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1.
Phytomedicine ; 80: 153398, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33130474

RESUMO

BACKGROUND: Celastrol, a pentacyclic triterpenoid quinonemethide isolated from several spp. of Celastraceae family, exhibits anti-inflammatory activities in a variety of diseases including arthritis. PURPOSE: This study aims to investigate whether the inhibition of NLRP3 inflammasome is engaged in the anti-inflammatory activities of celastrol and delineate the underlying mechanism. METHODS: The influence of celastrol on NLRP3 inflammasome activation was firstly studied in lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) and phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells treated with nigericin. Reconstituted inflammasome was also established by co-transfecting NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in HEK293T cells. The changes of inflammasome components including NLRP3, ASC, pro-caspase-1/caspase-1 and pro-IL-1ß/IL-1ß were examined by enzyme-linked immunosorbent assay (ELISA), western blotting and immunofluorescence. Furthermore, Propionibacterium acnes (P. acnes)/LPS-induced liver injury and monosodium urate (MSU)-induced gouty arthritis in mice were employed in vivo to validate the inhibitory effect of celastrol on NLRP3 inflammasome. RESULTS: Celastrol significantly suppressed the cleavage of pro-caspase-1 and pro-IL-1ß, while not affecting the protein expressions of NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in THP-1 cells, BMDMs and HEK293T cells. Celastrol suppressed NLRP3 inflammasome activation and alleviated P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis. Mechanism study revealed that celastrol could interdict K63 deubiquitination of NLRP3, which may concern interaction of celastrol and BRCA1/BRCA2-containing complex subunit 3 (BRCC3), and thereby prohibited the formation of NLRP3, ASC and pro-caspase-1 complex to block the generation of mature IL-1ß. CONCLUSION: Celastrol suppresses NLRP3 inflammasome activation in P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3, which presents a novel insight into inhibition of celastrol on NLRP3 inflammasome and provides more evidences for its application in the therapy of inflammation-related diseases.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite Gotosa/tratamento farmacológico , Fígado/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Triterpenos/farmacologia , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Células HEK293 , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/microbiologia , Fígado/patologia , Lisina/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Propionibacterium acnes/patogenicidade , Células THP-1 , Ubiquitinação/efeitos dos fármacos , Ácido Úrico/toxicidade
2.
Nat Commun ; 11(1): 6316, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33298911

RESUMO

The transcription factor MYC is deregulated in almost all human cancers, especially in aggressive lymphomas, through chromosomal translocation, amplification, and transcription hyperactivation. Here, we report that high expression of tribbles homologue 3 (TRIB3) positively correlates with elevated MYC expression in lymphoma specimens; TRIB3 deletion attenuates the initiation and progression of MYC-driven lymphoma by reducing MYC expression. Mechanistically, TRIB3 interacts with MYC to suppress E3 ubiquitin ligase UBE3B-mediated MYC ubiquitination and degradation, which enhances MYC transcriptional activity, causing high proliferation and self-renewal of lymphoma cells. Use of a peptide to disturb the TRIB3-MYC interaction together with doxorubicin reduces the tumor burden in MycEµ mice and patient-derived xenografts. The pathophysiological relevance of UBE3B, TRIB3 and MYC is further demonstrated in human lymphoma. Our study highlights a key mechanism for controlling MYC expression and a potential therapeutic option for treating lymphomas with high TRIB3-MYC expression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Linfoma não Hodgkin/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , RNA-Seq , Proteínas Repressoras/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 117(44): 27412-27422, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33087562

RESUMO

Nuclear receptor Nur77 participates in multiple metabolic regulations and plays paradoxical roles in tumorigeneses. Herein, we demonstrated that the knockout of Nur77 stimulated mammary tumor development in two mouse models, which would be reversed by a specific reexpression of Nur77 in mammary tissues. Mechanistically, Nur77 interacted and recruited corepressors, the SWI/SNF complex, to the promoters of CD36 and FABP4 to suppress their transcriptions, which hampered the fatty acid uptake, leading to the inhibition of cell proliferation. Peroxisome proliferator-activated receptor-γ (PPARγ) played an antagonistic role in this process through binding to Nur77 to facilitate ubiquitin ligase Trim13-mediated ubiquitination and degradation of Nur77. Cocrystallographic and functional analysis revealed that Csn-B, a Nur77-targeting compound, promoted the formation of Nur77 homodimer to prevent PPARγ binding by steric hindrance, thereby strengthening the Nur77's inhibitory role in breast cancer. Therefore, our study reveals a regulatory function of Nur77 in breast cancer via impeding fatty acid uptake.


Assuntos
Neoplasias da Mama/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , PPAR gama/metabolismo , Fenilacetatos/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ácidos Graxos/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Metabolismo dos Lipídeos/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Camundongos , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , PPAR gama/agonistas , Cultura Primária de Células , Prognóstico , Proteólise/efeitos dos fármacos , Análise Serial de Tecidos , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação/efeitos dos fármacos
4.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
5.
Sci Rep ; 10(1): 15543, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968148

RESUMO

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ácido Oleanólico/análogos & derivados , Fatores de Transcrição/metabolismo , Azepinas/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Triazóis/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
6.
Phytomedicine ; 78: 153318, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32896707

RESUMO

BACKGROUND: Hypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood. PURPOSE: The aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α. STUDY DESIGN: We investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities. MATERIALS AND METHODS: We performed the hypoxia-responsive element promoter-reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis. RESULTS: We observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin. CONCLUSION: Our results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzopiranos/farmacologia , Butiratos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Células A549 , Animais , Apoptose/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos Endogâmicos C57BL , Hipóxia Tumoral/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Am J Physiol Cell Physiol ; 319(5): C933-C944, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32936699

RESUMO

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.


Assuntos
Caveolina 1/genética , ATPases Transportadoras de Cobre/genética , Células Endoteliais/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase/genética , Animais , Aorta/citologia , Aorta/metabolismo , Caveolina 1/deficiência , Cobre/farmacologia , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Masculino , Artérias Mesentéricas/citologia , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/metabolismo , Ubiquitinação/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
8.
Toxicol Appl Pharmacol ; 408: 115253, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991915

RESUMO

Exposure to arsenic, which occurs via various routes, can cause reproductive toxicity. However, the mechanism for arsenic-induced reproductive disorders in male mice has not been extensively investigated. Here, 6-week-old male mice were dosed to 0, 5, 10, or 20 ppm sodium arsenite (NaAsO2), an active form of arsenic, in drinking water for six months. For male mice exposed to arsenite, fertility was lower compared to control mice. Moreover, for exposed mice, there were lower sperm counts, lower sperm motility, and higher sperm malformation ratios. Further, the mRNA and protein levels of the gonadotropin-regulated testicular RNA helicase (DDX25) and chromosome region maintenance-1 protein (CRM1), along with proteins associated with high mobility group box 2 (HMGB2), phosphoglycerate kinase 2 (PGK2), and testicular angiotensin-converting enzyme (tACE) were lower. Furthermore, chronic exposure to arsenite led to lower H2A ubiquitination (ubH2A); histone H3 acetylation K18 (H3AcK18); and histone H4 acetylations K5, K8, K12, and K16 (H4tetraAck) in haploid spermatids from testicular tissues. These alterations disrupted deposition of protamine 1 (Prm1) in testes. Overall, the present results indicate that the ubiquitination and acetylation of histones is involved in the spermiogenesis disorders caused by chronic exposure to arsenite, which points to a previously unknown connection between the modification of histones and arsenite-induced male reproductive toxicity.


Assuntos
Arsenitos/toxicidade , Histonas/metabolismo , Reprodução/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Feminino , Reabsorção do Feto , Masculino , Troca Materno-Fetal , Camundongos Endogâmicos C57BL , Gravidez , Análise do Sêmen , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/anormalidades , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Testículo/anormalidades , Testículo/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 117(37): 23106-23112, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32848052

RESUMO

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.


Assuntos
Hipnóticos e Sedativos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Teratogênios/metabolismo , Talidomida/farmacologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(35): 21588-21597, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817432

RESUMO

Proteasome inhibitors, such as bortezomib (BTZ), are highly effective and widely used treatments for multiple myeloma. One proposed reason for myeloma cells' exceptional sensitivity to proteasome inhibition is that they produce and continually degrade unusually large amounts of abnormal immunoglobulins. We, therefore, hypothesized that, heat shock may also be especially toxic to myeloma cells by causing protein unfolding, increasing further the substrate load on proteasomes, and, thus, putting further stress on their capacity for protein homeostasis. After a shift from 37 to 43 °C, all four myeloma lines studied underwent extensive apoptosis in 4 h, unlike 13 nonmyeloma cell lines, even though the myeloma cells induced heat-shock proteins and increased protein degradation similar to other cells. Furthermore, two myeloma lines resistant to proteasome inhibitors were also more resistant to 43 °C. Shifting myeloma cells to 43, 41, or 39 °C (which was not cytotoxic) dramatically increased their killing by proteasome inhibitors and inhibitors of ubiquitination or p97/VCP. Combining increased temperature with BTZ increased the accumulation of misfolded proteins and substrate load on the 26S proteasome. The apoptosis seen at 43 °C and at 39 °C with BTZ was mediated by caspase-9 and was linked to an accumulation of the proapoptotic Bcl-2-family member Noxa. Thus, myeloma cells are exceptionally sensitive to increased temperatures, which greatly increase substrate load on the ubiquitin-proteasome system and eventually activate the intrinsic apoptotic pathway. Consequently, for myeloma, mild hyperthermia may be a beneficial approach to enhance the therapeutic efficacy of proteasome inhibitors.


Assuntos
Resposta ao Choque Térmico/fisiologia , Mieloma Múltiplo/metabolismo , Proteostase/fisiologia , Antineoplásicos/farmacologia , Apoptose/fisiologia , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Temperatura Alta/uso terapêutico , Humanos , Mieloma Múltiplo/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
11.
Nat Chem Biol ; 16(11): 1199-1207, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32747809

RESUMO

Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.


Assuntos
Acetamidas/química , Antibacterianos/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Doxiciclina/farmacologia , Hidrazinas/química , Indóis/química , Proteólise/efeitos dos fármacos , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Humanos , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
12.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(1): 1-19, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32621419

RESUMO

Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, ß-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.


Assuntos
Antineoplásicos , Descoberta de Drogas , Ubiquitina-Proteína Ligases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Neoplasias/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
13.
Nat Commun ; 11(1): 3660, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694521

RESUMO

High expression or aberrant activation of epidermal growth factor receptor (EGFR) is related to tumor progression and therapy resistance across cancer types, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) are first-line therapy for NSCLC. However, patients eventually deteriorate after inevitable acquisition of EGFR TKI-resistant mutations, highlighting the need for therapeutics with alternative mechanisms of action. Here, we report that the elevated tribbles pseudokinase 3 (TRIB3) is positively associated with EGFR stability and NSCLC progression. TRIB3 interacts with EGFR and recruits PKCα to induce a Thr654 phosphorylation and WWP1-induced Lys689 ubiquitination in the EGFR juxtamembrane region, which enhances EGFR recycling, stability, downstream activity, and NSCLC stemness. Disturbing the TRIB3-EGFR interaction with a stapled peptide attenuates NSCLC progression by accelerating EGFR degradation and sensitizes NSCLC cells to chemotherapeutic agents. These findings indicate that targeting EGFR degradation is a previously unappreciated therapeutic option in EGFR-related NSCLC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Adulto , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Taxa de Sobrevida , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Med Chem ; 63(15): 8554-8566, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32678592

RESUMO

In preclinical and phase I and II clinical studies, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) displays a potent and long-lasting inhibition of HIV-1 infection. To investigate its mechanism of action, we compared it with the well-documented lamivudine (3TC). Pharmacokinetic studies revealed that the intracellular retention of FNC triphosphate in peripheral blood mononuclear cells was markedly longer than that of the 3TC triphosphate. FNC selectively enters and is retained in HIV target cells, where it exerts long-lasting prevention of HIV-1 infection. In addition to inhibition of HIV-1 reverse transcription, FNC also restores A3G expression in CD4+ T cells in FNC-treated HIV-1 patients. FNC binds to the Vif-E3 ubiquitin ligase complex, enabling A3G to avoid Vif-induced ubiquitination and degradation. These data reveal the mechanisms underlying the superior anti-HIV potency and long-lasting action of FNC. Our results also suggest a potential clinical application of FNC as a long-lasting pre-exposure prophylactic agent capable of preventing HIV infection.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Azidas/uso terapêutico , Desoxicitidina/análogos & derivados , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Lamivudina/uso terapêutico , Animais , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/farmacologia , Azidas/farmacocinética , Azidas/farmacologia , Desoxicitidina/farmacocinética , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Infecções por HIV/metabolismo , HIV-1/fisiologia , Humanos , Lamivudina/farmacocinética , Lamivudina/farmacologia , Macaca mulatta , Modelos Moleculares , Inibidores da Transcriptase Reversa/farmacocinética , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/uso terapêutico , Ubiquitinação/efeitos dos fármacos
15.
Endocrinology ; 161(9)2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32609836

RESUMO

Invasive lobular breast carcinoma (ILC) accounts for 10% to 15% of breast cancers diagnosed annually. Evidence suggests that some aspects of endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. We analyzed The Cancer Genome Atlas dataset and observed lower levels of ESR1 mRNA (P = 0.002) and ERα protein (P = 0.038) in ER+ ILC (n = 137) compared to IDC (n = 554), and further confirmed the mRNA difference in a local UPMC cohort (ILC, n = 143; IDC, n = 877; P < 0.005). In both datasets, the correlation between ESR1 mRNA and ERα protein was weaker in ILC, suggesting differential post-transcriptional regulation of ERα. In vitro, 17ß-estradiol (E2) decreased the rate of degradation and increased the half-life of ERα in ILC cell lines, whereas the opposite was observed in IDC cell lines. Further, E2 failed to induce robust ubiquitination of ERα in ILC cells. To determine the potential clinical relevance of these findings, we evaluated the effect of 2 selective estrogen receptor downregulators (SERDs), ICI 182,780 and AZD9496, on ERα turnover and cell growth. While ICI 182,780 and AZD9496 showed similar effects in IDC cells, in ILC cell lines, AZD9496 was not as effective as ICI 182,780 in decreasing ERα stability and E2-induced proliferation. Furthermore, AZD9496 exhibited partial agonist activity in growth assays in ILC cell lines. Our study provides evidence for a distinct ERα regulation by SERDs in ILC cell lines, and therefore it is important to include ILC models into preclinical and clinical testing of novel SERDs.


Assuntos
Neoplasias da Mama , Carcinoma Lobular , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Invasividade Neoplásica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
16.
Life Sci ; 256: 117893, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502539

RESUMO

AIMS: To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF). MATERIALS AND METHODS: Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation. KEY FINDINGS: Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result. SIGNIFICANCE: Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.


Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Pulmão/embriologia , Meliteno/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/sangue , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiprolina/metabolismo , Proteínas de Neoplasias/sangue , Proteínas Nucleares/sangue , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação/efeitos dos fármacos , Vimentina/metabolismo
17.
Biochim Biophys Acta Mol Basis Dis ; 1866(10): 165888, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32599142

RESUMO

Nucleus pulposus (NP) degeneration plays pivotal roles in intervertebral disc degeneration. The effect and mechanism of oxidative stress and epigenetics in NP degeneration is still unclear. We performed this study to evaluate the function of oxidative stress in NP and to explore the potential mechanism of ROS induced expression of matrix metalloproteinases (MMPs). We tested four methyltransferases, KMT2A, KMT2B, KMT2C and KMT2D in human NP samples, only KMT2D was significantly up-regulated in the severe degeneration samples. Knockdown of Kmt2d by siRNA significantly down-regulated the expression levels of catabolic enzymes including Mmp3, Mmp9 and Mmp13. Moreover, an interaction between KMT2D and ubiquitination was confirmed, and the application of H2O2 abrogated this process. Co-IP assay confirmed that H2O2 induced the phosphorylation of KMT2D to block the ubiquitination degradation, which was mainly mediated by phosphorylation of p38/MAPK. Further investigation suggested that ROS induced the alteration in levels of methylation is linked to H3K4me1 and H3K4me2, but not me3. However, usage of OICR-9429 (OICR) also suppressed the expression levels of Mmp3, Mmp9 and Mmp13. In an ex vivo model, application of OICR-9429 (OICR) also attenuated the degeneration of NP according to the H&E and Safranin-O/Fast Green staining assay, and the protein levels of MMP3, MMP9 and MMP13 were down-regulated, as well. In conclusion, we approved that oxidative stress induced ROS production promote the process of NP degeneration by enhancing KMT2D mediated transcriptional regulation of matrix degeneration related genes during NP degeneration.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Degeneração do Disco Intervertebral/patologia , Proteínas de Neoplasias/metabolismo , Núcleo Pulposo/patologia , Animais , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Di-Hidropiridinas/farmacologia , Di-Hidropiridinas/uso terapêutico , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Neoplasias/genética , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Ubiquitinação/efeitos dos fármacos , Regulação para Cima
18.
Oncogene ; 39(27): 5001-5014, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32523091

RESUMO

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.


Assuntos
Antineoplásicos/farmacologia , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Células da Medula Óssea/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Interferência de RNA , RNA Interferente Pequeno/genética , Índice Terapêutico do Medicamento , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos
19.
Mol Pharmacol ; 98(3): 222-233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32587095

RESUMO

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Nucleosídeos/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Linhagem Celular , DNA/metabolismo , DNA Topoisomerases Tipo II/química , Humanos , Células K562 , Proteínas de Ligação a Poli-ADP-Ribose/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
20.
Biochim Biophys Acta Mol Cell Res ; 1867(10): 118761, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32485270

RESUMO

Evasion from apoptosis is one of the hallmarks of cancer. X-linked inhibitor of apoptosis protein (XIAP) is known to modulate apoptosis by inhibiting caspases and ubiquitinating target proteins. XIAP is mainly found at the cytoplasm, but recent data link nuclear XIAP to poor prognosis in breast cancer. Here, we generated a mutant form of XIAP with a nuclear localization signal (XIAPNLS-C-term) and investigated the oncogenic mechanisms associated with nuclear XIAP in breast cancer. Our results show that cells overexpressing XIAPΔRING (RING deletion) and XIAPNLS-C-term exhibited XIAP nuclear localization more abundantly than XIAPwild-type. Remarkably, overexpression of XIAPNLS-C-term, but not XIAPΔRING, conferred resistance to doxorubicin and increased cellular proliferative capacity. Interestingly, Survivin and c-IAP1 expression were not associated with XIAP oncogenic effects. However, NFκB expression and ubiquitination of K63, but not K48 chains, were increased following XIAPNLS-C-term overexpression, pointing to nuclear signaling transduction. Consistently, multivariate analysis revealed nuclear, but not cytoplasmic XIAP, as an independent prognostic factor in hormone receptor-negative breast cancer patients. Altogether, our findings suggest that nuclear XIAP confers poor outcome and RING-associated breast cancer growth and chemoresistance.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lisina/metabolismo , Análise Multivariada , Proteínas Mutantes/metabolismo , Mutação/genética , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Prognóstico , Domínios Proteicos , Receptores de Superfície Celular/metabolismo , Análise de Sobrevida , Ubiquitinação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química
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