinais , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
2.
J Pharm Biomed Anal ; 173: 40-46, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31108422

RESUMO

Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.


Assuntos
Proteínas Reguladoras de Apoptose/isolamento & purificação , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Mitocondriais/isolamento & purificação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Proteínas Mitocondriais/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
3.
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
4.
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
5.
Int J Oncol ; 55(1): 191-202, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115498

RESUMO

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2­year survival rate of 30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X­linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF­related apoptosis­inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase­8 and co­occurred with caspase­8 and caspase­3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase­8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL­mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Benzoquinonas/farmacologia , Tumor Rabdoide/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Tumor Rabdoide/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
6.
Bioconjug Chem ; 30(5): 1395-1404, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30888797

RESUMO

The X-linked inhibitor of apoptosis protein baculovirus IAP repeat (XIAP BIR3) domain is a promising therapeutic target for cancer treatment. For the mirror-image screening campaign to identify drug candidates from an unexplored mirror-image natural product library, a facile synthetic protocol for XIAP BIR3 domain synthesis was established by a native chemical ligation strategy using conserved cysteines present among BIR domains. The native and mirror-image XIAP BIR3 domains with an appropriate functional group for labeling were prepared using the established protocol. Taking advantage of the resulting synthetic proteins, several bioassay systems were developed to characterize inhibitors of the protein-protein interaction between the XIAP BIR3 domain and the second mitochondria-derived activator of caspases.


Assuntos
Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Sequência de Aminoácidos , Bioensaio , Humanos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
7.
Turk Neurosurg ; 29(1): 26-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29384188

RESUMO

AIM: To investigate a new anti-tumor treatment method using stem cells transfected with specific genes and proteins that induce apoptosis in tumor cells. MATERIAL AND METHODS: We used glioblastoma (GBM) cells and human adipose tissue-derived mesenchymal stem cells (ADMSCs) in this study. The AD-MSCs were transfected with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To overcome apoptosis resistance in tumor cells, we used suberoylanilide hydroxamic acid (SAHA) as the histone deacetylase inhibitor and embelin as the X-linked inhibitor of apoptosis protein (XIAP). In addition, we silenced the XIAP gene on GBM cells with the shXIAP plasmid. Following the determination of half-maximal effective concentration (EC50%) doses of SAHA and embelin, GBM cells were incubated with them for 24 hours. XIAP-silenced and XIAP-non-silenced GBM cells were cultured with TRAIL-nontransfected and TRAIL-transfected stem cells for 24 hours. Viability and cell cycle analysis of all groups were determined using annexin V/propidium iodide and cell cycle method via flow cytometry. RESULTS: TRAIL-transfected AD-MSCs, XIAP silencing, embelin, and SAHA induced apoptosis in GBM cells and decreased their proliferation, whereas TRAIL-non-tranfected AD-MSCs did not. CONCLUSION: Engineered stem cell therapies and molecular studies show promise in developing combination therapies for effective treatment of GBM.


Assuntos
Antineoplásicos/farmacologia , Glioblastoma/patologia , Células-Tronco Mesenquimais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Adulto , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Inativação Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Vorinostat/farmacologia
8.
Mol Cancer Ther ; 18(2): 364-375, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30530769

RESUMO

Ovarian granulosa cell tumors (GCT) are characterized by indolent growth and late relapse. No therapeutic modalities aside from surgery have proven effective. We previously reported overexpression of the nuclear receptor, peroxisome proliferator-activated receptor-gamma (PPARγ), and constitutive activity of the NFκB and AP1 signaling pathways in GCT. PPARγ presents as a potential therapeutic target as it impedes proliferation and promotes terminal differentiation of granulosa cells. However, resistance to the actions of PPARγ is caused by NFκB transrepression in GCT-derived cell lines, KGN and COV434. We showed that abrogation of NFκB signaling in GCT cells enables PPARγ agonists to initiate apoptosis. In addition, we observed overexpression of an NFκB-induced gene, X-linked inhibitor of apoptosis protein (XIAP), in GCT and GCT-derived cells. XIAP is an attractive therapeutic target due to its role in inhibiting the apoptotic pathway. We investigated the antitumor effects of combined XIAP inhibition using Smac-mimetics and PPARγ activation using thiazolidinediones (TZD) in the GCT-derived cells. Transactivation assays revealed that NFκB transrepression of PPARγ can be relieved by NFκB or XIAP inhibition. Combined Smac-mimetic and TZD significantly induced apoptosis, reduced cell viability and proliferation in KGN cells in monolayer and 3D spheroid culture, and in GCT explant models. The Smac-mimetic and TZD cotreatment also delayed cell invasion, upregulated proapoptotic genes, and compromised cell metabolism in KGN cells. This study provides evidence that PPARγ and XIAP cotreatment has antineoplastic effects in GCT. As therapeutics that target these proteins are already in clinical or preclinical use, expedient translation to the clinic is possible.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Materiais Biomiméticos/farmacologia , Tumor de Células da Granulosa/metabolismo , Neoplasias Ovarianas/metabolismo , Tiazolidinedionas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tumor de Células da Granulosa/tratamento farmacológico , Humanos , Neoplasias Ovarianas/tratamento farmacológico , PPAR gama/antagonistas & inibidores , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
9.
Sci Rep ; 8(1): 17862, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30552344

RESUMO

The poor prognosis of ovarian cancer (it is the leading cause of death from gynecological cancers) is mainly due to the acquisition of resistance to carboplatin. Among the possible resistance pathways, resistance to apoptosis and especially the overexpression of inhibitor of apoptosis proteins (IAP) cIAP1 and X-linked IAP (XIAP), have been implicated. DEBIO 1143, a SMAC (second mitochondria-derived activator of caspase) mimetic, belongs to a new class of targeted agents currently being evaluated in clinical trials, which activate apoptotic cell death and block pro-survival signaling in cancer cells. Here, we demonstrate that DEBIO 1143 in vitro inhibits the cell viability of two carboplatin-sensitive cell lines (IGROV-1 and A2780S) as well as three carboplatin-resistant cell lines (A2780R, SKOV-3 and EFO-21). Of note, DEBIO 1143 is able to reverse resistance to carboplatin by inducing cell death either by apoptosis or necroptosis depending on the cell lines. To identify a biomarker able to predict the sensitivity of the cell lines to DEBIO 1143 treatment we analyzed the expression of the DEBIO 1143 targets cIAP1 and XIAP, and one of their downstream targets, caspase 9. These proteins did not constitute a marker of DEBIO 1143 sensitivity/resistance. Importantly, we confirmed these findings in vivo in SKOV-3 xenograft models where DEBIO 1143 highly potentiated carboplatin treatment.


Assuntos
Antineoplásicos/farmacologia , Azocinas/farmacologia , Compostos Benzidrílicos/farmacologia , Carboplatina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Azocinas/administração & dosagem , Compostos Benzidrílicos/administração & dosagem , Carboplatina/administração & dosagem , Caspase 9/análise , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Proteínas Inibidoras de Apoptose/análise , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Resultado do Tratamento , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise
10.
J Cancer Res Ther ; 14(Supplement): S648-S655, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30249882

RESUMO

Background: Embelin is an active compound identified as a novel X-linked inhibitor of apoptosis protein (XIAP) inhibitor from the Embelia ribes that exhibits various medicinal effects including anti-inflammatory and anticancer activities. However, the therapeutic effect of Embelin to human osteosarcoma is not yet determined. Objectives: In this study, we evaluated the sensitizing potential of Embelin on promoting apoptosis to cause osteosarcoma cell death and inhibiting its invasion. Methods: We uesd 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to detect the survival rates of osteosarcoma cells, Western blot to detect the expression of proteins in U-2 OS and MG63 cells, and fluorescence microscope to observe the morphology of apoptotic cells. Results: The survival of osteosarcoma cells decreased, When Embelin was used. Obvious condensed and flared fluorescence was observed, when used high-dose Embelin. There was an increase of caspase-3, cleaved caspase-3, caspase-8, and caspase-9 in Embelin group, while PI3K, AKt, p-AKt, X-linked inhibitor of apoptosis protein, and MMP-9 were downregulated. The invasion of Embelin application was significantly lower than that of the control application. Conclusion: Embelin promoted apoptosis via XIAP and PI3K/Akt signaling pathway. XIAP inhibitor Embelin inducing apoptosis could cause osteosarcoma cell death and inhibit its invasion.


Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Osteossarcoma/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Benzoquinonas/química , Proliferação de Células/efeitos dos fármacos , Embelia/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Osteossarcoma/genética , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
11.
Biomed Pharmacother ; 107: 1010-1019, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30257312

RESUMO

The treatment for leukemic malignancies remains a challenge despite the wide use of conventional chemotherapies. Therefore, new therapeutic approaches are highly demanded. TNF-related apoptosis-inducing ligand (TRAIL) represents a targeted therapy against cancer because it induces apoptosis only in tumor cells. TRAIL is currently under investigation for the treatment of leukemia. Preclinical studies evaluated the potential therapeutic efficacy of TRAIL on cell lines and clinical samples and showed promising results. However, like most anti-cancer drugs, resistance to TRAIL-induced apoptosis may limit its clinical efficacy. It is critical to understand the molecular mechanisms of TRAIL. Therefore, rational therapeutic drug combinations for clinical trials of TRAIL-based therapies might be achieved. In a variety of leukemic cells, overexpression of X-linked inhibitor of apoptosis protein (XIAP), a negative regulator of apoptosis pathway, has been discovered. Implication of XIAP in the ineffective induction of cell death by TRAIL in leukemia has been explored in several resistant cell lines. XIAP inhibitors restored TRAIL sensitivity in resistant cells and primary leukemic blasts. Moreover, TRAIL resistance in leukemic cells could be overcome by the effects of several anti-leukemic agents via the mechanisms of XIAP downregulation. Here, we discuss targeting XIAP, a strategy to restore TRAIL sensitivity in leukemia to acquire more insights into the mechanisms of TRAIL resistance. The concluding remarks may lead to identify putative ways to resensitize tumors.


Assuntos
Leucemia/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia/genética , Leucemia/patologia , Terapia de Alvo Molecular , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem
12.
J Med Chem ; 61(16): 7314-7329, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30091600

RESUMO

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).


Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Piperazinas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Canal de Potássio ERG1/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Piperazinas/química , Piperazinas/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Med Chem ; 61(14): 6350-6363, 2018 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-29940121

RESUMO

Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP. We report for the first time that it is possible to target such residue covalently to attain potent and selective agents. Preliminary cellular studies in various models of leukemia, multiple myeloma, and pancreatic cancers suggest that the derived agents possess a potentially intriguing pattern of activity, especially for cell lines that are resistant to the pan-IAP antagonist and clinical candidate LCL161.


Assuntos
Desenho de Drogas , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Linhagem Celular , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Termodinâmica , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
14.
SLAS Discov ; 23(9): 951-959, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29852073

RESUMO

Native electrospray ionization mass spectrometry (ESI-MS) was applied to analyze the binding of compounds generated during fragment-based drug discovery (FBDD) campaigns against two functionally distinct proteins, the X-linked inhibitor of apoptosis protein (XIAP) and cyclin-dependent kinase 2 (CDK2). Compounds of different molecular weights and a wide range of binding affinities obtained from the hits to leads and lead optimization stages of FBDD campaigns were studied, and their dissociation constants (Kd) were measured by native ESI-MS. We demonstrate that native ESI-MS has the potential to be applied to the stages of an FBDD campaign downstream of primary screening for the detection and quantification of protein-ligand binding. Native ESI-MS was used to derive Kd values for compounds binding to XIAP, and the dissociation of the complex between XIAP and a peptide derived from the second mitochondria-derived activator of caspases (SMAC) protein induced by one of the test compounds was also investigated. Affinities of compounds binding to CDK2 gave Kd values in the low nanomolar to low millimolar range, and Kd values generated by MS and isothermal titration calorimetry (ITC) followed the same trend for both proteins. Practical considerations for the application of native ESI-MS are discussed in detail.


Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Fenômenos Químicos , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/química , Termodinâmica
15.
Mol Cancer Ther ; 17(7): 1381-1391, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29695633

RESUMO

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I-II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381-91. ©2018 AACR.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Mimetismo Molecular , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cryobiology ; 82: 112-117, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29605544

RESUMO

Cryo-injury of mammalian blastocysts occurs during cryopreservation and induces apoptosis in trophoblast cells. This damage affects subsequent embryo development or may even cause death before implantation. X-linked inhibitor of apoptosis (XIAP) is an anti-apoptosis gene that has been widely studied in cancer research. However, only a few studies have investigated the activity of XIAP in cryopreservation. In this study, we investigate the role of XIAP in frozen and thawed murine blastocysts. A total of 1630 blastocysts were divided into fresh and freeze-thaw groups, and XIAP expression was investigated using qPCR, Western blot and confocal analyses. In addition, the effect of the embelin (a XIAP inhibitor) was also evaluated by co-culturing 390 dormant blastocysts. XIAP protein is primarily localized to the mitochondria of trophoblastic cells. Gene and protein expression is significantly down-regulated in blastocysts after cryopreservation, whereas embelin has negative effect on their survivals. These findings further broaden the understanding of mammalian embryonic cryopreservation.


Assuntos
Apoptose/fisiologia , Blastocisto/metabolismo , Criopreservação/métodos , Embrião de Mamíferos/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Benzoquinonas/farmacologia , Implantação do Embrião , Transferência Embrionária , Feminino , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
17.
IUBMB Life ; 70(5): 445-457, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29537730

RESUMO

Altered activity of the proteolytic machine-the 26S proteasome is observed in many disease conditions. Hence, either inhibition or activation of the 26S proteasome is thought to be a novel therapy for treatment of certain diseases such as cancer and neurodegenerative disorders. In this study, we tested the potential of cinnamon and one of its active ingredients, procyanidin-B2 (PCB2), in inhibiting the catalytic activities of the proteasome and suppressing prostate cancer cell growth. Proteasome activities were measured using fluorogenic substrates specific for the different enzymatic activities of the 26S proteasome by flourometry. Cell viability was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide assay, while apoptosis was examined by Hoechst and propidium iodide staining and caspase-3 activity. Both, the cinnamon extract and its PCB2-enriched F2 fraction inhibited the catalytic activities of the purified proteasome and the proteasome in cancer cells but not in normal cells. Furthermore, cinnamon and its active component decreased cell proliferation of human prostate cancer cells but not normal lung cells, decreased expression of anti-apoptotic and angiogenic markers in prostate cancer cell lysates. These results demonstrate that cinnamon extract and its PCB2-enriched fraction act as proteasome inhibitors and have prospects as anti-cancer agents. © 2018 IUBMB Life, 70(5):445-457, 2018.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Cinnamomum zeylanicum/química , Regulação Neoplásica da Expressão Gênica , Proantocianidinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Biflavonoides/isolamento & purificação , Catequina/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios Enzimáticos , Humanos , Concentração Inibidora 50 , Masculino , Especificidade de Órgãos , Extratos Vegetais/química , Proantocianidinas/isolamento & purificação , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/isolamento & purificação , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Survivina/antagonistas & inibidores , Survivina/genética , Survivina/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
18.
Theranostics ; 8(6): 1494-1510, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29556337

RESUMO

Rationale: Nasopharyngeal carcinoma (NPC) is the most frequent head and neck tumor in South China. The presence of cancer stem cells (CSCs) in NPC contributes to tumor maintenance and therapeutic resistance, while the ability of CSCs to escape from the apoptosis pathway may render them the resistant property to the therapies. Inhibitor of apoptosis proteins family proteins (IAPs), which are overexpressed in nasopharyngeal carcinoma stem cells, may play an important role in maintaining nasopharyngeal cancer stem cell properties. Here, we develop a novel CSC-targeting strategy to treat NPC through inhibiting IAPs. Methods: Human NPC S-18 and S-26 cell lines were used as the model system in vitro and in vivo. Fluorescence activated cell sorting (FACS) assay was used to detect nasopharyngeal SP cells and CD44+ cells. The characteristics of CSCs were defined by sphere suspension culture, colony formation assay and cell migration. The role of XIAP on the regulation of Sox2 protein stability and ERK1-mediated phosphorylation of Sox2 signaling pathway were analyzed using immunoblotting, immunoprecipitation, immunofluorescence, phosphorylation mass spectrometry, siRNA silencing and plasmid overexpression. The correlation between XIAP and Sox2 in NPC biopsies and their role in prognosis was performed by immunohistochemistry. APG-1387 or chemotherapies-induced cell death and apoptosis in S-18 and S-26 were determined by WST, immunoblotting and flow cytometry assay. Results: IAPs, especially X chromosome-linked IAP (XIAP), were elevated in CSCs of NPC, and these proteins were critically involved in the maintenance of CSCs properties by enhancing the stability of Sox2. Mechanistically, ERK1 kinase promoted autophagic degradation of Sox2 via phosphorylation of Sox2 at Ser251 and further SUMOylation of Sox2 at Lys245 in non-CSCs. However, XIAP blocked autophagic degradation of Sox2 by inhibiting ERK1 activation in CSCs. Additionally, XIAP was positively correlated with Sox2 expression in NPC tissues, which were associated with NPC progression. Finally, we discovered that a novel antagonist of IAPs, APG-1387, exerted antitumor effect on CSCs. Also, the combination of APG-1387 with CDDP /5-FU has a synergistic effect on NPC. Conclusion: Our study highlights the importance of IAPs in the maintenance of CSCs in NPC. Thus, XIAP is a promising therapeutic target in CSCs and suggests that NPC patients may benefit from a combination treatment of APG-1387 with conventional chemotherapy.


Assuntos
Azepinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Fatores de Transcrição SOXB1/genética , Sulfonamidas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Sinergismo Farmacológico , Fluoruracila/farmacologia , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/mortalidade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/mortalidade , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cell ; 69(4): 551-565.e7, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29452636

RESUMO

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.


Assuntos
Aminoquinolinas/farmacologia , Inflamação/prevenção & controle , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Sulfonas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Células Cultivadas , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Fosforilação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores
20.
Eur Rev Med Pharmacol Sci ; 22(2): 382-387, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29424923

RESUMO

OBJECTIVE: Cisplatin is an important anti-cancer drug. However, the molecular mechanism of cisplatin on inhibition of the proliferation of liver cancer cells is unclear. Thus, we aimed to investigate the regulatory role of cisplatin on the growth and apoptosis of hepatoma LM3 cells. MATERIALS AND METHODS: LM3 cells were treated with cisplatin (2 µmol/L) for 48 h. MTT assay, flow cytometry, and caspase-3 activity assay were used to detect the growth, proliferation and apoptosis of LM3 cells. Western blot was applied to detect the expression of the inhibitor of apoptosis protein, XIAP. siRNA was used to knockdown the level of XIAP followed by cisplatin (2 µmol/L) treatment, and then the apoptosis of LM3 cells was measured. RESULTS: The treatment of cisplatin significantly inhibited the growth but induced the apoptosis of LM3 cells. Cisplatin also downregulated the expression of XIAP. The downregulation of XIAP by using siRNA enhanced the apoptosis of LM3 cells induced by cisplatin. CONCLUSIONS: Downregulation of XIAP enhanced the proapoptotic effect of cisplatin on LM3 cells, suggesting that XIAP might be used as a potential target in the treatment of liver cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
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