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1.
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
2.
PLoS One ; 14(2): e0211746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30716099

RESUMO

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).


Assuntos
Fármacos Anti-HIV/farmacologia , Azocinas/farmacologia , Compostos Benzidrílicos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , HIV-1/fisiologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Latência Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Cell Chem Biol ; 26(3): 331-339.e3, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639259

RESUMO

Protein- and cell-based immunotherapeutic agents have revolutionized cancer treatment. However, small-molecule immunomodulators with favorable pharmacological properties for reaching intracellular targets remain to be developed. To explore the vast chemical space, a robust method that recapitulates the complex cancer-immune microenvironment in a high-throughput format is essential. To address this critical gap, we developed a high-throughput immunomodulator phenotypic screening platform, HTiP, which integrates the immune and cancer cell co-culture system with imaging- and biochemical-based multiplexed readouts. Using the HTiP platform, we have demonstrated its capability in modeling an oncogenic KRAS mutation-driven immunosuppressive phenotype. From a bioactive chemical library, multiple structurally distinct compounds were identified, all of which target the same class of proteins, inhibitor of apoptosis protein (IAP). IAP has demonstrated roles in cancer immunity. Identification of IAP antagonists as potent anti-tumor immune enhancers provides strong validating evidence for the use of the HTiP platform to discover small-molecule immunomodulators.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Fatores Imunológicos/química , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Humanos , Fatores Imunológicos/farmacologia , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Interferon gama/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismo
4.
Cancer Lett ; 440-441: 126-134, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312727

RESUMO

Multidrug resistance (MDR) in cancer patients undergoing chemotherapy is preventing effective treatment of multiple cancer types including pediatric tumors. Resistance to chemotherapeutic drugs in cancer cells is frequently associated with high expression of p-glycoprotein, a transporter in the plasma membrane that can mediate cellular drug export. Here, we generated pediatric cancer cells with acquired resistance to the chemotherapeutic drug vincristine (VCR). In these cells, acquired resistance is associated with increased expression of p-glycoprotein. VCR-resistant cells display an MDR phenotype and have acquired resistance to multiple other chemotherapeutic drugs including doxorubicin (DOXO) and etoposide (ETO). Notably, we discovered that these cells also display cross-resistance with several Smac mimetics, a novel class of experimental cancer therapeutics designed to induce apoptosis by inhibiting Inhibitor of Apoptosis (IAP) proteins. Resistance to Smac mimetics is reversible in the presence of p-glycoprotein inhibitors, highlighting Smac mimetics as novel substrates for p-glycoprotein. The identification of Smac mimetics as substrates for p-glycoproteins may influence the design of future clinical trials to prevent usage of Smac mimetics in the context of MDR or, alternatively, combine Smac mimetics with p-glycoprotein inhibitors to maximize their efficiency.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Materiais Biomiméticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Neuroblastoma/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oligopeptídeos/farmacologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Tiazóis/farmacologia , Regulação para Cima , Vincristina/farmacologia
5.
Chem Pharm Bull (Tokyo) ; 67(3): 203-209, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30369550

RESUMO

Targeted protein degradation by small molecules is an emerging modality with significant potential for drug discovery. We previously developed chimeric molecules, termed specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which induce the ubiquitylation and proteasomal degradation of target proteins. This degradation is mediated by the IAPs; the target proteins include bromodomain-containing protein 4 (BRD4), an epigenetic regulator protein. The SNIPER that degrades this particular protein, SNIPER(BRD)-1, consists of an IAP antagonist LCL-161 derivative and a bromodomain and extra-terminal (BET) inhibitor, (+)-JQ-1. SNIPER(BRD)-1 also degrades a cellular inhibitor of apoptosis protein 1 (cIAP1) and an X-linked inhibitor of apoptosis protein (XIAP), the mechanisms of which are not well understood. Here, we show that the degradation of cIAP1 and XIAP by SNIPER(BRD)-1 is induced via different mechanisms. Using a chemical biology-based approach, we developed two inactive SNIPERs, SNIPER(BRD)-3 and SNIPER(BRD)-4, incapable of degrading BRD4. SNIPER(BRD)-3 contained an N-methylated LCL-161 derivative as the IAP ligand, which prevented it from binding IAPs, and resulted in the abrogated degradation of cIAP1, XIAP, and BRD4. SNIPER(BRD)-4, however, incorporated the enantiomer (-)-JQ-1 which was incapable of binding BRD4; this SNIPER degraded cIAP1 but lost the ability to degrade XIAP and BRD4. Furthermore, a mixture of the ligands, (+)-JQ-1 and LCL-161, induced the degradation of cIAP1, but not XIAP and BRD4. These results indicate that cIAP1 degradation is triggered by the binding of the IAP antagonist module to induce autoubiquitylation of cIAP1, whereas a ternary complex formation is required for the SNIPER-induced degradation of XIAP and BRD4.


Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Proteólise , Azepinas/química , Proteínas de Ciclo Celular , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Ligantes , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteólise/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/química , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
6.
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
7.
Cell Death Dis ; 9(11): 1112, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385739

RESUMO

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
9.
Cell Death Dis ; 9(11): 1081, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30349042

RESUMO

Expression of tumor necrosis factor-α (TNFα) in the serum of prostate cancer patients is associated with poorer outcome and progression to castrate-resistant (CRPC) disease. TNFα promotes the activity of NFκB, which regulates a number of anti-apoptotic and proinflammatory genes, including those encoding the inhibitor of apoptosis proteins (IAPs); however, in the presence of IAP antagonists, TNFα can induce cell death. In the presence of recombinant or macrophage-derived TNFα, we found that IAP antagonists triggered degradation of cIAP1 and induced formation of Complex-IIb, consisting of caspase-8, FADD and RIPK1 in CRPC models; however, no, or modest levels of apoptosis were induced. This resistance was found to be mediated by both the long (L) and short (S) splice forms of the caspase-8 inhibitor, FLIP, another NFκB-regulated protein frequently overexpressed in CRPC. By decreasing FLIP expression at the post-transcriptional level in PC3 and DU145 cells (but not VCaP), the Class-I histone deacetylase (HDAC) inhibitor Entinostat promoted IAP antagonist-induced cell death in these models in a manner dependent on RIPK1, FADD and Caspase-8. Of note, Entinostat primarily targeted the nuclear rather than cytoplasmic pool of FLIP(L). While the cytoplasmic pool of FLIP(L) was highly stable, the nuclear pool was more labile and regulated by the Class-I HDAC target Ku70, which we have previously shown regulates FLIP stability. The efficacy of IAP antagonist (TL32711) and Entinostat combination and their effects on cIAP1 and FLIP respectively were confirmed in vivo, highlighting the therapeutic potential for targeting IAPs and FLIP in proinflammatory CRPC.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Animais , Caspase 8/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , NF-kappa B/metabolismo , Células PC-3 , Neoplasias de Próstata Resistentes à Castração/metabolismo , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo
10.
Eur J Med Chem ; 159: 282-291, 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30296687

RESUMO

Chalcones and 1,2-benzothiazines are two important classes of bioactive compounds, each scaffold endowed with diverse pharmacological activities. Combining both of these pharmacophores in a single molecule was aimed to yield multi-modal agents. Herein, we report a series of hybrid compounds 3a-3o derived from chalcones and 1,2-benzothiazine cores. They were synthesized from commercially available sodium saccharin, and the resulting 1,2-benzothiazine-derived ketone was then condensed with aromatic aldehydes in an aldol condensation to obtain the respective chalcones. The compounds were characterized using different analytical techniques including FT-IR, NMR spectroscopy, mass spectrometry and X-ray crystallography. Some synthesized chalcones revealed potent and/or selective inhibitory properties towards alkaline phosphatase isozymes transiently expressed in COS-7 cells. A detailed structure-activity and selectivity study was carried out with regard to the effect of different substituents at ortho-, meta- and para-positions of the phenyl residue. Compound 3c was the most effective human intestinal alkaline phosphatase (h-IAP) inhibitor (IC50 value of 1.04 µM), while it was not active against human tissue non-specific alkaline phosphatase (h-TNAP) isozyme. In contrast, 3i was a selective inhibitor of h-TNAP with IC50 values of 0.25 ±â€¯0.01 µM. The possible binding interactions of the most effective inhibitors of h-TNAP and h-IAP were obtained from molecular docking studies.


Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Chalcona/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Tiazinas/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Células COS , Chalcona/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Tiazinas/química
11.
Proc Natl Acad Sci U S A ; 115(40): E9317-E9324, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30181285

RESUMO

Protooncogene c-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.


Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Antineoplásicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
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.
FEBS J ; 285(17): 3286-3298, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30055105

RESUMO

Inhibitor of Apoptosis Proteins (IAPs) is highly conserved negative regulators of apoptosis overexpressed in many cancer cells. Based on their endogenous antagonist, Smac/DIABLO, mimic compounds (Smac-mimetics, SMs) have been developed to inhibit IAPs prosurvival activity, showing promising effects in advanced phases of clinical trials. Since different IAP homologs play distinctive roles in cancer cell survival and immunomodulation, SM-induced apoptosis proceeds through diverse mechanisms. After binding to their BIR3 domain, SMs have been shown to rapidly induce auto-ubiquitylation and degradation of cellular IAPs (cIAPs), thus leading to cell death mainly by activation of the noncanonical NF-κB pathway. For this reason, we started the BIR3-driven design of compounds selective for cIAP1 and with reduced affinity for X-linked IAP (XIAP), in order to focus SMs antitumor activity on cIAPs degradation. In this work, we describe the crystal structures of the BIR3 domains of cIAP1 and XIAP, each in complex with a cIAP1-selective SM (SM130 and SM114, respectively). The two SMs displayed 23- and 32-fold higher affinity for cIAP1-BIR3 over XIAP-BIR3 in molecular displacement experiments based on fluorescence polarization. In vitro cell-based assays confirmed that both selective SMs triggered apoptosis in cancer cells with different efficiencies by inducing caspases-3, -8, and -9-independent cIAP1 degradation. The design of cIAPs-selective compounds represents an innovative approach in the field of anticancer drugs development, being useful to elucidate different prosurvival mechanisms and to reduce the adverse effects of pan-IAPs compounds in cancer therapy. DATABASE: Structural data are available in the Protein Data Bank database under the accession codes 6EXW (cIAP1-BIR3/SM130 complex) and 6EY2 (XIAP-BIR3/SM114 complex).


Assuntos
Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Neoplasias da Mama/patologia , Desenho de Drogas , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Mitocondriais , Sequência de Aminoácidos , Antineoplásicos/química , Apoptose , Proteínas Reguladoras de Apoptose , Materiais Biomiméticos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Sobrevivência Celular , Cristalografia por Raios X , Feminino , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Células Tumorais Cultivadas
14.
Anticancer Res ; 38(7): 4281-4288, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29970562

RESUMO

BACKGROUND/AIM: Survivin expression has been shown to be associated with cancer progression, poor prognosis, and drug resistance. The aim of this study was to examine whether survivin knock-down could enhance paclitaxel-induced apoptosis in breast cancer cells in vitro. MATERIALS AND METHODS: MCF-7 cells were infected with an siRNA-expressing adenovirus vector against survivin (Adv-siSurv) or Renilla luciferase as a control (Adv-siRL). After treatment with paclitaxel, cells were analyzed by apoptotic, cell cycle and immunoblotting assays. RESULTS: Of cells treated with paclitaxel alone, only 20.2±2.08% showed apoptotic features. An increase in the paclitaxel dose was associated with increased survivin expression. In contrast, Adv-siSurv infection resulted in a marked increase in apoptotic cell death in paclitaxel-treated MCF-7 cells (49.9±7.70%). The percentage of cells in the G2M phase was lower (23.9±1.64%) in Adv-siSurv-infected cells than that of Adv-siRL-treated cells (40.0±2.43%). Adv-siSurv infection reduced survivin, procaspase-9, and procaspase-3 levels in paclitaxel-treated MCF-7 cells. CONCLUSION: Loss of survivin expression enhanced paclitaxel-induced apoptosis in MCF-7 breast cancer cells in vitro.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Terapia Genética/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Adenoviridae , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Farmacorresistência Fúngica/fisiologia , Feminino , Técnicas de Silenciamento de Genes/métodos , Vetores Genéticos , Humanos , Células MCF-7 , Paclitaxel/farmacologia , Survivina
15.
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
16.
Biochem Biophys Res Commun ; 503(1): 249-256, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29885833

RESUMO

Malignant melanoma has shown increased incidence and high mortality rate in the last three decades. In this study, we investigated whether combination therapy with ch282-5 (a novel BH3 mimetic) and microwave hyperthermia could display synergistic antitumor effects against melanoma. Our results indicated that combination therapy reduced the viability and proliferation of melanoma cells. Through inhibiting the expression of anti-apoptotic proteins of Bcl-2 and IAP family and activating MAPK proteins, combined hyperthermia enhanced ch282-5-induced apoptosis. Combination therapy also synergistically disturbed the mTOR/p70S6k signaling pathway, which is important for cell survival and migration. Moreover, our results showed that combination therapy remarkably suppressed melanoma cell migration in vitro and significantly reduced experimental pulmonary metastasis in vivo. In conclusion, our results indicate that combination therapy with ch282-5 and hyperthermia has synergistic antitumor effects and provides a possible therapeutic strategy for advanced melanoma.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Hipertermia Induzida , Melanoma/terapia , Neoplasias Cutâneas/terapia , Animais , Apoptose/efeitos dos fármacos , Biomimética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Terapia Combinada , Gossipol/análogos & derivados , Gossipol/farmacologia , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Micro-Ondas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
17.
Biochem Biophys Res Commun ; 501(1): 253-258, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29727601

RESUMO

High expression levels of survivin in KRAS-mutant lung adenocarcinomas are linked with unfavorable patient outcomes, suggesting that survivin is a promising target for tumor treatment. We found that trametinib, a MEK inhibitor, downregulates survivin expression in the RB1-positive KRAS-mutant lung adenocarcinoma cell lines H358 and H441. In these cell lines, trametinib treatment induced p21 expression and dephosphorylated RB1, leading to sustained suppression of survivin. Knockdown of p21 or RB1 restored survivin expression in trametinib-treated cells, at least partially, which supports the contribution of these molecules to trametinib-mediated survivin suppression. In RB1-negative KRAS-mutant lung adenocarcinoma H2009 cells, survivin downregulation by trametinib was only slight and transient, and trametinib-resistant (TR) cells developed within 1 month of treatment. H2009 TR cells depended much more on survivin for survival than its parental cells, as evidenced by apoptosis induction when survivin was depleted. These findings collectively suggest that trametinib is effective for the treatment of RB1-positive KRAS-mutant lung adenocarcinomas through sustained survivin suppression, but not for RB1-negative lung adenocarcinomas. Thus, the RB1 status could be a biomarker for trametinib application in KRAS-mutant lung adenocarcinomas.


Assuntos
Adenocarcinoma/tratamento farmacológico , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridonas/farmacologia , Pirimidinonas/farmacologia , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Ligação a Retinoblastoma/antagonistas & inibidores , Proteínas de Ligação a Retinoblastoma/genética , Survivina , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética
18.
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
19.
Biochem Biophys Res Commun ; 501(1): 48-54, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29678577

RESUMO

Breast cancer is the most common cancer among women worldwide. Chemoresistance remains to be a considerable obstacle in breast cancer therapy and it is often involves dysregulation of a variety of microRNAs (miRNAs). miR-485-5p functions as a tumor suppressor in several types of human cancers. However, its role in breast cancer chemosensitivity have not been determined. In the present study, we demonstrated that overexpression of miR-485-5p suppresses breast cancer progression and enhances chemosensitivity both in vitro and in vivo. Further study demonstrated that miR-485-5p directly targeted the 3'-untranslated region of survivin and overexpression of survivin overcomes the miR-485-5p induced effects on breast cancer. In conclusion, our study identified that miR-485-5p suppresses cancer progression and enhances the chemosensitivity by targeting survivin. Targeting survivin by miR-485-5p may provide a potential approach to reverse chemosensitivity in breast cancer cells.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/uso terapêutico , Regiões 3' não Traduzidas , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Genes Supressores de Tumor , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Células MCF-7 , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Survivina , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
20.
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
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